higher biology pcr essay

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AP®︎/College Biology

Course: ap®︎/college biology > unit 6.

Introduction to genetic engineering
Intro to biotechnology
DNA cloning and recombinant DNA
Overview: DNA cloning

Polymerase chain reaction (PCR)

Gel electrophoresis
DNA sequencing
Applications of DNA technologies
Biotechnology

Key points:

Polymerase chain reaction , or PCR , is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism).
PCR relies on a thermostable DNA polymerase, Taq polymerase , and requires DNA primers designed specifically for the DNA region of interest.
In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.
PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.

What is PCR?

Taq polymerase, pcr primers, the steps of pcr.

Denaturation ( 96 ° C ‍ ): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
Annealing ( 55 ‍ - ‍ 65 ‍ ° C ‍ ): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension ( 72 ° C ‍ ): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

Using gel electrophoresis to visualize the results of PCR

Applications of pcr, sample problem: pcr in forensics.

(Choice A) Suspect 1 ‍ A Suspect 1 ‍
(Choice B) Suspect 2 ‍ B Suspect 2 ‍
(Choice C) Suspect 3 ‍ C Suspect 3 ‍
(Choice D) None of the suspects D None of the suspects
Crime scene DNA: homozygous 200 ‍ bp allele
Suspect 1 ‍ : homozygous 300 ‍ bp allele
Suspect 2 ‍ : heterozygous
Suspect 3 ‍ homozygous 200 ‍ bp allele

More about PCR and forensics

Attribution:, works cited:.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Forensic evidence and genetic profiles. (10th ed., pp. 430-431). San Francisco, CA: Pearson.

References:

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3.5: Polymerase Chain Reaction (PCR)

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Ross Hardison
The Pennsylvania State University

The polymerase chain reaction ( PCR) is now one of the most commonly used assays for obtaining a particular segment of DNA or RNA. It is rapid and extremely sensitive. By amplifying a designated segment of DNA, it provides a means to isolate that particular DNA segment or gene. This method requires knowledge of the nucleotide sequence at the ends of the region that you wish to amplify. Once that is known, one can make large quantities of that region starting with miniscule amounts of material, such as the DNA within a single human hair. With the availability of almost complete or complete sequences of genomes from many species, the range of genes to which it can be applied is enormous. The applications of PCR are numerous, from diagnostics to forensics to isolation of genes to studies of their expression.

The power of PCR lies in the exponential increase in amount of DNA that results from repeated cycles of DNA synthesis from primers that flank a given region, one primer designed to direct synthesis complementary to the top strand, the other designed to direct synthesis complementary to the bottom strand (Figure \(\PageIndex{1}\)). When this is done repeatedly, there is roughly a 2-fold increase in the amount of synthesized DNA in each cycle. Thus it is possible to generate a million-fold increase in the amount of DNA from the amplified region with a sufficient number of cycles. This exponential increase in abundance is similar to a chemical chain reaction, hence it is called the polymerase chain reaction.

The events in the polymerase chain reaction are examined in more detail in Figure \(\PageIndex{2}\). The several panels show what happens in each cycle. Each cycle consists of a denaturation step at a temperature higher than the melting temperature of the duplex DNA (e.g. 95 o C ), then an annealing step at a temperature below the melting temperature for the primer-template (e.g. 55 o C), followed by extension of the primer by DNA polymerase using dNTPs provided in the reaction. This is done at the temperature optimum for the DNA polymerase (e.g. 70 o C for a thermostable polymerase). Thermocylers are commercially available for carrying out many cycles quickly and reliably (Figure \(\PageIndex{3}\)).

The template supplied for the reaction is the only one available in the first cycle, and it is still a major template in the second cycle. At the end of the second cycle, a product is made whose ends are defined by primers. This is the desired product, and it serves as the major template for the remaining cycles. The initial template is still present and can be used, but it does not undergo the exponential expansion observed for the desire product.

If n is the number of cycles, the amount of desired product is approximately 2 n-1 –2 times the amount of input DNA (between the primers). Thus in 21 cycles, one can achieve a million-fold increase in the amount of that DNA (assuming all cycles are completely efficient). A sample with 0.1 pg of the segment of DNA between the primers can be amplified to 0.1 mg in 21 cycles, in theory. In practice, roughly 25-35 cycles are done in many PCR assays.

The ease if doing PCR was greatly increased by the discovery of DNA polymerases that were stable at high temperatures. These have been isolated from bacteria that grow in hot springs, such as those found in Yellowstone National Park, such as Thermus aquaticus . The Taq polymerase from this bacterium will retain activity even at the high temperatures needed for melting the templates, and it is active at a temperature between the melting and annealing temperature. This particular polymerase is rather error-prone, and other thermostable polymerases have been discovered that are more accurate.

Schools & departments

PCR in Action

This video has been designed to support Higher Biology and Higher Human Biology learners to develop their knowledge and understanding of PCR.

Our interactive video series Lab Techniques in Action shows commonly used lab techniques that are explored in the Scottish Curriculum for Excellence. These videos form part of the National e-Learning Offer by Education Scotland and are available to support your learners to develop their knowledge and understanding of these techniques.

This video shows the laboratory technique Polymerase Chain Reaction, known as PCR, which is used by scientists; to make many copies of a specific section of DNA. It has been designed specifically for pupils studying the Scottish Curriculum for Excellence Higher Biology & Higher Human Biology courses.

This video is not an instructional video and should not be used as a resource to carry out the techniques in your school. If you do wish to carry out PCR in your school you can visit the SSERC website for more information. If you would like your Higher pupils to get hands-on with PCR then you can bring them to us to take part in our PCR Masterclass: A Question of Taste workshop.

Watch PCR in Action

Online Pupil Assessment

PCR Simulation Activity

How to Build a Bone Critical Reading Text

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Safety notice:

The experiments described in this video by the University of Edinburgh are potentially hazardous and require a high level of safety training, specialist facilities, equipment and supervision by highly trained individuals. These videos should not be used as instructional videos for you to carry out or attempt to carry out the technique described in the video. The University of Edinburgh is not liable for the actions or activity of any person who uses the information in this resource for anything other than educational demonstration purposes. The University of Edinburgh assumes no liability with regard to injuries or damage to property that may occur as a result of using the information within this video and carrying out the practical activities contained in this resource or in any of the suggested further resources, if you use them for anything other than educational demonstration purposes.

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Polymerase Chain Reaction (PCR) Fact Sheet

Polymerase chain reaction (PCR) is a technique used to "amplify" small segments of DNA.

What is PCR?

Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993.

What is PCR used for?

Once amplified, the DNA produced by PCR can be used in many different laboratory procedures. For example, most mapping techniques in the Human Genome Project (HGP) relied on PCR. PCR is also valuable in a number of laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders.

How does PCR work?

To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

Last updated: August 17, 2020

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Validating Real-Time Polymerase Chain Reaction (PCR) Assays

For many years, the development of assays took place in the laboratories where the test was required. Whilst more and more commercially developed tests have become available, novel assays continue to be developed in academic hospital laboratories. Although commercial kits are more expensive than LDTs, they enable the rapid introduction of assays, with the advantage of being CE marked or FDA approved. However, this approval does not necessarily guarantee the rigorous validation of the assay. Furthermore, commercial assays have to be commercially attractive, which is unlikely for small-scale, specialist tests targeting rarely occurring infectious pathogens and thus there will continue to be a need for laboratory-developed tests.

Introduction

For many years, the development of assays took place in the laboratories where the test was required, the so-called in-house or LDT. Although more and more commercially-developed tests have become available, novel assays continue to be developed in academic hospital laboratories. These laboratories are more able to respond quickly to new and re-emerging infections and crucially have the samples necessary to develop the test. This was the case with the new human coronavirus causing clusters of pneumonia epidemiologically linked to a seafood market in Wuhan, China in December 2019. Researchers from Shanghai, Wuhan, Beijing and Sydney sequenced a sample from a patient who had worked at the market; the sequence was deposited in the publicly available GenBank database on 10th January. Further sequencing showed the virus shared 85% identity with a SARS-like coronavirus found in bats. The first LDT was published on 23rd January 2020, followed a month later by the first of the commercial tests.

Although often more expensive than LDTs, commercial kits enable the rapid introduction of new tests, with the further advantage of being CE marked or FDA approved, providing some form of reassurance to the laboratory. However, CE marking is only a declaration of compliance with European legislative requirements; it does not necessarily guarantee the rigorous validation of the assay, or give any details of how and when a test should be used. Furthermore, commercial assays, by definition, have to be commercially attractive. Therefore, specialist, small-scale tests for rarely occurring infectious pathogens will not be cost-effective. Thus, there will continue to be a need for laboratory-developed assays.

The Need for Validation

In-house assays.

Over the years many laboratories have established methodologies for validating their assays. However, the literature continues to show a lack of detail in some critical areas, e.g., optimization of extraction techniques, methods used in primer and probe design, no evidence of amplicon sequencing to confirm specificity, imprecise estimates of sensitivity and specificity and assays that do not include internal or extraction controls. Such lack of detailed experimental information makes assessing the clinical utility of the assay difficult. Some of these problems lie with the scientific literatures’ approach to publishing, where space limitations restrict the amount of detail permitted in a paper. These difficulties have led to a number of papers calling for an improvement in the standards of reporting diagnostic assays, including the STARD initiative (standards for reporting of diagnostic accuracy) and the MIQE guidelines (the minimum information for publication of quantitative real-time PCR experiments; Bustin et al ., 2009 ). In addition to the fundamental requirements of good scientific practice, there are a number of regulatory bodies that require assays to be validated to certain standards, such as the FDA and CLIA requirements in the USA and the IVD Regulations (EU) 2017/7462017 in Europe. There is also an obligation on health institutions to be accredited according to the ISO 15189 standard. There has been an ongoing debate in Europe regarding CE marking of LDTs, similarly in the USA the FDA announced its intention to shift from a policy of enforcement discretion to exercising regulatory oversight at a future date on LDTs. However, the imposition of rigorous controls on the development of assays must not stifle innovation and the ability of front-line laboratories to respond quickly to new and emerging threats. Under these circumstances it makes sense for laboratories developing and publishing assays to ensure that they produce rigorously documented experimental proof to support any potential CE-marking/IVDD requirements that may be necessary in the future.

