Nucleic Acid Amplification Techniques

1. Introduction

Nucleic acid amplification techniques are based on 2 different approaches: 1.) amplification of a target nucleic acid sequence using, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), or isothermal ribonucleic acid (RNA) amplification, 2.) amplification of a hybridisation signal using, for example, for deoxyribonucleic acid (DNA), the branched DNA (bDNA) method. In this case signal amplification is achieved without subjecting the nucleic acid to repetitive cycles of amplification. In this general chapter, the PCR method is described as the reference technique.  Alternative methods may be used, if they comply with the quality requirements  described below.

2. Scope

This section establishes the requirements for sample preparation, in vitro amplification of DNA sequences and detection of the specific PCR product. With the  aid of PCR, defined DNA sequences can be detected. RNA sequences can also be  detected following reverse transcription of the RNA to complementary DNA (cDNA)  and subsequent amplification.

3. Principle of the method

PCR is a procedure that allows specific in vitro amplification of segments of DNA or  of RNA after reverse transcription into cDNA. Following denaturation of double-stranded DNA into single-stranded DNA, 2 synthetic  oligonucleotide primers of opposite polarity, anneal to their respective  complementary sequences in the DNA to be amplified. The short double-stranded  regions which form as a result of specific base pairing between the primers and the  complementary DNA sequence, border the DNA segment to be amplified and serve  as starting positions for in vitro DNA synthesis by means of a heat-stable DNA  polymerase. Amplification of the DNA occurs in cycles consisting of: —heat denaturation of the nucleic acid (target sequence) into 2 single strands; —specific annealing of the primers to the target sequence under suitable reaction conditions; —extension of the primers, which are bound to both single strands, by DNA polymerase at a suitable temperature (DNA synthesis). Repeated cycles of heat denaturation, primer annealing and DNA synthesis results  in an exponential amplification of the DNA segment limited by the primers. The specific PCR product known as an amplicon can be detected by a variety of  methods of appropriate specificity and sensitivity. Multiplex PCR assays use several primer pairs designed for simultaneous  amplification of different targets in one reaction.


4. Test material

Because of the high sensitivity of PCR, the samples must be protected against  external contamination with target sequences. Sampling, storage and transport of  the test material are performed under conditions that minimise degradation of the  target sequence. In the case of RNA target sequences, special precautions are  necessary since RNA is highly sensitive to degradation by ribonucleases. Care must  be taken since some added reagents, such as anticoagulants or preservatives, may  interfere with the test procedure.

5. Test method

5.1. Prevention of contamination

The risk of contamination requires a strict segregation of the areas depending on the  material handled and the technology used. Points to consider include movement of  personnel, gowning, material flow and air supply and decontamination procedures. The system should be sub-divided into compartments such as: —master-mix area (area where exclusively template-free material is handled, e.g. primers, buffers, etc.), —pre-PCR (area where reagents, samples and controls are handled), —PCR amplification (amplified material is handled in a closed system), —post-PCR detection (the only area where the amplified material is handled in an open system).

5.2. Sample preparation

When preparing samples, the target sequence to be amplified needs to be efficiently  extracted or liberated from the test material in a reproducible manner and in such a  way that amplification under the selected reaction conditions is possible. A variety of  physico-chemical extraction procedures and/or enrichment procedures may be  employed. Additives present in test material may interfere with PCR. The procedures described  under 7.3.2. must be used as a control for the presence of inhibitors originating from  the test material. In the case of RNA-templates, care must be taken to avoid ribonuclease activity.

5.3. Amplification

PCR amplification of the target sequence is conducted under defined cycling  conditions (temperature profile for denaturation of double-stranded DNA, annealing  and extension of primers; incubation times at selected temperatures; ramp rates).  These depend on various parameters such as: —the length and base composition of primer and target sequences; —the type of DNA polymerase, buffer composition and reaction volume used for the amplification; —the type of thermocycler used and the thermal conductivity rate between the apparatus, reaction tube and reaction fluid.

