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14.6 NUCLEIC ACID AMPLIFICATION TECHNIQUES

INTRODUCTION​

      Nucleic acid amplification techniques are based on two 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 hybridization 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 appendix, the PCR method is described as the reference technique. Alternative methods may be used, if they comply with the quality requirements described below.

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.

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, two 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 two 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 after or during amplification by a variety of methods of appropriate specificity and sensitivity. However, only post-PCR detection will be described herein.

TEST MATERIAL

      Because of the high sensitivity of PCR, the samples must be optimally protected against external contamination with target sequences. Sampling, storage and transport of the test material are performed under conditions that minimize 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.

TEST METHOD

      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 subdivided 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).

      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 physicochemical extraction procedures and/or enrichment procedures may be employed.

      Additives present in test material may interfere with PCR. The procedures described under Internal control 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.

      Amplification

      PCR amplification of the target sequence is conducted under optimized 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.

      Detection

      The amplicon generated by PCR may be identified by size, sequence, chemical modification or a combination of these parameters. Characterization by size may be achieved by gel electrophoresis (using agarose or polyacrylamide slab gels or capillary electrophoresis) or column chromatography (for example, HPLC). Characterization by sequence composition may be achieved by the specific hybridization of probes having a sequence complementary to the target sequence or by cleavage of the amplified material reflecting target-specific restriction-enzyme sites. Characterization 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 radio isotopic or immuno-enzyme-coupled detection.

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.

QUALITY ASSURANCE

      Validation of the PCR assay system

      The validation programme must include validation of instrumentation and the PCR method employed.

      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. 

      During validation the positive cut-off point must be determined. The positive cut-off point is defined as the minimum number of target sequences per volume of the 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.

      Quality control of reagents

      All reagents crucial for the methodology used have to be controlled prior to use in routine applications. Their acceptance or withdrawal is based on predefined quality criteria.

      Primers are a crucial component of the PCR assay and as such their design, their purity and the validation of their use in a PCR assay require careful attention. Each new batch of primers is tested for specificity, amplification efficiency and absence of inhibitory impurities before acceptance. 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.

      Run controls

      EXTERNAL CONTROLS

      In order to minimize the risk of contamination and to ensure adequate sensitivity, the following external controls are included in each PCR test:

– positive control: this contains a defined number of target-sequence copies, the number being determined individually for each assay system and indicated as a multiple of the positive cut-off value of the test system;

– negative control: a sample of the same matrix already proven to be free of the target sequences;

      INTERNAL CONTROLS

      Internal controls are defined as nucleic acid sequences containing the primer binding sites. Internal controls must be amplified with similar efficacy as the target sequence to be tested, but the amplicons must be clearly discernible. Internal controls must be of the same type of nucleic acid (DNA or 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).

      External quality assessment

      Participation in external quality assessment programmes is an important PCR quality assurance procedure for each laboratory and each operator.