Commercial Assays

Although commercial assays are typically CE marked or FDA approved this does not necessarily mean the assay has been validated for all applications. Furthermore, although the assay may perform acceptably in the commercial developer’s laboratory, a number of factors can affect the assay’s performance elsewhere, e.g., staff competences and workflow systems, where not all laboratories may have separate working areas recommended to reduce contamination. There may also be differences in equipment maintenance schedules, including anything from freezers and pipettes to thermal cyclers, which can fundamentally affect the assay. Therefore, it is necessary for laboratories to verify the stated criteria. Although there are no formal requirements in the UK for laboratories to evaluate newly-introduced commercial assays, in the USA, FDA-approved, FDA-modified (i.e., tests modified from the manufacturer’s instructions but with performance characteristics determined by the user laboratory, consistent with CLIA requirements) and LDTs require verification before introducing the test into the clinical laboratory. CLIA stipulates that prior to implementation of an FDA-cleared test laboratories must verify the manufacturer’s performance specifications. Where a FDA-modified test or LDT is to be introduced, CLIA stipulates that, additionally, analytical sensitivity and specificity must be established.

In an attempt to clarify the situation, it is the intention in this article to provide a set of basic working guidelines for the validation process. These guidelines can be applied throughout the continuous process of maintaining the validated status of an assay. The key concepts in the process of validating an assay are illustrated in Fig. 1 .

Assay validation: Key concepts.

The Validation Process: Consultation Stage

The process begins with the development of a validation plan and involves decisions based on the clinical need for the assay, e.g., epidemiological studies, infection control or screening. As discussed previously, there are differences between the levels of validation laboratories perform when introducing commercial or LDTs. As a minimum, the laboratory introducing a commercial test must establish that the manufacturer’s performance claims can be reproduced. Once the performance characteristics of an assay have been met, whether commercial or LDT, the validation exercise must continue on a daily basis (see Fig. 1 ). This involves continually monitoring the levels of internal and external positive controls to ensure the validation status of the assay is maintained.

Preliminary Considerations

The initial step is to define the purpose of the assay; all the subsequent steps in the validation process are guided by this decision. The following three variables that can affect an assay’s performance must be considered at this stage:

(1) The sample-type and the host/pathogen interactions that determine whether a qualitative or quantitative assay is required.
(2) The assay system: the biological, technical and operator-related factors that affect the assay’s ability to detect the target in the specific sample-type. All assays are considered multiplex, since they must include a co-amplified extraction control.
(3) The result: will the result accurately predict the status of an individual or population in regard to the analyte detected?

The sample-type (e.g., tissue, whole blood, CSF) may contain inhibitors that affect the activity of the polymerase in the PCR. Therefore, the extraction process needs to be evaluated and necessary alternatives considered. Quantification assays are useful in the management of patients, where pathogen load can be monitored during therapy. Some viral pathogens, such as EBV and CMV can establish a primary low-level latent infection and subsequently become reactivated, switching to a productive lytic infection. Is more than one pathogen to be identified in a multiplex assay? Another significant factor is the availability of sufficient numbers of well-characterized positive control samples to enable the validation. In order to maintain objectivity, the method chosen to resolve discrepant results must be established as part of the validation plan before testing begins.

A quality assurance plan must be established for the assay. Consideration must also be given to the availability of external QA reagents. This can be a problem with new assays targeting rare pathogens, where QA panels are unlikely to be available; the laboratory may need to consider working with the providers to produce suitable reagents. Once the requirement for a test has been established, the next decision is whether to use a commercial assay or develop a LDT. In the USA the CLIA specify that laboratories using FDA-cleared assays must verify that the manufacturer’s performance specifications for accuracy, precision, reportable range and reference intervals can be replicated. For modified commercial assays the following should also be tested:

(1) Analytical Sensitivity (LOD).
(2) Analytical Specificity to include inhibitory substances.
(3) Any other performance characteristics required for test performance.

When assessing a commercial assay, the extraction process also needs to be verified. In most cases the manufacturer’s protocol includes details on the extraction process to use. However, the assay may need to be validated with a number of different extraction methods, depending on the type of equipment available.

The Validation plan

Verification : the process of establishing whether the individual components of an assay meet the analytical performance requirements established at the start of the development process.

Validation : the process of ensuring that the completed assay conforms to the users’ needs, requirements, and/or specifications under defined operating conditions.

There are numerous aspects of an assay that need to be continuously monitored throughout its use in the laboratory. Micro-organisms, particularly viruses, often mutate. This means that the efficiency of the PCR needs to be monitored for potential false-negative results, as this may be the first sign that the primers and/or probe need to be updated and revalidated. Manufacturers continually develop new buffers, enzymes and extraction kits, these must be assessed and if adopted, re-verified, and the assay revalidated. New types of probe chemistry are constantly being introduced and these, too, need testing to maintain or improve the assay’s performance.

Although each new assay may present its own particular challenges, the basic criteria for validation, such as specificity, sensitivity and reproducibility apply and any specific differences can be incorporated without difficulty. The first steps concern decisions based on the clinical need for the test, i.e., the scope, purpose and application of the assay and whether a commercial or a LDT will be required. For all in-house developments it is strongly recommended that the comprehensive MIQE guidelines are followed.

Analytical Verification Stage

Reference materials and sample numbers.

The first question that arises when developing a LDT, particularly for rare and emerging infectious disease pathogens, is the availability of samples. If sufficient samples are not available, are they obtainable elsewhere? If not, it may be necessary to construct test samples by spiking various concentrations of the analyte into a suitable matrix. Other sources of suitable samples may be other clinical/research laboratories or commercial standards, quality control materials or proficiency panels. Typically, 100 samples of 50–80 positive and 20–50 negative specimens are used. Consideration also needs to be given to potential inhibitory substances likely to be found in the specimens tested. Therefore, paired control specimens should be prepared by adding low concentrations of the analyte, with and without the known inhibitors, to suitable negative samples. However, such artificially constructed samples are unlikely to have the same properties as clinical samples. Therefore, when sufficient numbers of genuine samples become available, the assay and extraction methods will need to be re-assessed. Where samples are available, more than one specimen type may be required, e.g., respiratory pathogens can be extracted from nasopharyngeal aspirates, bronchoalveolar lavages or nose and throat swabs. A literature search should always be carried out to determine the type of specimens to be assessed and the most efficient extraction method for each of the sample type determined.

When sufficient samples numbers are available, the numbers need to be calculated. Ideally this should be determined by statistical analyzes, where the sample size required to detect a significant difference is determined by the standard deviation from the difference of the means of paired samples used in the test under development and the gold standard comparator. Alternatively, they can be estimated from Table 1 below. This method allows for either a 2% or a 5% calculated error in diagnostic sensitivity and specificity. So, for example, if a 2% error is assumed, the number of samples required for an assay with 99% confidence and 99% estimated sensitivity/specificity is 164. However, if the same assay achieved a 95% estimated sensitivity/specificity then the samples required to achieve a high confidence (99%) would be 788.

Theoretical number of samples from subjects of known infection status required for establishing diagnostic specificity and sensitivity estimates using likely estimated specificity/sensitivity value and desired error margin and confidence

Template, Primers and Probes: In-Silico Design

A thorough literature review is first carried out to identify a suitable locus to be amplified. It may be necessary to consider using degenerate primers and possibly probes to cover sequence variants that occur in different strains of the pathogen. Thorough sequence searches with BLAST (Basic Local Alignment Search Tool; See “Relevant Websites section”) are required to ensure as many variants as possible are included in the design. A local database can be constructed and aligned using one (or more) of the sequence alignment program available, such as CLUSTALW (See “Relevant Websites section”). Optimal primer and probe function is critical to successful PCR. There a number of software packages available to assist in primer and probe design, such as Primer Express (Applied Biosystems) and OLIGO (See “Relevant Websites section”). If the assay is a multiplex, the sets of primers and probes must be checked for cross-reactivity, to eliminate any inhibition due to co-amplification issues. Multiplexing is a useful method of syndromic testing and reducing the cost diagnoses by targeting more than one pathogen in a sample. Furthermore, most, if not all assays will be multiplexed, due to the co-amplified EC.

Any matches with other sequences, particularly from other pathogens likely to be encountered in the sample types under investigation will necessitate redesign. It must be stressed that this preliminary specificity check against the databases is not conclusive evidence. The assay itself must be tested against the range of pathogens likely to be encountered in the sample types under investigation.

Choice of the Quantification Standards

There are two main options for quantifying by a real time PCR, either absolute or relative. In most diagnostic assays absolute quantification is chosen. A set of separately amplified, log-diluted previously quantified templates are used to form a standard curve, derived from Ct values plotted against the log concentrations of each standard. The copy number of any sample is estimated from its Ct intercept on the standard curve. The accuracy of the standards can be confirmed by testing against external QA controls. The National Institute for Biological Standards and Control in the UK produce over 300 WHO International standards. Several standards designed for nucleic acid based assay testing are also commercially available, including HIV, hepatitis, CMV, EBV, and B19 parvovirus and are reportable in international units/milliliter (IU/ml).

Reaction Controls

The LDT must include positive controls (PC), negative, no template controls (NTC) and extraction controls (EC). The Ct values of the positive control must be recorded as part of the routine quality assurance. Typically, a single positive control for each specific pathogen, a NTC after every eight samples, and a further NTC as the last sample on the run is used to monitor the assay. Placing a NTC at the start of the set-up could give a false impression of any contamination problems. The PC can be produced from extracted clinical samples or from commercial sources and should be diluted to be reproducibly amplified at the lowest detectable level (typically a Ct of 30). An internal PCR or amplification control (IC) is an absolute requirement for any diagnostic assay. Nucleic acid from a suitable non-target pathogen can be spiked into each extracted specimen prior to the PCR and amplified and detected with a separate set of primers and probe. Preferably, an EC, using a non-related virus or bacterium is used to monitor extraction efficiency. Using this approach, the EC is spiked into the sample before extraction. This has the advantage over using naked nucleic acid as an EC, because it controls for the all the steps in the extraction process. Phocine herpesvirus is a useful EC for assays detecting viral DNA pathogens and for RNA viruses, MS2 phage or mengovirus can be used. The use of an RNA template in RT-PCR controls for both the critical reverse transcription step and the PCR. Inhibition can be assessed by comparing the Ct values of the amplified EC in samples with the Ct value of the control extracted in a negative matrix, e.g., water or elution buffer. A difference, typically greater than three Cts (approximately equivalent to one log) indicates inhibition and a potentially false negative result. It is necessary to establish that the primers and probes and the amount of control added to the reaction does not interfere with the amplification of the pathogen target.

