There has been a marked improvement in the diagnosis of viral infections over the last decade with the application of the polymerase chain reaction (PCR), and more recently real-time PCR. The employment of real-time PCR for virus detection offers the advantage of high sensitivity and reproducibility combined with an extremely broad dynamic range. However, the greatest impact of real-time PCR has undoubtedly been its ability to quantify the viral load in clinical specimens. The results can be expressed in absolute terms with reference to quantified standards, or in relative terms compared to another target sequence present in the sample. In addition, quantitative PCR (qPCR) tests offer the possibility to determine the dynamics of viral proliferation, monitor the response to treatment, and distinguish between latent and active infections.

Many qPCR protocols for the detection of viral targets have now been published and commercial assays are available for a number of clinically important viruses, including human immunodeficiency virus (Kumar et al., 2002; O'Doherty et al, 2002), hepatitis B and C viruses (Berger et al, 1998), and cytomegalovirus (Boeckh and Boivin, 1998). However, the number of commercial assays available is still limited and this has led to the development and introduction of in-house real-time qPCR protocols.

Current approaches to the development of viral real-time PCR assays commonly employ fluorescent chemistry to effect the kinetic measurement of product accumulation. These may include non-specific compounds such as the DNA intercalating dye SYBR® Green or sequence specific oligoprobes which employ fluorescent resonance energy transfer (FRET). In practice, our experience has been that the specific approach has yielded better results for the detection of viral pathogens in clinical specimens, and particularly so in the determination of viral loads.

Recently, technical improvements in sequence detection systems have enabled extensive characterization of the viral genome, including determination of viral subtype, genotype, variants mutants and genotypic resistance patterns. However, such characterization has demonstrated that many regions of the viral genome are heterogeneous, displaying sequence variation not only between genera, but also variants of the same virus isolated at a single geographic location. Such variation has profound implications for the design of sensitive and specific real-time PCR protocols, particularly for RNA viruses where genomic polymorphism is widespread.

Also, in order to obtain meaningful results, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples, and careful optimization of the PCR conditions are required to obtain consistent results. This is particularly important in the design and performance of qPCR. Even so, other factors, inherent to the target virus, also can affect the results of qPCR. Recent studies in our laboratory have demonstrated that sequence variation in the primer or probe target sites has a significant and major impact of the validity of qPCR results in determining viral load (Whiley and Sloots, 2005b). This chapter will examine the effect of sequence variation of primer and probe target sites on assay performance, and its effect on quantification of viral loads.

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