Additional comments

There are many other factors concerning viral qPCR standards that should be considered. First, the use of plasmids as external standards has been criticized, because, although these may be well characterized, little may be known about the composition of the clinical samples being analyzed. Thus, the use of plasmids may erroneously assume comparable amplification characteristics to the viral target in test samples. Niesters and Puchhammer-Stockl (2004) emphasize that standards should use whole virus diluted in the same appropriate matrix as the clinical sample, and nucleic acids must be isolated using the same extraction procedure. Multiple replicates of the standards should be analyzed to determine confidence limits based on inter-assay and intra-assay variation. The problems concerning standards are further complicated by a lack of universally accepted international standards for most clinically significant viruses (Niesters and Puchhammer-Stockl, 2004), and thus, qPCR results may vary between different laboratories. It is essential therefore, that clinicians are aware of these limitations and their potential for variation in the qPCR results. Recognition of these does not devalue the overall utility of qPCR, but rather will allow for a proper assessment of the results within a relevant clinical context.

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