Reverse Transcription PCR

Reverse transcription PCR is used to detect RNA viruses, but the use of differential gene-expression assays is becoming more widespread to distinguish between infection and colonization in other infectious micro-organisms. Reverse transcription-PCR can be carried out as a two-step reaction, where the RT step is carried out separately and an aliquot of the reaction transferred to the PCR. More usually, in infectious disease assays, a one-step reaction is employed, where the reverse transcription and PCR occur in the same tube on the thermocycler. The one-step is quicker and more suited to high-throughput diagnostic laboratories, since the potential for contamination is lessened by number of steps that require opening tubes. The choice of priming the RT step needs to be considered; typically, the downstream, anti-sense primer is used. However, random hexamers may be more efficient. Often the choice is down to a sensitivity/efficiency balance between the hexamers ability to bind and hence copy all the RNA species in the sample and the sensitivity provided by the sequence-specific binding of the downstream primer.

Westgard Rules

For each run the Ct values of controls are plotted on Levy-Jennings or Shewhart charts to monitor the performance of the assay; visual presentation of the control data is helpful in spotting variations in the operation of the assay (see Table 2 ). Westgard rules can also be applied to determine when a corrective action should be taken and give some indication of the nature of the analytical error. The use of Westgard rules and Shewhart charts must form part of the ongoing monitoring of the assay’s validated status.

The Basic Westgard Rules

Experimental Optimization

Optimal primer and probe concentrations must be verified, together with the PCR program itself. A well characterized, stable positive control must be used throughout this verification process to provide an invariant baseline from which the effects of changes to the various components can be measured. The formation of primer dimers and the degree of non-specific amplification can be assessed by analytical agarose gel electrophoresis which also provides evidence of the size of the amplicon. To optimize primer and probe concentrations, ideally two log-dilutions of the positive control are used in a checkerboard layout of varying primer/probe combinations. The optimal concentrations and thermal cycling conditions are those that provide the lowest Ct values, the greatest ΔRn and a consistent C t difference of approximately three cycles between the two log-diluted samples. Once the basic parameters of the assay have been established it will be necessary to confirm the amplicon product by sequencing and BLAST analysis. Due to the short amplicon lengths in real-time PCR, the fragment may need to be cloned into a suitable plasmid vector.

Normalization

Expression analyzes with reverse transcription PCRs is not normally used in diagnostic PCR. However, the range of diseases and their associated pathogens being diagnosed by RT-PCR is increasing. In some cases, e.g., with fungal species, there is a need to distinguish between carriage, environmental contamination and infection. Therefore, assays capable of detecting and quantifying differentially expressed genes indicative of the disease condition are needed. This requires carefully selected reference genes to control for variations in extraction, reverse-transcription and amplification efficiencies, so that comparisons across different mRNA concentrations can be made and fold changes in expression levels determined. A detailed literature search for suitable reference gene mRNA targets must be made and the selected candidates experimentally tested for stable expression in both diseased and non-diseased samples.

Analytical Specificity and Sensitivity

Analytical specificity : the LDT’s ability to detect the target it was designed for and not cross-react with other analytes in the sample.

Specificity is demonstrated either by spiking samples with a range of different pathogens prior to extraction, or adding the extracted nucleic acid from these pathogens to the extracted sample under investigation. All reactions should be analyzed by agarose gel electrophoresis to ensure that amplification has not occurred in any of the samples expected to be negative. In some cases, primers may amplify non-target templates, which may not be detected by the probe. This will compromise the sensitivity of the assay and must be eliminated, either by re-evaluating the PCR conditions (typically raising the annealing temperature), or preferably by redesigning the primers.

Analytical sensitivity : The LDT’s ability to detect very low levels of a given analyte in a biological specimen.

This is synonymous with the assay’s LOD, i.e., the lowest concentration of the analyte consistently detected in ≥95% of samples tested with acceptable precision. The LOD is usually determined for diagnostic assays by using a set of log-diluted controls, such as patient samples, a suitable cell line, or proficiency panels. It is important to include a NTC in the LOD study to ensure that the PCR does not generate a signal that could interfere with true low-level positive signals from a sample.

There is no hard and fast rule as to how many samples to use, The Clinical and Laboratory Standards Institute suggests a minimum of 60 data-points (12 separate measurements from each of 5 samples) are required from a manufacturer to establish the LOD. This seems a reasonable figure to adopt as a standard approach for both LDTs and commercially produced assays and will provide the necessary intra-assay variation measurements. This measurement should also be carried out on a second thermocycler to establish machine-to-machine variation.

Estimation of the LOD is usually carried out by probit analysis, a type of regression used to analyze binomial response variables, where a sigmoid dose-response curve is transformed into a straight line that can be analyzed by regression either through least squares or maximum likelihood. For PCR assays the lowest concentration of analyte that can be detected with a stated probability can be determined by plotting the data from positive replicate results versus the analyte concentration.

Inhibition Study

The factors that inhibit or prevent the amplification of nucleic acids by PCR can be present in the extracts from a number of sources. Inhibitors typically act by: (1) interference with the cell lysis necessary for extraction of nucleic acids, (2) interference by degrading the nucleic acid or inhibiting its capture, (3) inhibiting polymerase activity during amplification of the target DNA, resulting in false-negative results or inaccurate quantification. Different sample types present different problems due to the range of endogenous inhibitors found, for example in fecal samples, complex polysaccharides, breakdown products from hemoglobin, and bile acids can all be present and careful thought needs to be given to the method of extraction. Other potential inhibitors include metabolites resulting from pathological conditions such as diabetes mellitus and homeostatic hepatitis, or from medications used in treatment.

Manufacturers continue to develop their PCR reagents specifically to overcome many of the common endogenous inhibitors found in clinical specimens and it is recommended that a number of different commercial amplification kits are evaluated for their performance in this respect. As discussed previously, inhibitors can be detected using an IC. If the IC fails to amplify or exhibits suppression below the acceptable threshold, the amplification of the intended target sequence may also be inhibited. To avoid reducing the sensitivity of the assays, the IC should be used at the lowest concentrations that can be reproducibly amplified to minimize any competition between its amplification and that of the target pathogen.

The IC must demonstrate inhibition by the substances expected to be found in the sample, for which an inhibition study needs to be carried out. The study must first identify what types of inhibitors are likely to be present in the sample types used in the assay. The IC can be tested in negative samples with and without the interfering substance(s) in parallel and the effect on the C t values monitored. This test must be repeated with the full assay (including the pathogen detection primers and probes) using paired samples, with and without the target analyte to fully validate the assay. Once interference is found, of either or both the IC and target, samples can be serially diluted to test the limit at which the target will amplify. In most cases dilution of inhibited samples provides a straightforward method of enabling amplification. The assay must then be tested against clinical samples; this is because artificially constructed inhibition control samples may not accurately reflect the assays performance in routine use.

PCR Efficiency

The efficiency of the PCR can have a significant impact on the robustness and precision of the assay. The efficiency of the reaction is determined by a number of factors, including primer design, cycling conditions and the reagents used in the reaction mixture. PCR efficiency is particularly important in assays reporting fold changes of mRNA for target genes relative to those of reference genes, where both templates must be amplified with equal efficiencies. PCR amplification efficiency is typically established using calibration curves and it makes sense to use the dilution series (covering 5–6 orders of magnitude) from the LOD analyzes for this purpose. The equation of the linear regression line, together with Pearson’s correlation coefficient (r) and the coefficient of determination (r 2 ) are used to determine amplification efficiency. Amplification efficiency itself is determined from the slope of the log-linear portion of the calibration curve and is given by Eq. (1) :

Ideally the amount of template will double during each round of exponential amplification, this translates to a reaction efficiency of 2, therefore, using equation 1: 2 = 10 (−1/slope) which gives a slope for the standard curve of −3.32. This ideal figure of 3.32 also represents the difference in cycle number for each log dilution in the series. The efficiency can also be expressed as a percentage of the template amplified in each cycle using Eq. (2) :

In the ideal example given above; % Efficiency = (2−1) × 100% = 100%.

The experimental measurement should be carried out in triplicate on at least two thermal cyclers and the information included in the validation documentation. In practice acceptable assays should achieve efficiencies of 90%–100%. Reaction efficiencies lower than this may be caused by suboptimal reaction conditions or poor primer design. Reaction efficiencies of more than 100% could be due to measurement errors e.g., in preparation of the dilution series, or co-amplification of primer-dimers. Improving specificity will improve the sensitivity and increase the dynamic range of the assay, by reducing competition between the specific and non-specific amplification products.

Linear Dynamic Range or Reportable Range

The linear dynamic range can be determined during the LOD analysis by extending the range of the dilution series. The dilution series is often set at the range one would expect the analyte to be found in clinical specimens, although this may not always be known, especially for a novel pathogen and so initial testing can be carried out from 1 up to 10 7 or higher copies/ml of target. If using a generic assay to detect a range of pathogens, e.g., a pan-fungal PCR, each of the species the assay is designed to detect must be tested. Similarly, for multiplex assays, the LOD must be determined individually for each target analyte in the assay and must include the individual targets in the multiplex at high and low concentrations to ensure competitive amplification does not prevent all targets being identified. The reportable range is defined as the lowest and highest results (in suitable units of concentration) reliably detected in the assay. The linearity of the range can be established by calibration curves, where an r 2 value (coefficient of determination) of 1.00 indicates a perfect fit of the data points. Generally, in qPCR assays an r 2 of not less than 0.99 is considered acceptable. The linearity and reportable range should be carried out on at least 5 log-dilutions of the target nucleic acid extracted from an appropriate sample type (serum, urine, NPA etc.,) in triplicate, ideally on two different thermocyclers. Quantitative results from the test can only be reported when they fall within the linear range of the assay. A reportable range is not applicable to qualitative assays, since the result is simply positive or negative (below the LOD). However, a log-diluted panel should be tested, because the information derived is useful in assessing both the efficiency and clinical utility of the test and the results (as C t values) reported as part of the supporting documentation for the assay.

Clinical Validation Stage

Precision, accuracy and trueness.

Precision : a measure of the closeness of agreement between independent test results obtained under defined conditions.

Accuracy : the level of agreement between the true value of an analyte and the value obtained by the new test.

Trueness : defines the level of agreement between the average value obtained from a large series, the test and the accepted reference value.

Precision experiments are designed to measure the random error of an assay over a pre-determined period of time by multiple measurements of an aliquot derived from a homogeneous sample. For qualitative assays random error can be established by testing the PC and NC material in triplicate over a period of 10 days on two different thermocyclers. For qPCR assays estimates can be determined from the quantification standards and two positive controls at the lower and upper ends of the assay’s reportable range.

