The majority of diagnostic PCR assays reported to date have been used in a qualitative, or ‘yes/no’ format. The development of real-time PCR has brought true quantitation of target nucleic acids out of the pure research laboratory and into the diagnostic laboratory.
Determining the amount of template by PCR can be performed in two ways: as relative quantitation and as absolute quantitation. Relative quantitation describes changes in the amount of a target sequence compared with its level in a related matrix. Absolute quantitation states the exact number of nucleic acid targets present in the sample in relation to a specific unit (71
). Generally, relative quantitation provides sufficient information and is simpler to develop. However, when monitoring the progress of an infection, absolute quantitation is useful in order to express the results in units that are common to both scientists and clinicians and across different platforms. Absolute quantitation may also be necessary when there is a lack of sequential specimens to demonstrate changes in virus levels, no suitably standardised reference reagent or when the viral load is used to differentiate active versus persistent infection.
A very accurate approach to absolute quantitation by PCR is the use of competitive co-amplification of an internal control nucleic acid of known concentration and a wild-type target nucleic acid of unknown concentration, with the former designed or chosen to amplify with an equal efficiency to the latter (72
). However, while conventional competitive PCR is relatively inexpensive, real-time PCR is far more convenient, reliable and better suited to quick decision making in a clinical situation (77
). This is because conventional, quantitative, competitive PCR (qcPCR) requires significant development and optimisation to ensure reproducible performance and a predetermined dynamic range for both the amplification and detection components (79
Although a comparison of absolute standard curves, relative standard curves and CT
values produces similar final values (80
), the general belief remains that an internal control in combination with replicates of each sample are essential for reliable quantitation by PCR (38
). Unfortunately, real-time PCR software with the ability to calculate the concentration of an unknown by comparing signals generated by an amplified target and internal control is only beginning to emerge. This issue will hopefully be addressed in upcoming commercial releases (81
). Therefore, the next best approach to quantitation by PCR is the use of an external standard curve. This approach relies upon titration of an identically amplified template, in a related sample matrix, within the same experimental run. While the external standard curve is the more commonly described approach, it suffers from uncontrolled and unmonitored inter-tube variations. Because of this omission, such experiments should be described as semi-quantitative. Despite this sub-optimal approach, fluorescence data is generally collected from PCR cycles that span the linear amplification portion of the reaction where the fluorescent signal and the accumulating DNA are proportional. Because the emissions from fluorescent chemistries are temperature dependent, data is generally acquired only once per cycle at the same temperature in order to monitor amplicon yield (45
). The CT
of the sample at a specific fluorescence value can then be compared with similar data collected from a series of standards by the calculation of a standard curve. The determination of the CT
depends upon the sensitivity and ability of the instrument to discriminate specific fluorescence from background noise, the concentration and nature of the fluorescence-generating component and the amount of template initially present.
Real-time PCR offers significant improvements to the quantitation of viral load because of its enormous dynamic range that can accommodate at least eight log10
copies of nucleic acid template (33
). This is made possible because the data are chosen from the linear phase (LP; Fig. B) of amplification where conditions are optimal, rather than the end-point where the final amount of amplicon present may have been affected by inhibitors, poorly optimised reaction conditions or saturation by inhibitory PCR by-products and double-stranded amplicon. The result of taking data from the end-point is that there may not be a relationship between the initial template and final amplicon concentrations.
Real-time PCR is also an attractive alternative to conventional PCR for the study of viral load because of its low inter-assay and intra-assay variability (77
) and its equivalent or greater analytical sensitivity in comparison with traditional viral culture, or conventional single-round, and nested PCR (77
). Real-time PCR has been reported to be at least as sensitive as Southern blot (92
). However, these reports could be an over-estimate due to the choice of smaller targets, which amplify more efficiently, or due to the use of different or improved primers for the real-time assays because the use of software to design optimised primers and oligoprobes is more common.
When this increased sensitivity and broad dynamic range are combined, it is possible to quantitate template from samples containing a large range of concentrations, as is often the case in patient samples. This avoids the need for dilution of the amplicon prior to conventional detection or repeat of the assay using a diluted sample because the first test result falls outside the limits of the assay. These are problems encountered when using some conventional qcPCR assay kits, which cannot encompass high viral loads whilst maintaining suitable sensitivity (52
). The flexibility of real-time PCR is also demonstrated by its ability to detect one target in the presence of a vast excess of another target in duplexed assays (84
Viral load is also a useful indicator of the extent of active infection, virus–host interactions and the response to antiviral therapy, all of which can play a role in the treatment regimen selected (100
). Conventional quantitative PCR has already proven the benefits of applying nucleic acid amplification to the monitoring of viral load as a useful marker of disease progression and as a component of studies into the efficacy of antiviral compounds (74
). The severity of some diseases has been shown to correlate with the viral load making real-time PCR quantitation useful to study not simply the presence of a virus but the role of viral reactivation or persistence in the progression of disease (78
An example of the benefits which real-time PCR has brought to the quantitative detection of human cytomegalovirus (CMV) is seen in patients who are immunosuppressed following solid organ or bone marrow transplantation. Although qualitative detection of CMV DNA by PCR has been used as an indicator for the success of antiviral therapy, quantitative assays are preferred in order to monitor patient’s therapeutic responses. Moreover, since it has been postulated that the monitoring of viral replication over time is a more reliable indicator of a developing viral disease than the determination of absolute viral amounts at a single point of time, several quantitative assays have been established and evaluated to increase diagnostic accuracy. Quantitative competitive assays based on end-point analysis have displayed detection limits of 5 × 101
genome equivalents (ge) per assay and a dynamic range of 5 × 101
–5 × 104
). Hybridisation-based assays covered approximately the same dynamic range of four orders of magnitude with detection limits of 20 ge/assay (114
). Although these assays possess dynamic ranges that may be sufficient for most clinical applications, they display a high inter- and intra-assay variability, up to 40% (115
In contrast, one of the first published real-time PCR assays for the detection of CMV DNA could be performed in <90 min, spanned a dynamic range of six to seven orders of magnitude with a detection limit of at least 10 ge/assay and an inter-assay and intra-assay variability of <10% and <5%, respectively, using plasma samples from bone marrow transplant patients (116