5.4. Detection

The amplicon generated by PCR may be identified by size, sequence, chemical  modification or a combination of these parameters. Detection and characterisation by  size may be achieved by gel electrophoresis (using agarose or polyacrylamide slab  gels or capillary electrophoresis) or column chromatography (for example, liquid  chromatography). Detection and characterisation by sequence composition may be  achieved by the specific hybridisation of probes having a sequence complementary  to the target sequence or by cleavage of the amplified material reflecting target-specific restriction-enzyme sites. Detection and characterisation by chemical  modification may be achieved, for example, by incorporation of a fluorophore into the  amplicons and subsequent detection of fluorescence following excitation. Detection of amplicons may also be achieved by using probes labelled to permit a  subsequent radioisotopic or immuno-enzyme-coupled detection.

6. Evaluation and interpretation of results

A valid result is obtained within a test only if the positive control(s) is unambiguously  positive and the negative control(s) is unambiguously negative. Due to the very high  sensitivity of the PCR method and the inherent risk of contamination, it is necessary  to confirm positive results by repeating the complete test procedure in duplicate,  where possible on a new aliquot of the sample. The sample is considered positive if  at least one of the repeat tests gives a positive result. As soon as a measurable  target threshold is defined, a quantitative test system is required.

7. Quality assurance

7.1. Validation of the PCR assay system

The validation programme must include validation of instrumentation and the PCR  method employed. Reference should be made to the ICH guidelines (topic Q2B)  Validation of Analytical Method: Methodology. Appropriate official working reference preparations or in-house reference preparations  calibrated against International Standards for the target sequences for which the test  system will be used are indispensable for validation of a PCR test.

7.1.1. Determination of the positive cut-off point

During validation of qualitative tests, the positive cut-off point must be determined.  The positive cut-off point is defined as the minimum number of target sequences per  volume sample which can be detected in 95 per cent of test runs. The positive cut-off  point depends on interrelated factors such as the volume of the sample extracted  and the efficacy of the extraction methodology, the transcription of the target RNA  into cDNA, the amplification process and the detection. To define the detection limit of the assay system, reference must be made to the  positive cut-off point for each target sequence and the test performance above and  below the positive cut-off point.

7.1.2. Quantitative assay systems

For a quantitative assay, the following parameters are determined during validation:  accuracy, precision, specificity, quantitation limit, linearity, range and robustness.

7.2. Quality control of reagents

All reagents crucial for the methodology used have to be controlled prior to use in  routine applications. Their acceptance/withdrawal is based on pre-defined quality  criteria. Primers are a crucial component of the PCR assay and as such their design, purity  and the validation of their use in a PCR assay require careful attention. Primers may  be modified (for example, by conjugation with a fluorophore or antigen) in order to  permit a specific method of detection of the amplicon, provided such modifications do  not inhibit accurate and efficient amplification of the target sequence.

7.3. Run controls

7.3.1. External controls

In order to minimise the risk of contamination and to ensure adequate sensitivity, the  following external controls are included in each PCR assay: —positive control: this contains a defined number of target-sequence copies, the number being close to the positive cut-off value, and determined individually for each assay system and indicated as a multiple of the positive cut-off value of the assay system; —negative control: a sample of a suitable matrix already proven to be free of the target sequences.

7.3.2. Internal control

Internal controls are defined nucleic acid sequences containing, unless otherwise  prescribed, the primer binding sites. Internal controls must be amplified with defined  efficacy, and the amplicons must be clearly discernible. Internal controls must be of  the same type of nucleic acid (DNA/RNA) as the material to be tested. The internal  control is preferably added to the test material before isolating the nucleic acid and  therefore acts as an overall control (extraction, reverse transcription, amplification,  detection).

7.3.3. Threshold control

The threshold control for quantitative assays is a test sample with the analyte at a  concentration which is defined as the threshold not to be exceeded. It contains the  analyte suitably calibrated in IU and is analysed in parallel in each run of a  quantitative assay.