Accuracy refers to the closeness in agreement between a single measurement and the true value of the analyte under investigation. Trueness is determined by analyzing the average value obtained from a series of measurements with the new assay and the true value of the analyte (if an international standard is available) or a reference method (if a standard is not available). There are two approaches to evaluating trueness ( Burd, 2010 ):

(1) A comparison of methods study, where split samples are tested in parallel with an appropriate gold standard method.
(2) A recovery study, where proficiency samples, or other verified sample types are compared with the assay under development.

Although either of these methods is acceptable, a split sample, comparative study using clinical samples is preferred. However, in some cases, e.g., when developing an assay for a novel pathogen, a suitable gold standard assay may not available and a recovery study will have to suffice.

The Gold Standard Comparative Study

The comparative study is the cornerstone of the validation exercise. Estimates of sensitivity and specificity are derived from comparisons between the LDT and an established gold standard, which, ideally, is the same type of test and has an assumed sensitivity and specificity of 100%. By definition the gold standard assay is an error-free diagnostic method, which, rarely (if ever) exists. Consequently, an earlier PCR assay, or a culture-based method is often used.

Samples for the comparative study should be representative of the disease and population being investigated and also suitably distributed across both age range and gender. Where only limited samples are available, it may be necessary to use archived positive and negative specimens, or cultured material spiked into negative clinical samples. Other sources may be commercial standards, quality control materials or proficiency panels. Sample numbers can be determined as described previously. However, the sample numbers are more often selected for more pragmatic reasons, such as cost and feasibility and therefore, less than the statistically optimal number will be tested. This is often the case for a new or re-emerging infectious disease.

The difficulty comes in interpreting the results of a study where the comparative test is not a “perfect” gold standard, (i.e., the alloyed standard). This is particularly so if the comparative method is cell culture, which is thought to be 100% specific, but provides less than optimal sensitivity. The strength of the PCR lies in its sensitivity (theoretically a single DNA copy). Thus a newly developed PCR assay may produce many more positive results than the reference method, leading to a misleading, lower estimate of specificity. The researcher is then left with the problem of deciding whether these are true or false positives. A thorough and detailed assessment of the sample derivation, clinical history and sequencing can help to determine the true status of any sample, together with discrepant analysis. In discrepant analysis the discordant results are resolved by a third test, such as another PCR directed to a different gene-target and the results from this resolver test are used to definitively assign the final results. It is important to include a number of randomly selected samples with concordant results for discrepant analysis so as to reduce bias.

Further statistical analysis can be carried out to improve confidence in the results from the new LDT, particularly when the true disease status of the samples is difficult to evaluate. The agreement between the new test and comparator test can be expressed as the kappa value. The kappa statistic is a generic term for a number of similar measures of agreement applied to categorical data and is a measure of the proportion of agreement between results beyond chance. It is used in assessing the degree to which two or more raters, (i.e., tests) examining the same data, (i.e., specimens) agree in assigning the data to categories (positive or negative results). A kappa value of 1.00 indicates perfect agreement; a value of 0.00 indicates no agreement above that expected by chance, and a kappa value of −1.00 indicates complete disagreement. Generally, kappa statistics above 0.80 are considered almost perfect. The kappa value is calculated from the results in the standard 2×2 contingency table, used to record the results from the comparative test (see Fig. 2 ).

Fig. 2 2×2 Contingency Table.

K = Kappa value.
OP = observed proportion of agreement.
EP = expected proportion of agreement.
TP = true positive.
FP = false positive.
TN = true negative.
FN = false negative.
n = total number of samples.

Diagnostic sensitivity and specificity (DSe and DSp) are also calculated from the results in the contingency table.

Two further important probabilities can be calculated from the data derived from the trueness study, the positive and negative predictive values (PPV and NPV). The PPV is a measure of a positive result from the test to truly predict the presence of disease/infection, while the NPV is the probability that a negative result accurately indicate a non-diseased/uninfected status.

The Study Without a Gold Standard

In many cases, particularly for novel pathogens, a comparative method will not be available. Under these circumstances the gold standard may be a diagnosis determined by accepted clinical methods. It may also be necessary to incorporate results from more than one method to provide the comparative results. Whatever the approach chosen, estimating the performance characteristics of a new assay without a true comparative test is a challenging task. Typically, agreement is measured as the overall percentage or fraction of samples that have the same result (i.e., both either positive or negative). The difficulty with these simple agreement measures is that they do not take agreement by chance into account. A number of models have been proposed, based on latent class analysis, log-linear modeling and other techniques. Latent class analysis involves multiple imperfect tests that are used to construct a gold standard. Such models are based on the concept that the observed results of different, imperfect, tests for the same disease are influenced by a common but unobserved (latent) variable i.e., the true disease status.

Inter-Laboratory Testing

The ultimate evidence of an assay’s fitness for purpose is its successful integration into other laboratories. The LDT reagents and samples used for the comparative study should be sent to a minimum of three laboratories willing to participate in testing. This will also provide additional data on the assay’s reproducibility and robustness .

Maintaining the Validated Status and Re-Validation

The validated assay must be monitored consistently for repeatability through the performance of the run controls to evaluate any potential changes in the assay’s precision and accuracy. All reagents should be assigned batch numbers and new batches tested with the controls and a suitable number of positive and negative samples from a previous run to ensure consistent efficiencies between batches. If more than one thermal cycler is used, each machine should be referenced and each test carried out on it identified. The results of the PCR controls and quantification standards must be plotted daily on Shewhart charts and assessed by Westgard rules to ensure that reaction efficiencies are maintained.

Participation in regular IQA and EQA schemes is fundamental to maintaining an assay’s validated status. The purpose of EQA testing is not solely to monitor the performance of the assay, but asses all aspects of the test procedure, including the extraction method and reporting accuracy. If an external panel is not available, alternatives can include blind sample testing, sample exchange with other laboratories, or clinical note reviews.

Technical modifications continually arise as manufacturers develop and improve their reagents for extracting nucleic acids, the PCR and instrumentation. Assessment of new probe chemistry or the transfer of an assay to full or semi-automated instrumentation, normally only requires a methods comparison study. In this way any changes to the diagnostic sensitivity and specificity can be assessed quickly and accurately and the updated assay introduced with the minimum delay.

Over time it is likely that an assay will require some degree of revalidation, either for purely technical reasons, or because of changes in the nature of the analyte detected. Regular monitoring of amplification efficiency together with clinical information will give an early warning of any changes in the sequence of the amplicon detected in an assay. Point mutations are common in the genomes of many infectious microorganisms, particularly RNA viruses. New viral lineages may also be introduced into the population being tested due to travel from abroad. Under such circumstances, significant modifications to the primers and probe may be required, necessitating reverification and revalidation of the assay. Amplicon sequencing should be part of the validation plan and an integral part of routine trouble-shooting algorithms, since it is capable of resolving errors due to non-specific binding of primers and probes.

Acknowledgment

This article, including tables and figure, is based on author’s article “The Validation of Real-time PCR Assays for Infectious Diseases”, published in Real-Time PCR: Advanced Technologies and Applications (2013), edited by: Nick A. Saunders and Martin A. Lee, Health Protection Agency, Colindale, UK and Porton Consulting Research Ltd, Salisbury, UK (respectively), Caister Academic Press.

Abbreviations

Burd E.M. Validation of laboratory-developed molecular assays forinfectious diseases. Clinical Microbiology Reviews. 2010; 32 :550–576. [ PMC free article ] [ PubMed ] [ Google Scholar ]
Bustin S.A., Benes V., Garson J.A., et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry. 2009; 55 (4):611–622. [ PubMed ] [ Google Scholar ]

Relevant Websites

https://www.ebi.ac.uk/Tools/msa/clustalo/ –Clustal Omega.
http://www.ncbi.nlm.nih.gov –National Center for Biotechnology Information.
http://www.oligo.net/ –oligo.net.

Predictive Biomarkers in Oncology pp 63–73 Cite as

Overview of PCR-Based Technologies and Multiplexed Gene Analysis for Biomarker Studies

Yesim Gökmen-Polar 3

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Polymerase chain reaction (PCR) has become an invaluable tool for the assessment of the presence and type of nucleic acids in tissues and body fluids. It is the in vitro enzymatic synthesis and amplification of specific DNA sequences. It can amplify one molecule of DNA or RNA into billions of copies in a few hours. This enables mutation tracking for management of any cancer, which is particularly crucial in targeted therapies. Novel applications include analysis of blood for circulating DNA for tumor-associated mutations. RNA analysis has been extensively used for quantification of gene expression. This forms the basis of multiple gene expression assays including multigene panels that are being developed for prognostic and predictive purposes. This chapter will provide a brief overview of the basics of PCR and the current applications in clinical oncology.

Polymerase chain reaction

Endpoint PCR
Quantitative PCR and reverse transcription-qPCR
Clinical oncology

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Abbreviations

Deoxyribonucleic acid

Deoxyribonucleotide triphosphates

Quantitative real-time PCR

Reverse transcriptase quantitative real-time PCR

Reverse transcriptase polymerase chain reaction

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Gökmen-Polar, Y. (2019). Overview of PCR-Based Technologies and Multiplexed Gene Analysis for Biomarker Studies. In: Badve, S., Kumar, G. (eds) Predictive Biomarkers in Oncology. Springer, Cham. https://doi.org/10.1007/978-3-319-95228-4_5

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Home > Books > Biotechnology to Combat COVID-19

Molecular Biology of PCR Testing for COVID-19 Diagnostics

Submitted: 11 January 2021 Reviewed: 25 January 2021 Published: 13 February 2021

DOI: 10.5772/intechopen.96199

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COVID-19 cases were first reported in December 2019, and since then it has spread quickly to create a global pandemic. This respiratory disease is caused by the SARS-CoV-2 virus. A major contributing factor for the fast spread of this virus is that the infectivity by the asymptomatic carriers is similar to symptomatic patients. Thus, to identify the asymptomatic individuals and to provide the essential treatment and care to COVID-19 patients, we rely heavily on diagnostic assays. Efficient, reproducible and accessible diagnostic tests are crucial in combatting a pandemic. Currently, there are few key detection tests which have been successfully employed to field-use. However, there are constant efforts to enhance their efficacy and accessibility. This chapter aims at explaining the basic principles of the current molecular diagnostic tests, which determine the presence of the virus through the detection of its genetic material. This chapter will aid the readers in understanding the basic workings of these molecular diagnostic tests.

molecular diagnostic tests
CRISPR-based assays

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Vinita chittoor-vinod *.

