A. PCR METHODS USED IN ROUTINE MOLECULAR DIAGNOSTICS
The principles of the PCR
Polymerase chain reaction (PCR) implies that there is an enzyme-based amplification reaction in the assay. The term ‘chain reaction’ refers to several cycles of copying a specified stretch of DNA or target nucleic acids (nucleotides), in this case from the genome of an infectious agent. The region to be amplified is defined by two (or more) short nucleotide sequences, termed primer sites, that flank the target sequence. Primers, short oligonucleotides that are complementary to the primer sites, bind to the DNA strand to be copied. Using a polymerase, which is not denatured during heat cycling, it is possible to copy the target sequence by joining free nucleotides to the primers. By repeating the heat-cycling regime 20–40 times, the amount of copied target DNA gained is enough for further operations, such as detection, cloning or sequencing. The diagnostic sensitivity of the PCR is very high because several million copies of the selected target are produced. The specificity may also be very high, as determined by the specific nucleotide sequences of the selected target, as well as primer design. The primers can be designed to detect very specific nucleotide sequences in the genomes of the selected target infectious agents, or can be designed to be complementary to very common sequences occurring in nature. These nonspecific primers, termed universal primers, are able to bind to a wider range of DNA and can be used to detect members within a family or genus of infectious agent.
If the genome of the infectious agent is DNA, the amplification is performed directly, with or without previous purification of the target DNA. In many cases, use of DNA extracted and purified from the material to be tested (e.g. blood) will result in increased analytical and diagnostic sensitivity.
RNA amplification (reverse-transcription PCR)
The genomes of many infectious agents contain ribonucleic acid (RNA) that cannot be amplified directly by the PCR. For PCR amplification, a single-stranded DNA target is necessary, and this is not available in the case of RNA viruses. This problem can be solved by the addition of a step before the PCR is begun. Using reverse transcriptase it is possible to transcribe the RNA into complementary DNA (cDNA), which is double-stranded DNA and hence can be used in a PCR assay (the procedure is termed reverse transcriptase PCR: RT-PCR). Traditionally, the reverse transcription reaction was performed in a separate reaction vessel and the cDNA produced is then transferred to a new tube for the PCR. However, heat-stable DNA polymerases with reverse transcriptase activity and specific buffers in which RT and DNA polymerases are active are now readily available. Both allow an RT-PCR reaction to take place in the same tube and in direct sequence without any further handling with less chance of carry-over contamination. In most cases, it will be necessary to extract and purify RNA prior to reverse transcription; however, in some cases, it is sufficient to boil the sample before RT-PCR (e.g. faecal samples in PBS).
PCR amplicon detection
The PCR product, or amplicon, can be detected using a variety of procedures. The most common include nonspecific detection of the PCR product based on amplicon size using electrophoresis in agarose gel and staining of the DNA with a nonspecific dye, such as ethidium bromide, or specific recognition of the amplified target sequence using Southern blot transfer of the DNA followed by hybridisation with oligonucleotide probes complementary to the target sequence. Hybridisation probes can be enzyme, chemiluminescent, or radionucleotide-labelled to allow visual detection of the specific target sequence.
Some examples of PCR methods currently used are given below.
‘Conventional PCR’ (or simply PCR) uses one pair of oligonucleotide primers to amplify a small part of the genome of the infectious agent. Analytical sensitivity is typically high with a minimum number of 100 to 1000 copies of the target DNA detectable. Analytical specificity can be high, dependent on target selection, primer design, and assay optimisation. Both analytical sensitivity and specificity can be further improved by applying nested PCR (see point 3 below). Detection methods, such as Southern blotting followed by hybridisation probes, can further improve sensitivity and specificity, but are time-consuming, require laboratory handling of amplified DNA, and interpretation of results can be technically subjective. Based on complexity and expense, these detection methods are not generally considered suitable procedures for common practice in diagnostic laboratories today.
Nested PCR assays use two sets of amplification cycles with four primers, termed external and internal primers. In general, nested PCR assays provide higher analytical sensitivity and specificity compared with conventional PCR assays; however, there is a substantial increased risk of cross contamination. The lower limit of detection with the nested PCR is typically <10 genomic copies of the target DNA, and analytical specificity is also enhanced because in the nested PCR, four oligonucleotide primers have to bind specifically to the selected targets in order to yield a positive reaction (2).
Real-time PCR differs from standard PCR in that the amplified PCR products are detected directly during the amplification cycle using hybridisation probes, which enhance assay specificity. Various real-time methods, such as TaqMan, Scorpions, FRET, or Molecular Beacons assays, have become popular tools for detection of infectious agents. Real-time PCR has been used for the detection of bacteria, viruses or parasites from a range of animal species (2, 6, 8). These new assays have several advantages over the ‘classical’ conventional or nested PCR methods. Only one primer pair is used, providing sensitivity often close or equal to traditional nested PCR but with a much lower risk of contamination. Fluorescence, indicating the presence of the amplified product, is measured through the lid or side of the reaction vessel thus there is no need for post-PCR handling of the amplified DNA. These procedures are considerably less time-consuming compared with traditional post-amplification PCR product detection in agarose gels followed by ethidium bromide staining and again, the risk of contamination is reduced. The use of a 96-well microtitre plate format, without the need for nested PCR, allows the procedure to be automated and suitable for large-scale testing (5). Diagnosis can be further automated by using robots for DNA/RNA extractions and pipetting. Compared with classical amplification methods, a further advantage of the real-time PCR is that it is possible to perform quantitative assays (6).
PCR reactions using multiple primers directed at different targets in a single assay are referred to as multiplex PCR assays. In multiplex PCR, various infectious agents can be detected and differentiated in a single reaction vessel at the same time. The different PCR targets amplified in a standard PCR assay are identified based on PCR product size. The use of ‘classical’ nested PCR methods for the construction of a multiplex assay is complicated by the need for targets of different sizes, as well as primers that may ‘compete’ with each other in the same reaction mix, both of which can negatively impact PCR efficiency. In contrast, the concept of real-time PCR (single primer pairs) provides excellent possibilities for the construction of highly sensitive multiplex systems (2, 4) based on more uniform target size, uniform amplification conditions, and differential detection of targets using specific hybridisation probes labelled with different fluorophores.