7.4. External quality assessment

Participation in external quality assessment programmes is an important PCR  quality assurance procedure for each laboratory and each operator. The following section is published for information.


1. Scope

The majority of nucleic acid amplification analytical procedures are qualitative  (quantal) tests for the presence of nucleic acid with some quantitative tests (either in-house or commercial) being available. For the detection of HCV RNA  contamination of plasma pools, qualitative tests are adequate and may be  considered to be a limit test for the control of impurities as described in the  Pharmeuropa Technical Guide for the elaboration of monographs, December 1999,  Chapter III “Validation of analytical procedures”. These guidelines describe methods  to validate only qualitative nucleic acid amplification analytical procedures for  assessing HCV RNA contamination of plasma pools. Therefore, the 2 characteristics  regarded as the most important for validation of the analytical procedure are the  specificity and the detection limit. In addition, the robustness of the analytical  procedure should be evaluated. However, this document may also be used as a basis for the validation of nucleic  acid amplification in general. For the purpose of this document, an analytical procedure is defined as the complete  procedure from extraction of nucleic acid to detection of the amplified products. Where commercial kits are used for part of or the complete analytical procedure,  documented validation points already covered by the kit manufacturer can substitute  for the validation by the user. Nevertheless, the performance of the kit with respect to  its intended use has to be demonstrated by the user (e.g. detection limit,  robustness, cross contamination).

2. Specificity

Specificity is the ability to unequivocally assess nucleic acid in the presence of  components which may be expected to be present. The specificity of nucleic acid amplification analytical procedures is dependent on  the choice of primers, the choice of probe (for analysis of the final product) and the  stringency of the test conditions (for both the amplification and detection steps). When designing primers and probes, the specificity of the primers and probes to  detect only HCV RNA should be investigated by comparing the chosen sequences  with sequences in published data banks. For HCV, primers (and probes) will  normally be chosen from areas of the 5′ non-coding region of the HCV genome which  are highly conserved for all genotypes. The amplified product should be unequivocally identified by using one of a number of  methods such as amplification with nested primers, restriction enzyme analysis,  sequencing or hybridisation with a specific probe. In order to validate the specificity of the analytical procedure, at least 100 HCV RNA-negative plasma pools should be tested and shown to be non-reactive. Suitable  samples of non-reactive pools are available from the European Directorate for the  Quality of Medicines. The ability of the analytical procedure to detect all HCV genotypes will again depend  on the choice of primers, probes and method parameters. This ability should be  demonstrated using characterised reference panels. However, in view of the difficulty  in obtaining samples of some genotypes (e.g. genotype 6), the most prevalent  genotypes (e.g. genotype 1 and 3 in Europe) should be detected at a suitable level.

3. Detection limit

The detection limit of an individual analytical procedure is the lowest amount of  nucleic acid in a sample which can be detected but not necessarily quantitated as  an exact value. The nucleic acid amplification analytical procedure used for the detection of HCV  RNA in plasma pools usually yields qualitative results. The number of possible  results is limited to two, either positive or negative. Although the determination of the  detection limit is recommended, for practical purposes, a positive cut-off point should  be determined for the nucleic acid amplification analytical procedure. The positive  cut-off point (as defined in the General Chapter (2.6.21)) is the minimum number of  target sequences per volume sample which can be detected in 95 per cent of test  runs. This positive cut-off point is influenced by the distribution of viral genomes in  the individual samples being tested and by factors such as enzyme efficiency and  can result in different 95 per cent cut-off values for individual analytical test runs. In order to determine the positive cut-off point, a dilution series of a working reagent  or of the hepatitis C virus BRP, which has been calibrated against the WHO HCV  International Standard 96/790, should be tested on different days to examine  variation between test runs. At least 3 independent dilution series should be tested  with a sufficient number of replicates at each dilution to give a total number of 24 test  results for each dilution to enable a statistical analysis of the results. For example, a laboratory could test 3 dilution series on different days with 8  replicates for each dilution, 4 dilution series on different days with 6 replicates for  each dilution, or 6 dilution series on different days with 4 replicates for each dilution.  In order to keep the number of dilutions at a manageable level, a preliminary test  (using, for example, log dilutions of the plasma pool sample) should be done in order  to obtain a preliminary value for the positive cut-off point (i.e. the highest dilution  giving a positive signal). The range of dilutions can then be chosen around the  predetermined preliminary cut-off point (using, for example, a dilution factor of 0.5 log  or less and a negative plasma pool for the dilution matrix). The concentration of HCV  RNA which can be detected in 95 per cent of test runs can then be calculated using  an appropriate statistical evaluation. These results may also serve to demonstrate the intra-assay variation and the day-to-day variation of the analytical procedure.