, Stanford University, Palo Alto, California, United States

*Address all correspondence to: [email protected]

1. Introduction

In December 2019 primary cases of COVID-19 were reported in Wuhan (China), which was later declared as a pandemic by WHO in March 2020 [ 1 ]. The COVID-19 is caused by the contagious virus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), belonging to the same family as MERS-CoV (Middle East respiratory syndrome coronavirus) and SARS-CoV-1 [ 2 ]. Typical symptoms of COVID-19 span a wide range, including fever, dry cough, sore throat and shortness of breath [ 3 ]. These symptoms are similar to flu and other respiratory illnesses. In order to provide appropriate care and treatment for COVID-19, it is critical to diagnose SARS-CoV-2 (referred to as CoV-2 hereafter) infections distinctly from other similar diseases. Another complexity to this is that some CoV-2 infected individuals do not exhibit exaggerated symptoms, termed as asymptomatic carriers [ 4 ]. It has been shown that the asymptomatic carriers can spread the virus to the same extent as the symptomatic individuals, in the absence of appropriate precautions [ 4 ]. Thus, it is imperative to identify all infections efficiently, quarantine and treat appropriately to cease the spread of CoV-2. To achieve this, many diagnostic tests (assays) have been developed and improved by multiple referring (participating) laboratories and CDC (Centers for Disease Control and Prevention). This chapter is focused on simplifying the basic principles of these diagnostic assays. Many scientific review articles which dive deep into the science of CoV-2 diagnostic testing have been published [ 1 , 3 , 5 , 6 , 7 ]. This chapter, however, aims at explaining the same in non-technical terms, intended for a general audience.

2. Biology of SARS-CoV-2

SARS-CoV-2 belongs to the family Coronaviridae , similar to SARS-CoV-1. CoV-2 consists of a spherical protein structure of ~80–160 nm diameter [ 8 ] ( Figure 1 ). This sphere is composed of two lipid layers placed face-to-face close to each other (bilayer). This bilayer is embedded with proteins, collectively referred to as the structural proteins. They include the envelope (E), membrane (M) and spike (S) proteins [ 2 ]. The E, M and S-proteins are embedded in the lipid bilayer forming the sphere [ 9 ]. The S-proteins stick out of the sphere prominently, giving a spiked crown-like appearance to the virus and thereby conferring the name ‘corona’ (crown) [ 2 ]. They bind to the ACE-2 (human angiotensin-converting enzyme 2) receptors on the host cell membrane to initiate fusion, and therefore are key in the invasion of host cells [ 10 , 11 , 12 ]. Encapsulated inside this sphere is the virus genome, which in the case of CoV-2, is a plus (or positive) single-stranded (ss) RNA (ribonucleic acid, ss RNA) [ 2 ]. This genetic material is spooled around the nucleocapsid (N) proteins, which are also accounted as structural proteins [ 2 ].

Cartoon representing the human SARS-CoV-2 virus structure. (Left) Sphere showing the outside of the virus. (Right) A cross-section of the virus sphere depicting the membrane structural proteins (S, M and E), along with the ss RNA genetic material which is wound around the nucleocapsid (N) proteins.

The genome of CoV-2 was successfully sequenced by January 2020 [ 13 ]. The ss RNA is ~30 kb (kilobases) in length [ 2 ], encompassing all the information for protein syntheses and assembly of the new virus particles (virions). There is another group of non-structural proteins (NSPs) which are involved in non-structural functions such as genetic material replication or the assembly of virions [ 2 ]. There are 16 such NSPs identified in the CoV-2 genome, which include an RNA-dependent RNA polymerase (RdRP), ExoN (exonuclease) and ORF proteins [ 2 ]. RdRP synthesizes new viral genetic material, while ExoN is responsible for genome stability and for removing any errors in the newly synthesized genetic RNA sequence. ORF proteins act as accessory proteins [ 2 ].

Viruses have been traditionally categorized in a separate class, from the biotic or living organisms. This is due to their inability to replicate in the absence of a host. In general, the virus invades the host cell, releases its genetic material and hijacks the host machinery for synthesizing its own macromolecules (nucleic acid and proteins) [ 14 ]. Upon assembly of virions (new viruses), the host cell is lysed (broken open) to release the new infectious particles [ 14 ]. The host cells try to combat the invasion by evoking an immune response specific to the virus (adaptive immunity) [ 15 ]. This is attempted through the presentation of fragments of the foreign macromolecules on the host cell surface. These non-indigenous particles, called antigens, are sensed by the immune cells which initiate the production of antibodies against them [ 15 ]. Thereafter, any particle resembling the antigen is attacked and cleared by the host immune cells. Antibodies are broadly classified into 5 main immunoglobulin (Ig) classes: G, A, M, E and D. They differ in their structures, capacity to recognize the antigen and occurrence in the course of immune response [ 15 ]. These antibodies encompass a structural region which is specific to binding the antigen (called the variable region). The immune response and components are much more complicated than this simple excerpt presented here, and the readers are encouraged to refer to other reviews on the immune system [ 15 ].

3. Detection of SARS-CoV-2

The observation that the virus from asymptomatic carriers is equally infectious as those exhibiting clear symptoms of COVID-19 [ 4 , 16 ], makes it imperative to identify the asymptomatic individuals in order to take appropriate measures for their seclusion and treatment. This is highly dependent on the reliability and accuracy of the diagnostic tests. Further, these assays also permit the recognition of patients with CoV-2 infections at hospitals where it is crucial for their segregation into the COVID-19 specific wards. This is important to prevent further transmission of the virus to admitted and highly vulnerable patient populations. Reliability of a diagnostic test depends on its specificity and sensitivity. Specificity is the ability of the test to correctly detect the negative samples as negative, thus reducing false-positive results [ 17 ]. On the other hand, sensitivity is the ability of the test to correctly identify the positive cases as positive, thereby decreasing false-negative results [ 17 ]. It is essential that a test is dependable for both these features. False results, either way, will aid in the spread of the virus, and misdirect contact tracing.

High sensitivity

High specificity

Easy read-out method

Rapid turn-around-time (TAT, time to get the results)

Easy transport and storage

High reproducibility

Present diagnostic assays for CoV-2 have been categorized as shown in Figure 2 .

Categorization of available CoV-2 diagnostic tests. The present diagnostic assays for CoV-2 can be segregated into two broad classes- molecular and serological. Molecular tests determine the presence of the virus by detecting its genome. For molecular assays, specimens can be collected from multiple relevant areas, such as the nasal swab, BAL, etc. Serological tests detect either the presence of the antigen (a protein that is only expressed by the virus) or the antibodies (generated by the host’s immune system in response to the infection) in blood samples. While PCR (molecular) and antigen tests detect the presence of the virus at the time of testing, antibody assays mainly determine previous infection. All these tests can identify both, symptomatic and asymptomatic carriers of the virus. The PCR assays include reverse transcription-PCR (RT-PCR), recombinase polymerase amplification (RPA), reverse transcription-loop mediated isothermal amplification (RT-LAMP), and CRISPR-Cas based tests. These PCR assays will be discussed in this chapter.

4. Molecular testing

These diagnostic assays detect the virus through the presence of their genetic material, which is amplified to produce a detectable signal. To fully comprehend the mechanism of these assays, it is essential to first understand the central dogma ( Figure 3 ). The common genetic material is DNA (deoxyribonucleic acid), a comparatively more stable nucleic acid than RNA. Generally, DNA is a double-stranded (ds) molecule composed of plus (+) and minus (−) strands [ 18 ]. It is made of deoxyribose sugar molecules as backbone and are attached with bases or nucleotides A (adenine), T (thymine), G (guanine) or C (cytosine) [ 18 ]. The complementary nature of the bases, i.e. their ability to pair specifically, provides the ds structure. The same feature allows faithful replication of the DNA and syntheses of RNA, thus enabling truthful relaying of the message. The base pairings are A-T and C-G [ 18 ]. The deoxyribose (and ribose) sugars provide a directionality to the nuclei acids with their chemical groups, 5′ end and 3′ end [ 19 ]. All DNA strands are synthesized by DNA polymerases in 5′ - > 3′ direction [ 19 ]. The two strands, however, run in opposite directions i.e. the 5′ end of plus strand is closest to the 3′ end of minus strand, while the 3′ end of the plus strand is opposite to the 5′ end of the minus end [ 19 ]. Based on the ATGC code (sequence) carried by the plus strand, the minus complementary strand is built [ 19 ]. For example, 5′-AGGCTC-3′ sequence on the plus strand will be paired with 5′-GAGCCT-3′.

Central dogma. The message in the double stranded DNA is converted to single stranded RNA by transcription, through complementary base pairing. The code in RNA is converted to proteins by a process called translation (three bases constitute a codon, which represents one amino acid). Amino acids are the building blocks of proteins. RNA can be converted back to double stranded DNA (cDNA) through reverse transcription.

4.1 DNA to RNA

The information in the genetic material needs to be converted to proteins, which act at the functional level. In this process, a key intermediate is the RNA. The code in the DNA is first converted to RNA through transcription by RNA polymerases [ 20 ]. The complementarity of the bases is used to transfer the information faithfully into RNA. Usually, RNA is a ss molecule, with the same complementary base pairings as the DNA. In RNA, T is replaced by U (uracil) which also pairs with A (A-U) [ 19 ]. In some viruses, ds RNA serves as the genetic material. However, the rules of complementation and the ss RNA intermediate for protein synthesis remain the same.

4.2 RNA to protein

The code carried by RNA is used for the synthesis of proteins, through translation [ 20 ]. Proteins are composed of amino acids building blocks. A codon in the RNA, which is composed of three bases in a specific order, codes for a particular amino acid [ 20 ]. So, the sequence of the amino acids in the protein are built according to the sequence of the codons in the respective RNA. Proteins are the macromolecules which acts as support structures, catalyze reactions, relay signaling information, and many other functions.

The dependency of protein synthesis on RNA has been ingeniously employed in the current Pfizer and Moderna vaccines [ 21 ]. These vaccines carry an mRNA (messenger RNA) which carries the code for an antigenic fragment of the CoV-2 S-protein [ 21 ]. The host cells produce the S-protein fragments which elicit an appropriate immune response. This leads to the production of antibodies that can identify the CoV-2 S-protein upon an actual infection.

5. Polymerase chain reaction (PCR)

PCR is the process of photocopying a specific region (target region) of the DNA, achieved through base complementation. This amplification process is used to produce a detectable signal, which can be correlated to the presence and amount of target DNA present in the reaction ( Figure 4 ). In PCR, a specific target region in the DNA sample is demarcated through primers, which are short stretches of DNA ranging from 8–20 nucleotide bases. Short DNA strands are termed as oligonucleotides, where primers are a sub-group which are used in PCR reactions. Primers are complementary to the boundaries of the target region in the DNA sample. In PCR, two primers are required to bind at the 5′ end boundaries, one each for the plus and minus strands. The primers provide a pre-requisite platform for the DNA polymerase to bind and extend the new complementary strands [ 22 ], one for each of the two original template strands ( Figure 4B ).

