Detection and monitoring of virus infections by real-time PCR

Abstract

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction

“…Both the polymerase chain reaction and the detection of amplicons are combined in this technique, so minimising the risk of contamination on the amplicon level and reducing the costs of analysis (Niesters, 2002;Gunson et al, 2006;Watzinger et al, 2006). This paper describes the development and evaluation of two real-time PCR assays for the detection of proviral SRLV sequences located in the untranslated leader-gag and LTR regions.…”

Real-time quantification of RNA polymerase activity using a “broken beacon”

“…Upon intercalation the fluorescence quantum yield of the dye increases substantially, and the resulting increase in fluorescence is proportional to the concentration of dsDNA. However, since intercalating dyes bind nonspecifically to DNA, amplification of mutations and contaminants can be problematic [1]. As a result, sequence-specific labeled probes are more prevalent in real-time detection.…”

“…The advantage of this method is due to the strict spatial limitation for observing FRET. In this case, non-bound probes in solution exhibit only a minimal background so that lower detection limits can be achieved [1,[3][4][5].…”

“…The result is an increase in fluorescence with an increase in the amount of DNA produced. The TaqMan® probes are now widely used for the detection of DNA amplification during PCR [1,[3][4]6]. However, this approach can not be easily used with RNA polymerases since the product is single-stranded RNA.…”

“…Opposite ends are labeled with a fluorophore and quencher, respectively. As shown in Figure 1C, the loop structure is complementary to the target so that when the beacon binds to the product, the fluorophore is separated from the quencher, and fluorescence increases in proportion to the amount of DNA produced [1,3,[6][7][8][9].…”