4. Robustness

The robustness of an analytical procedure is a measure of its capacity to remain  unaffected by small but deliberate variations in method parameters and provides an  indication of its reliability during normal usage. The evaluation of robustness should be considered during the development phase. It  should show the reliability of the analytical procedure with respect to deliberate  variations in method parameters. For NAT, small variations in the method parameters  can be crucial. However, the robustness of the method can be demonstrated during  its development when small variations in the concentrations of reagents (e.g. MgCl2,  primers or dNTP) are tested. To demonstrate robustness, at least 20 HCV RNA  negative plasma pools (selected at random) spiked with HCV RNA to a final  concentration of 3 times the previously determined 95 per cent cut-off value should  be tested and found positive. Problems with robustness may also arise with methods which use an initial  ultracentrifugation step prior to extraction of the viral RNA. Therefore, to test the  robustness of such methods, at least 20 plasma pools containing varying levels of  HCV RNA, but lacking HCV specific antibodies, should be tested and found positive. Cross contamination prevention should be demonstrated by the accurate detection of  a panel of at least 20 samples consisting of alternate samples of negative plasma  pools and negative plasma pools spiked with high concentrations of HCV (at least 102 times the 95 per cent cut-off value or at least 104 IU/ml).

5. Quality assurance

For biological tests such as NAT, specific problems may arise which may influence  both the validation and interpretation of results. The test procedures must be  described precisely in the form of standard operating procedures (SOPs). These  should cover: —the mode of sampling (type of container, etc.), —the preparation of mini-pools (where appropriate), —the conditions of storage before analysis, —the exact description of the test conditions, including precautions taken to prevent cross contamination or destruction of the viral RNA, reagents and reference preparations used, —the exact description of the apparatus used, —the detailed formulae for calculation of results, including statistical evaluation. The use of a suitable run control (for example, an appropriate dilution of hepatitis C  virus BRP or plasma spiked with an HCV sample calibrated against the WHO HCV  International Standard 96/790) can be considered a satisfactory system suitability  check and ensures that the reliability of the analytical procedure is maintained  whenever used. Technical qualification: an appropriate installation and operation qualification  programme should be implemented for each critical piece of the equipment used.  Confirmation of analytical procedure performance after change of critical equipment  (e.g. thermocyclers) should be documented by conducting a parallel test on 8  replicate samples of a plasma pool spiked with HCV RNA to a final concentration of  3 times the previously determined 95 per cent cut-off value. All results should be  positive. Operator qualification: an appropriate qualification programme should be  implemented for each operator involved in the testing. To confirm successful training  each operator should test at least 8 replicate samples of a plasma pool spiked with  HCV RNA to a final concentration of 3 times the previously determined 95 per cent  cut-off value. This test (8 replicate samples) should be repeated twice on two  separate days, i.e. a total of 24 tests performed on three different days. All results  should be positive.

(Appendix XIV L  The British Pharmacopoeia 2007)


2 Komentar

Filed under Biologi Molekuler

2 responses to “PCR

  1. Frengki

    He3…pingin nimbrung plus lihat2 pelkerjaaannya Bang Nadjibb
    Ga nyontek loh,,,,,

  2. pika

    pak, ku kopi pak…bwt refernsi

    OK semoga tugasnya cepat kelar……..

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