RT-PCR. (A) Viral ss RNA is converted to ds DNA by the reverse transcriptase enzyme. (B) In PCR, the cDNA acts the template (1). The two strands of the cDNA are separated (denatured) through high heat (2). This allows the annealing of the primers (forward and reverse) and the probe with their complementary regions on the template DNA strands (3). The DNA polymerase extends the primers to synthesize new DNA strands. In this process, the polymerase displaces and separates the fluorescent reporter (F) and the quencher (4). (C) Explanation of the read-out signal generation. At the ground state, the quencher is in close proximity to the reporter, and thus suppresses its emission. Upon cleavage by the polymerase (during extension step), the reporter is released. This relieves its inhibition thereby producing a fluorescent signal. Thus, with an increase in amplification of the template DNA, there is a corresponding increase in the detectable fluorescent signal.

When the starting material for PCR is RNA, as in the case of CoV-2, the RNA template is first reverse transcribed to complementary DNA (cDNA) ( Figure 4A ). The cDNA then serves as a template for PCR amplification using targeted primers. The conversion of RNA to DNA is achieved through naturally occurring RNA-dependent DNA polymerases, aptly called reverse transcriptase (RT). PCR reactions which depend on the RT enzyme are generally categorized as RT-PCR.

The amplified DNA region was traditionally detected using color or fluorescent agents which bind to ds DNA products [ 23 ]. Thus, with an increase in amplification there is an increase in the color/fluorescent signal. PCR read-outs are of two types: quantitative and qualitative. The former, quantitative, yields the amounts of template DNA, either absolute or relative values. On the other hand, qualitative PCR provides information on whether the template DNA is present in a sample. For CoV-2 RNA detection, a simple answer on its presence or absence is essential.

The general types of PCR techniques employed or developed for COVID-19 diagnosis are discussed below.

5.1 Reverse transcriptase-polymerase chain reaction (RT-PCR)

The first diagnostic kit developed to detect the CoV-2 infection was based on RT-PCR. As described above, the viral genomic RNA in the specimen is reverse transcribed to cDNA, followed by PCR amplification of a target gene using primers ( Figure 4 ). There are two variants of this reaction: 1-step and 2-steps [ 3 ]. In 1-step, reverse transcription and PCR are conducted in the same tube, in tandem. This minimizes the chances of contamination by reducing handling. The 2-steps variation separates the RT reaction which provides cDNA in a separate tube for retention. This is useful since it is easier to store and handle DNA than RNA, and the cDNA can be used for further testing of other genes, if needed.

Choosing the target gene and region within that gene (usually PCR is directed at amplifying only a small region within a gene) is important, as it needs to be specific to the virus, excluding any overlap with the host genome or any other parasite/virus. For CoV-2 PCR, regions within the N, E, RdRP, S and ORF1ab genes have been successfully used as targets for RT-PCR [ 3 ]. It was recommended to use PCRs directed at amplifying at least two target regions for higher specificity. In addition to the viral genes, the human RNase P gene which is present ubiquitously in all cells is also amplified separately [ 3 ]. Detection of RNase P ensures that the PCR reaction did receive the specimen. This is important especially in determining negative results for viral gene targets.

During the PCR, the two strands of DNA template (plus and minus strands) are separated using high temperature to break the complementary pairs (reversible). This step is termed as “denaturation” in PCR, which is required to expose the bases for the primers to bind. This is followed by an “annealing” step which is ambient for primer binding to the complementary regions. Entailing this step is “extension” of the primers by DNA polymerase to synthesize complementary product strands. These three key steps are repeated multiple times in the same order to amplify the signal (referred to as PCR cycles). The newly synthesized DNA fragments can then themselves act as templates in the following PCR cycles, thereby giving an exponential amplification pattern. Thus, even a small amount of starting DNA is sufficient to generate a positive signal. Although, the annealing and extension temperatures can be synchronized through appropriate designing of the primers, denaturation requires a higher temperature. This demands the use of thermocyclers for RT-PCR, which can change temperatures of the reaction cyclically. Further, RT-PCR read-out is generally a fluorescent signal which also requires a specific detection instrument. These limit the use of this assay at point-of-care (POC), i.e. use by medical practitioners for instant results to make informed and immediate decisions.

A variation of this conventional assay, TaqMan PCR, was employed as a primary technique for CoV-2 diagnostics [ 24 ]. It involves the addition of another oligonucleotide called the probe. This probe, which is complementary to the plus strand is usually positioned towards the center of the target region i.e. between the two opposing primers. Probe is flanked by a fluorescent reporter molecule at its 5′ end and a quencher molecule at the 3′ end (as explained earlier, all oligonucleotides have a direction imparted by the backbone sugar molecules). The fluorescent reporter signal is suppressed by the quencher due to their close proximity. When the probe binds to the plus strand of the template (after denaturation), the DNA polymerase starts synthesizing the new strand from the 3’end of the forward primer (the quencher molecule in the probe 3′ end will not allow the polymerase to start at the probe). In this process, the polymerase cleaves the probe and releases the fluorescent and quencher molecules separately. Due to this irreversible separation, the signal from the fluorescent reporter is uninhibited and detectable. Thus, the level of signal from the reaction is proportional to the amount of new DNA products. TaqMan PCR retains the need for a thermocycler and a fluorescence reader, but provides more specificity than the traditional technique.

During the early stages of the COVID-19 pandemic, samples from multiple individuals were pooled together to reduce the testing times [ 25 ]. Upon detecting a positive result, the samples from that pool were individually tested to identify the infected individual/s.

RT-PCR is a commonly used assay in most laboratories. Hence, it was easily absorbed as a CoV-2 diagnostic test during the start of this pandemic.

It has high specificity, determined by the rigor of the chosen primers/probes.

This assay can be easily modified to adapt the mutations of the virus, as reported for CoV-2 S-protein in UK in December 2020 [ 26 ].

This test has the capability to multiplex. It has been recently modified by the CDC to detect the presence of CoV-2, Influenza strains A and B [ 3 ].

There is no requirement for a purification step in RT-PCR.

This assay depends on a thermocycler and a fluorescence reader, limiting its use at POC.

The RT and PCR reactions yield a TAT of ~3–24 hours [ 27 ], depending on the number of samples and the handling capacity of the testing center.

5.2 Recombinase polymerase amplification (RPA)

This assay works on the same principle as the RT-PCR but bypasses the need for temperature variations for DNA amplification. It eliminates the denaturation high temperature step, and then combines the annealing and extension steps to a single temperature [ 28 ]. The assays which use a single temperature to complete all the reactions are termed as ‘isothermal’, thus eliminating the need for a thermocycler.

RPA achieves isothermal amplification through the inclusion of a few key components in the reaction mixture ( Figure 5 ). The first is the recombinase enzyme, which is incubated with the primers to form a complex, in the presence of a crowding agent (increases viscosity of the solution). The recombinase-primers complex is then added to the reaction with the cDNA derived from viral RNA. Thus, this assay also depends on RNA isolation and RT reaction. The recombinase allows the invasion of the ds cDNA by the primers to bind to their complementary regions. The ss DNA regions (or loops) that are created due to this invasion are stabilized by the binding of ss DNA-binding proteins (SSBPs). This prevents the re-binding of the original template strands (plus and minus). The recombinase enzyme is then displaced from the DNA by a strand-displacing DNA polymerase. This polymerase opens the template DNA structure as it synthesizes new DNA strands emanating from the primer (i.e. strand-displacing). All these components of RPA aim towards the elimination of the denaturation step in the PCR cycle, thus making it isothermal. This assay holds the capacity to be carried out in solid-phase, i.e. on a dry surface with immobilized components [ 28 ]. Although the load-of-detection (LOD) and time are compromised in solid-phase RPA [ 28 ], this feature can enable the designing of lyophilized kits with minimal storage and transport requirements.

Recombinase polymerase amplification (RPA). The primers and the recombinase enzyme are allowed to form a complex (1). The recombinase opens up the cDNA template, while the primers bind to their complementary regions (2). The ss DNA regions generated due to the opening of the template structure are stabilized through the binding of the single-strand binding proteins (SSBPs) (3). The DNA polymerase binds to the primers attached to the template DNA, detaching the recombinase (4). The polymerase extends the primers, while opening the ds template on its way. It also moves the SSBPs while synthesizing the new strands (5). New strands are synthesized by the polymerase (6). New ds DNA products which can act as templates for the following amplification cycles (7).

End-point detection of RPA has been vastly calibrated to fit the lateral flow assays (LFA) [ 28 ]. This assay yields rapid results in a visual read-out format ( Figure 6 ). To adapt RPA to LFA, three different oligonucleotides (2 primers and 1 probe) and a nfo nuclease are required. Similar to the TaqMan assay described above, the probe is flanked with a 5′ end antigenic label (usually 6-Carboxyfluorescein i.e. FAM) and a 3′ end blocking group [ 28 ]. The 3′ end group inhibits the DNA polymerase from extending the probe (remember that the polymerase can only add nucleotides at the 3′ end). In addition to these end groups, the probe is also equipped with an abasic nucleotide (tetrahydrofuran) which does not pair with any of the standard bases (A, T, G or C) [ 28 ]. This abasic nucleotide creates a fold in the probe that is bound to the complementary template DNA. The nfo nuclease recognizes this fold and nicks the probe at this position [ 28 ]. The abasic nucleotide is strategically positioned in the probe, such that the nick by the nuclease releases the 3′ end blocking group from the probe/template DNA complex [ 28 ]. This opens up the 3′ end for the polymerase to extend the new DNA strand from the probe. The reverse primer (the primer that will bind to the opposite strand) is tagged with a 5’end ligand (usually biotin) [ 28 ]. The main feature of these two 5′ end tags (FAM and biotin) is the availability of strong binding proteins or antibodies against them. The binding proteins or antibodies are immobilized on LFA strips (dipsticks) at two separate lines, test and control. The test band is coated with biotin-binding proteins (which will capture the 5’end tag of the reverse primer), while the control band is stacked with Ab 2 antibodies (which capture to unbound Ab 1 antibodies, see below). Once the stick is exposed to the sample (either through immersion of the sample pad or loading of the sample) on the sample pad, the sample moves across the conjugation pad which has lyophilized antibodies against FAM (Ab 1 ). Here the RPA ds DNA products which carry both the tags will be bound by Ab 1 . From the conjugation pad, the complex containing Ab 1 -RPA products will move further up the strip (due to capillary action) towards the test band. At this juncture, only DNA products which have the biotin tag will be captured by the biotin-binding proteins, showing a positive result. Further movement of these complexes is restricted as the binding proteins are immobilized onto the test line. Along with the complexes, the unbound free Ab 1 antibodies also move up from the conjugation pad. These free antibodies move further up from the test line as they do not carry any biotin. They are captured by the Ab 2 antibodies in the control line. Hence, a positive result should yield two distinct lines in the strip. In case of a negative sample, there is no fruitful conjugation of Ab 1 antibodies on the conjugation pad. However, due to capillary action of the sample, the Ab 1 move up the strip. Although these antibodies will not be captured at the test line, they will be immobilized by Ab 2 on the control line. Thus, a reliable negative result should show one control line on the strip. All other combinations would indicate inconclusive results.

