TaqMan Real-time RT-PCR
All real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, the signal of which increases in direct proportion to the amount of PCR product in a reaction. The real-time RT-PCR technique has been used to detect an amplicon that is amplified during the PCR cycling, in real time. The development of fluorogenic PCR utilizing 5'-3' nuclease activity of Taq DNA polymerase made it possible to eliminate post-PCR processing, such as visualization in agarose gels. All of these advantages are due to the nature of the amplification reaction and the availability of instruments able to perform both thermocycling and fluorescence detection.
Real-time assays are based on the same principles for RT and PCR amplification as described for standard RT-PCR. In addition to two virus- specific primers, a virus-specific oligonucleotide probe dual labeled with a fluorescent reporter dye and quencher molecule is used to facilitate the real-time monitoring of amplification. When the oligonucleotide probe is intact, either free in solution or bound to target, the reporter and quencher are in close proximity and the emission fluorescence from the reporter dye is quenched. The Taq polymerase enzymes utilized in these TaqMan assays (either Taq or Tth) possess a 5'-3' exonuclease activity and, as a result, during replication of one of the DNA strands the enzyme will encounter the bound probe and cleave it, resulting in the release of the reporter dye into solution. The release of the reporter dye and its physical separation from the quencher molecule results in an increase in fluorescence (Figure 1). The increased specificity of the TaqMan assay compared with standard RT-PCR is the result of the use of the virus-specific internal probe during the amplification. The hybridization of this probe to the target sequence and subsequent hydrolysis is detectable by the increase in fluorescence. This sequence-specific detection obviates any postamplification characterization of the amplified DNA. As a result, amplified DNA is not manipulated in the laboratory as in standard RT-PCR, thus greatly reducing the likelihood of amplicon contamination.
Principles and chemistry of SYBR® Green I- and TaqMan-based real-time assays. (A) SYBR Green I chemistry is a sequence-independent cost-effective method that relies on the intercalation of dsDNA-binding flurophores. (B) TaqMan chemistry is a sequence-specific reliable approach that utilizes fluorescent probes labeled with high energy dye on the 5' base, and a low energy quenching dye on the 3' base. h: With fluorescence; x: No fluorescence.
Pastorino et al. first developed a sensitive and specific one-step, direct TaqMan RT-PCR assay without any pretreatment step for RNA extraction and purification to detect and quantify West and Central African genotype CHIKV RNAs in infected cell supernatants and sera by targeting the E1 gene. In this study, attempts were made to reduce sample time processing and minimize viral loss during each processing step, by adapting a simple heat release of the viral RNA prior to real-time assays. The heat release of RNA was accomplished during the cDNA synthesis process itself, without preheating of the sample. The incubation of the sample at 50°C for 20 min during cDNA synthesis led to partial exposure of the viral genome. The thermal release of viral RNA by destabilization of the physical integrity of viruses by heating resulted in the release of viral RNA which is subsequently available for RT-PCR detection. It was also demonstrated that the direct TaqMan RT-PCR detection of CHIKV RNA was as sensitive as the real-time RT-PCR with an RNA extraction step having a detection limit of 27 synthetic RNA copies with spiked human serum samples. However, the temperature at which virus particles became disintegrated during heating differed significantly between virus types and physicochemical conditions. The assay system needs to be further validated with real-world clinical samples. The direct real-time RT-PCR protocol reported here has many advantages, including the requirement of a very small quantity of template and the ability to process a large number of samples in a very short time period with minimum potential viral RNA loss and risk of carry-over contamination, all of which should be advantageous for clinical analysis.
Subsequently, Edward et al. designed another E1-gene-based TaqMan real-time RT-PCR system for detection of the currently circulating strains of virus as well as other genotypes, with improved sensitivity compared with the existing TaqMan real-time RT-PCR assay. The real-time RT-PCR was tenfold more sensitive than a conventional block-based RT-PCR with a detection limit of as low as 20 copies of RNA transcript. The assay also had tenfold improved sensitivity in detecting the outbreak strain of virus when compared with the TaqMan assay of Pastrino. The evaluation of the system using a panel of 55 clinical serum samples and a synthetic RNA transcript as a positive control, demonstrated higher sensitivity by picking up additional positive cases that were missed by conventional RT-PCR, but a positive serology and virus isolation. The assay did not detect any of the other Alphaviruses, Flaviviruses or the Phlebovirus tested in the specificity panel.
Carleti et al. developed a fluorescence resonance energy transfer probe-based, quantitative, real-time RT-PCR (qRT-PCR) by targeting the NSP1 gene (with an overall mean genetic distance of 0.024) as a potential region for PCR, which is more conserved than E1. The validation of this assay by in vitro experiments in the presence of interferon, a well-known virus inhibitor, showed a dose-dependent inhibition of virus replication in Vero cells. This new, real-time RT-PCR was also used to measure viral load in serum samples from cases recently imported to Italy in which the viremia level in acute-phase serum samples ranged from 1.3 × 105 to 6 × 108 copies/ml.
Laurent et al. developed a dual-color TaqMan one-step RT-PCR assay in a LightCycler 2.0 system wherein a coextracted and coamplified chimerical RNA sequence was used as an internal control to eliminate false-negative results. The CHIKV-specific and internal control probes were labeled with 6-carboxyfluorescein (530 nm) and the wide-span dye DYXL (705 nm), respectively, eliminating the need for color compensation. A synthetic RNA was used as an external calibrator for CHIKV absolute quantification. The detection limit of 40-350 copies/ml was obtained by use of a larger plasma volume. The assay is rapid, CHIKV-specific, and highly sensitive and proved useful to detect and quantify CHIKV during the Reunion Island epidemic.
Future Virology. 2008;3(2):179-192. © 2008 Future Medicine Ltd.
Cite this: Rapid and Real-time Assays for Detection and Quantification of Chikungunya Virus - Medscape - Mar 01, 2008.