Real-time PCR in virology

Abstract

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Figure 1

Kinetic amplification. (A) An idealised plot of temperature versus time during a single PCR cycle comprised of the denaturation (D), primer and probe annealing (A) and primer extension (E) steps. At the indicated optimal temperature ranges, dsDNA is denatured (T_D), oligoprobes anneal (T_M-PROBE) and finally the primers anneal as a precursor to their extension (T_M-PRIMER). The actual temperature, shown as a dashed line, may overshoot the desired temperature to varying degrees, depending on the quality of the thermocycler employed. (B) The ideal amplification curve of a real-time PCR (bold), when plotted as fluorescence intensity against the cycle number, is a typical sigmoidal growth curve. Early amplification cannot be viewed because the detection signal is indistinguishable from the background. However, when enough amplicon is present, the assay’s exponential progress can be monitored as the rate of amplification enters a log-linear phase (LP). Under ideal conditions, the amount of amplicon increases at a rate of approximately one log₁₀ every three cycles. As primers and enzyme become limiting and products inhibitory to the PCR accumulate, the reaction slows, entering a transition phase (TP), eventually reaching the plateau phase (PP) where there is little or no further increase in product yield. The point at which the fluorescence passes from insignificant levels to clearly detectable is called the threshold cycle (C_T; indicated by an arrow), and this value is used in the calculation of template quantity during quantitative real-time PCR. Also shown are curves representing an optimal titration of template (grey), consisting of higher and lower starting template concentrations, which produce lower or higher C_T values, respectively. Data for the construction of a standard curve are taken from the LP, which subsequently allows the concentration of unknown samples to be determined.

Figure 2

Fluorogenic mechanisms. When a 5′ nuclease probe’s reporter (R) and quencher (Q, open) are in close proximity as in (A), the quencher ‘hijacks’ the emissions that have resulted from excitation of the reporter by the instrument’s light source. The quencher then emits this energy (solid arrows). When the fluorophores are separated beyond a certain distance, as occurs upon hydrolysis as depicted in (B), the quencher no longer exerts any influence and the reporter emits at a distinctive wavelength (dashed arrows) which is recorded by the instrument. In the reverse process as depicted in (C) using adjacent oligoprobes, the fluorophores begin as separated entities. A signal from the acceptor (A) can only be generated when the donor (D) comes into close proximity as shown in (D). This occurs as the result of adjacent hybridisation of the oligoprobes to the target amplicon. In (E) another form of quenching is shown, caused by the intimate contact of labels attached to hairpin oligoprobes (molecular beacon, sunrise or scorpion). The fluorophore (F) and a NFQ (Q, closed) interact more by collision than FRET, disrupting each other’s electronic structure and directly passing on the excitation energy which is dissipated as heat (jagged, dashed arrows). When the labels are separated by disruption of the hairpin, as is the case in (F), the fluorophore is free to fluoresce (dashed arrows).

Figure 3

Oligoprobe chemistries. (A) 5′ Nuclease oligoprobes. As the DNA polymerase (pol) progresses along the relevant strand, it displaces and then hydrolyses the oligoprobe via its 5′→3′ endonuclease activity. Once the reporter (R) is removed from the extinguishing influence of the quencher (Q, open), it is able to release excitation energy at a wavelength that is monitored by the instrument and different from the emissions of the quencher. Inset shows the NFQ and MGB molecule that make up the improved MGB nuclease-oligoprobes. (B) Hairpin oligoprobes. Hybridisation of the oligoprobe to the target separates the fluorophore (F) and non-fluorescent quencher (Q, closed) sufficiently to allow emission from the excited fluorophore, which is monitored. Inset shows a wavelength-shifting hairpin oligoprobe incorporating a harvester molecule. (C) Adjacent oligoprobes. Adjacent hybridisation results in a FRET signal due to interaction between the donor (D) and acceptor (A) fluorophores. This bimolecular system acquires its data from the acceptor’s emissions in an opposite manner to the function of nuclease oligoprobe chemistry. (D) Sunrise primers. The opposite strand is duplicated so that the primer’s hairpin structure can be disrupted. This separates the labels, eliminating the quenching in a similar manner to the hairpin oligoprobe. (E) Scorpion primers. The primer does not require extension of the complementary strand; in fact it blocks extension to ensure that the hairpin in the probe is only disrupted by specific hybridisation with a complementary sequence designed to occur downstream of its own, nascent strand. Inset shows a duplex scorpion that exchanges the stem–loop structure for a primer element terminally labelled with the fluorophore and a separate complementary oligonucleotide labelled with a quencher at the 5′ terminus.

Figure 4

Fluorescent melting curve analysis. At the completion of a real-time PCR using a pair of adjacent oligoprobes, the reaction can be cooled to a temperature below the expected T_M of the oligoprobes then heated above 85°C at a fraction of a degree per second (B). During heating, the emissions of the acceptor can be constantly acquired (C). Software calculates the negative derivative of the fluorescence over time, producing clear peaks that indicate the T_M of the oligoprobe-target melting transition (D). When one or more mutations are present under one or both oligoprobes (A), this T_M is shifted and this shift can be used diagnostically to discriminate single nucleotide polymorphisms in the template. Ideally, one of the oligoprobes, the anchor, is designed to bind to a stable sequence region, whereas the other, sensor, will span the mismatch. Mismatches near the centre of the probe and flanked by G:C pairs are more destabilising than mismatches near the ends of the oligoprobe flanked by A:T pairs.