Lateral flow assay. (A) The strip consists of the regions depicted in the figure. The sample is loaded onto the sample pad. The sample then moves upwards, towards the absorption pad due to capillary action. Sample comprises of the amplified ds DNA products with tags FAM and biotin at two separate ends. From the sample pad, the sample first moves to the conjugation pad which is pre-loaded with Ab 1 (antibodies against FAM). The Ab 1 antibodies bind to the FAM-DNA-biotin products. These complexes move upward and are captured at the test line by the immobilized biotin-binding proteins. This produces a visible test line. The unbound free Ab 1 antibodies (excess) from the conjugation pad also move upwards. These antibodies, however, move past the test line as they do not possess any biotin for interaction at this line. Upon reaching the control line, these antibodies are captured by Ab 2 (antibodies that can bind Ab 1 ). This interaction produces a visible control line. (B) The appearance of the positive, negative and invalid results. Positive result should show two lines, since it ensures the working of all components of the LFA strip. A conclusive negative result will only produce a control line which is generated by the interaction of immobilized Ab 2 with free unbound Ab 1 . All other results are considered invalid.

Solid-phase RPA can yield kits with minimal needs for transport and storage, thereby significantly improving diagnostics at POC. However, more research is required in improving its LOD and TAT.

The LFA compatibility is useful in non-laboratory settings, again increasing its usage at POC.

RPA can be easily modified to accommodate the new mutations in target regions.

At present, RPA kits are sold by one company. This restricts modifications at the user’s end.

Prior to LFA, there is a protein purification step to avoid impaired flow of the sample on the strip. This adds to TAT.

This assay still requires RNA isolation and reverse transcription steps. These add to the detection times.

5.3 LAMP (loop-mediated isothermal amplification)

LAMP is another isothermal amplification technique which produces long, self-complementary looping DNA strands to generate a detectable signal. This technique employs an engineered DNA polymerase Bst 2.0 with strand-displacing feature [ 29 ]. This enzyme can separate the two template DNA strands (plus and minus) as it builds the new strand, thus removing the denaturation step from PCR. LAMP is conducted at a single temperature (60–65 °C) [ 29 ], conducive to both the annealing and extension steps. Recent modifications include addition of the engineered RT enzyme along with the Bst 2.0 polymerase, thus making it a 1-step protocol [ 29 ]. Again, this assay still requires RNA isolation from the sample.

Generally, a set of 4 (or 6) specific primers are used in LAMP assay. These primers cover at least six distinct regions, flanking the entirety of the target region. A glimpse of the assay steps is described in Figure 7 . The self-complementary regions of the primers promote the formation of looped DNA products. This allows LAMP to yield concatemers of various lengths, which are long DNA strands with multiple copies of the target region aligned back to each other [ 29 ]. Concatemers multiply the read-out signal at a much faster rate than RT-PCR. The mechanism is explained in the Figure 7 . A review article by Thompson and Lei [ 29 ] is recommended for further reading on this assay. Here, I would like to highlight the modifications made to RT-LAMP to progress COVID-19 diagnostics.

LAMP. Six specific regions (A, B, C, D, E, F) flanking the target DNA are chosen to design the primers (1). The forwards inner primer (FIP) invades the target DNA. This primer encompasses a self-complementary region (C) on its 5′ end (2). The new strand synthesized with FIP is displaced by the forward primer (3). The DNA products synthesized by the forward primer is similar to the original cDNA template (4). Due to the self-complementarity region on the FIP, it folds on itself forming a loop. This strand is invaded by the backward inner primer (BIP) and backward primer (5). The product formed by BIP on the FIP strand leads to two loops at the ends, forming a dumbbell shaped structure (6). These strands can be extended to form concatemeric DNA products (7). Concatemeric products of variable lengths (8).

This detection assay was developed to provide a visible color read-out. A colorimetric detection was incorporated using a simple pH-sensitive dye such as phenol red [ 29 ]. The color of this dye is closer to red when the pH is neutral (pH 7.0), and changes towards the yellow spectrum with acidic pH (pH < 7.0). The byproducts of DNA synthesis are acidic, which reduce the pH of the solution. This color change can be easily noted by eye, without any instrument. However, this detection method is limited by the baseline differences in the pH of collected specimens. To circumvent this, fluorescent dyes such as GeneFinder have been employed [ 29 ]. This dye produces green color under blue light illuminator to report positive results. Another variation of this assay excluded RNA isolation step to find comparable amplification of the N-protein gene under laboratory conditions [ 29 ]. This modification still needs more testing and evaluation with patient samples, prior to field use. After calibration, this assay could significantly reduce the TAT.

Moreover, individual samples can be tagged with specific barcodes in LAMP assay which allow tracing in a pooled sample [ 29 ]. A common method of barcoding is the transposase Tn5-adapter system [ 30 ]. The original article on this barcoding method is recommended to readers for understanding its mechanism and potential in diagnostics [ 30 ]. The barcodes, however, have to be read through NGS (next-generation sequencing), which requires specific lab equipment. This adds time for obtaining the results. It is possible that the bargain between time saved by pooling samples and the time added by NGS could be a deciding factor for the field-use application of this assay.

An important feature of LAMP is its amenability to be paired with other PCR techniques to combine their advantages. So far, the efficacy of LAMP has been tested after merging with RPA, using lab samples [ 29 ]. The combined assay is found to have increased sensitivity. RT-LAMP when integrated with CRISPR-Cas12 assay (described below) was shown to reduce detection time considerably [ 3 ].

This assay is conducted at a single temperature, and thus without a thermocycler.

The detection rate is much shorter than other mentioned techniques [ 3 ].

LAMP offers flexibility to be paired with other assays for improving their detection abilities.

This assay can be adapted to lab instrument-free detection methods.

Specificity of LAMP is higher than RT-PCR [ 3 ].

LAMP relies on precisely designed primers. Hence, it is more complicated to accommodate new mutations as compared to RT-PCR [ 3 ].

Sensitivity of LAMP is lower than the conventional RT-PCR [ 3 ].

Similar to the previously mentioned techniques, LAMP also requires RNA isolation and reverse transcription.

5.4 CRISPR-based assays

CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated nuclease) technology has been recently tested for its potency in the field of diagnostics. This technique, which was given recognition through the Nobel Prize in Chemistry 2020, is a modified biological process of the bacterial (prokaryotic) adaptive immune system [ 1 ]. Here a ss guide RNA (sgRNA or crRNA) leads/guides the CRISPR-Cas complex to the target nucleic acid region [ 1 ], owing to its complementarity to this region ( Figure 8 ). Upon binding, Cas nuclease cleaves the template nucleic acid [ 1 ], along with non-specific cleavage of nearby ss DNA/RNA. This feature is called “collateral cleavage” activity [ 1 ], and has been used in designing read-out methods for diagnostic tests. This assay is equipped with ss DNA or RNA probes with a fluorescent or traceable reporter molecule, which produces a detectable signal only upon cleavage through collateral activity.

CRISPR-Cas cleavage system. Viral RNA is reverse transcribed to ds cDNA, which can be further transcribed to amplified RNA through an in vitro system. The cDNA can be processed either through Cas9 or Cas12a, while the ss RNA can be cleaved through Cas13a. The single-guide RNA (sgRNA) or crRNA is complementary to the region of interest, and thus guides the Cas nuclease to the site. After target cleavage, collateral cleavage activity cleave and releases the reporter from quencher inhibition. This produces a detectable fluorescent signal, which is proportional to the amount of target DNA in the reaction.

The target for CRISPR-Cas cleavage complex can be modified by changing the crRNA strand sequence, similar to the primers/probes in RT-PCR. Based on the Cas nuclease paired with the crRNA in the assay, the template can vary. For example, Cas13 targets ss RNA, while Cas9 or Cas12 target ds DNA [ 1 ]. Thus, in case of CoV-2, the virus ss RNA will need to be reverse transcribed to cDNA which will be either amplified as DNA products or through in vitro transcription as RNA products. CRISPR-Cas complexes cannot amplify nucleic acids, and hence rely on other amplification techniques for this (such as previously described methods).

A recently invented SHERLOCK (Specific high-sensitivity enzymatic reporter unlocking) technique includes the crRNA-Cas13a complex to target RNA molecules [ 1 ]. This technique uses RPA amplification assay and a non-targeting RNA strand tagged with a fluorescent dye [ 31 ]. Patchsung et al. [ 32 ] used SHERLOCK for CoV-2 diagnostics, targeting the S and ORF1ab genes. This assay has been further modified to suit LFA detection methods, i.e. using paper strips. Another variation of CRISPR-Cas technique is DETECTR, where it is combined with RT-LAMP amplification. This has been tested for CoV-2 E and N genes [ 33 ].

Ding et al. [ 34 ] developed an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay where all the reactions components are incubated at 37 °C together. This simplifies the diagnostic assay protocol. The AIOD-CRISPR was then modified for a visual color detection in LED blue light illuminator.

CRISPR-Cas reactions can be conducted at 37 °C temperature. This is an achievable temperature at POC.

This is an isothermal reaction, and thereby does not depend on a thermocycler.

The basic CRISPR-Cas technology has the flexibility to be paired with other assays to reap advantages from both techniques.

DETECTR assay has a shorter process time as compared to conventional RT-PCR tests [ 3 ].

The sensitivity of CRISPR-based assays is higher than the other mentioned tests.

These assays can be adapted to non-instrumental detection methods like LFA or visible color change under blue light [ 3 , 34 ].

Specificity of CRISPR-based assays is lower than that reported for RT-PCR.

These assays also require RNA extraction which adds to TAT.

Currently, there is no portable CRISPR-based devices which have been developed for CoV-2. Thus, more research in needed in this field.

Calibration of the assays is more complicated than the standard RT-PCR or RPA. Thus, although possible, it will take longer to modify these tests to detect new mutations of the target gene.

6. Conclusion

The current molecular diagnostic tests provide variable degrees of sensitivity and specificity to the detection of SARS-CoV-2. It is clear that at present there is no single assay which fits all the requirements. However, there are constant research efforts aimed at improving the efficiency and accessibility of these assays to meet the growing demand of this pandemic. The improved assays will increase our ability to combat COVID-19 spread, and enhance our preparedness for any future infectious agents by providing a strong platform for building new diagnostic tests.

Acknowledgments

All figures have been created with BioRender.com.

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30. Schmid-Burgk JL SR, Li D, Hollstein R, Ben-Shmuel A, Israeli O, Weiss S, Paran N, Wilbring G, Liebing J, Feldman D SM, Lippke B, Sib E, Borrajo J, Strecker J, Reinhardt J HP, Cleary B, Hölzel M, Nöthen MM, Exner M LK, Regev A, Zhang F. LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding. BioRxiv. 2020. https://doi.org/10.1101/2020.04.06.025635
31. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019;14(10):2986-3012. 10.1038/s41596-019-0210-2
32. Patchsung M, Jantarug K, Pattama A, Aphicho K, Suraritdechachai S, Meesawat P, Sappakhaw K, Leelahakorn N, Ruenkam T, Wongsatit T, Athipanyasilp N, Eiamthong B, Lakkanasirorat B, Phoodokmai T, Niljianskul N, Pakotiprapha D, Chanarat S, Homchan A, Tinikul R, Kamutira P, Phiwkaow K, Soithongcharoen S, Kantiwiriyawanitch C, Pongsupasa V, Trisrivirat D, Jaroensuk J, Wongnate T, Maenpuen S, Chaiyen P, Kamnerdnakta S, Swangsri J, Chuthapisith S, Sirivatanauksorn Y, Chaimayo C, Sutthent R, Kantakamalakul W, Joung J, Ladha A, Jin X, Gootenberg JS, Abudayyeh OO, Zhang F, Horthongkham N, Uttamapinant C. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-9. 10.1038/s41551-020-00603-x
33. Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan CY, Guevara H, Wadford DA, Chen JS, Chiu CY. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874. 10.1038/s41587-020-0513-4
34. Ding X, Yin K, Li Z, Liu C. All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus. bioRxiv. 2020. 10.1101/2020.03.19.998724

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Polymerase chain reaction (PCR) (article)
Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism). PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest. In PCR, the reaction is repeatedly cycled through a series ...
PDF Carrick Academy Higher Biology Essays
(ii) PCR (4) Replication of DNA 1. Double helix unwinds/hydrogen bonds break between bases 2. Primer anneals to DNA 3. Free complementary DNA nucleotides added to 3' end by DNA polymerase 4. DNA polymerase adds nucleotides from 5' to 3' end 5. Lead strand replication continuous AND lag strand replicated in fragments 6.
Polymerase chain reaction
Polymerase chain reaction. The Polymerase Chain Reaction (PCR) is a technique for the amplification of DNA in vitro (this describes experiments with cells outside their normal environment). PCR ...
Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq DNA.[1][2] In 1985, PCR was introduced by Mullis and colleagues for which they received a Nobel prize.[3] It is a ...
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PCR is the most sensitive and specific test for Helicobacter pylori, the disease organism now known to cause almost all stomach ulcers.Unlike previous tests, PCR can detect three different sexually transmitted disease organisms on a single swab (herpes, papillomaviruses, and chlamydia) and can even distinguish the particular strain of papillomavirus that predisposes to cancer, which other ...
3.5: Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is now one of the most commonly used assays for obtaining a particular segment of DNA or RNA. It is rapid and extremely sensitive. By amplifying a designated segment of DNA, it provides a means to isolate that particular DNA segment or gene. This method requires knowledge of the nucleotide sequence at the ...
Polymerase chain reaction
A strip of eight PCR tubes, each containing a 100 μL reaction mixture Placing a strip of eight PCR tubes into a thermal cycler. The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
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2. Basic Principles. Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR, is a modification of the PCR strategy which allows monitoring of the PCR progress in real-time PCR itself is an enzymatic process used in vitro for the amplification of a selected DNA region through several orders of magnitude, generating thousands to millions of copies of a specific DNA ...
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This video has been designed to support Higher Biology and Higher Human Biology learners to develop their knowledge and understanding of PCR. Our interactive video series Lab Techniques in Action shows commonly used lab techniques that are explored in the Scottish Curriculum for Excellence. These videos form part of the National e-Learning Offer by Education Scotland and are available to ...
Polymerase Chain Reaction (PCR) Fact Sheet
Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification ...
Validating Real-Time Polymerase Chain Reaction (PCR) Assays
Generally, in qPCR assays an r 2 of not less than 0.99 is considered acceptable. The linearity and reportable range should be carried out on at least 5 log-dilutions of the target nucleic acid extracted from an appropriate sample type (serum, urine, NPA etc.,) in triplicate, ideally on two different thermocyclers.
(PDF) Polymerase Chain Reaction
Polymerase chain reaction (PCR) is an efficient and one of the most common methods used in biological sciences for in vitro multiplication of a target DNA molecule. The technique has significantly ...
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PCR is a process used in molecular biology to. amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands. to millions of copies of a ...
Polymerase Chain Reaction (PCR): Stages, Types ...
ADVERTISEMENTS: Read this article to learn about the stages, primer design, types, sensitivity, factors affecting, applications and variations of polymerase chain reaction. PCR has been one of the most important techniques developed in recent years. The reason behind is its simplicity of the reaction and relative case of the practical manipulation steps. The PCR is […]
8.4.4 Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a common molecular biology technique used in most applications of gene technology, for example, DNA profiling (eg. identification of criminals and determining paternity) or genetic engineering; It can be described as the in vitro method of DNA amplification; It is used to produce large quantities of specific fragments of DNA or RNA from very small quantities ...
Higher Biology
Study with Quizlet and memorize flashcards containing terms like What does PCR stand for, What does PCR amplify, Where does PCR amplify DNA and more. ... Higher Biology - Essay 8 - PCR and uses. Flashcards; Learn; Test; Match; Q-Chat; Flashcards; Learn; Test;
Higher Biology: Unit 1.2
Amplifies the target region of DNA. Step 1 of PCR. DNA is heated to between 92 and 98 degrees to separate the strands (by breaking the hydrogen bonds between them) Step 2 of PCR. It is then cooled to between 50 and 65 degrees to allow the primers to bind to target sequences. Step 3 of PCR. The DNA is then heated to between 70 and 80 degrees for ...
Overview of PCR-Based Technologies and Multiplexed Gene ...
The major components of a PCR reaction consist of the template DNA, DNA polymerase, primers, deoxyribonucleotide triphosphates (dNTPs), magnesium ion (Mg 2+), and buffer (15 mM MgCl 2, 500 mM KCl, 100 mM Tris-HCl, pH 8.3 at 25 °C).Nowadays, most of these components are sold by commerci al vendors as "PCR master mix," which is a premixed concentrated solution with the components of a PCR ...
Polymerase Fidelity: What is it, and what does it mean for your PCR
The discovery and development of high-fidelity polymerases has for many years been a key focus at New England Biolabs (NEB). Highfidelity amplification is essential for experiments whose outcome depends upon the correct DNA sequence (e.g., cloning, SNP analysis, NGS applications). Whereas traditional fidelity assays are sufficient for Taq and ...
Molecular Biology of PCR Testing for COVID-19 Diagnostics
The sensitivity of CRISPR-based assays is higher than the other mentioned tests. These assays can be adapted to non-instrumental detection methods like LFA or visible color change under blue light [3, 34]. Limitations: Specificity of CRISPR-based assays is lower than that reported for RT-PCR. These assays also require RNA extraction which adds ...
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Higher Human Biology - PCR. Flashcards; Learn; Test; Match; ... 1 / 14. Flashcards; Learn; Test; Match; Q-Chat; Created by. jordanmcintyre999. Share. Share. Terms in this set (14) What does PCR stand for? Polymerase Chain Reaction. What is PCR? Amplification of a piece of DNA in vitro. What does the amplification of DNA involve.
Higher
What does PCR stand for? Click the card to flip 👆. Polymerase chain reaction
Higher Biology: PCR Flashcards
Study with Quizlet and memorize flashcards containing terms like What is PCR used for?, Describe Stage 0 (before replication) of PCR, Describe Stage 1 of PCR and more. Scheduled maintenance: March 23, 2024 from 11:00 PM to 12:00 AM

AP®︎/College Biology

Polymerase chain reaction (PCR)

Key points:

What is PCR?

Using gel electrophoresis to visualize the results of PCR

More about PCR and forensics

References:

3.5: Polymerase Chain Reaction (PCR)

PCR in Action

Terms and Conditions

Curriculum Links

Polymerase Chain Reaction (PCR) Fact Sheet

What is PCR?

What is PCR used for?

How does PCR work?

Validating Real-Time Polymerase Chain Reaction (PCR) Assays

Introduction

The Need for Validation

Commercial Assays

The Validation Process: Consultation Stage

Preliminary Considerations

The Validation plan

Analytical Verification Stage

Template, Primers and Probes: In-Silico Design

Choice of the Quantification Standards

Reaction Controls

Reverse Transcription PCR

Westgard Rules

Experimental Optimization

Normalization

Analytical Specificity and Sensitivity

Inhibition Study

PCR Efficiency

Linear Dynamic Range or Reportable Range

Clinical Validation Stage

The Gold Standard Comparative Study

The Study Without a Gold Standard

Inter-Laboratory Testing

Maintaining the Validated Status and Re-Validation

Acknowledgment

Abbreviations

Further Reading

Relevant Websites

Overview of PCR-Based Technologies and Multiplexed Gene Analysis for Biomarker Studies

Buying options

Abbreviations

Author information

Corresponding author

Editor information

Copyright information

About this chapter

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Molecular Biology of PCR Testing for COVID-19 Diagnostics

Biotechnology to Combat COVID-19

Author Information

1. Introduction

2. Biology of SARS-CoV-2

3. Detection of SARS-CoV-2

4. Molecular testing

4.1 DNA to RNA

4.2 RNA to protein

5. Polymerase chain reaction (PCR)

5.1 Reverse transcriptase-polymerase chain reaction (RT-PCR)

5.2 Recombinase polymerase amplification (RPA)

5.3 LAMP (loop-mediated isothermal amplification)

5.4 CRISPR-based assays

6. Conclusion

Acknowledgments

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