It should be noted that the successful validation of a PCR assay against specimens collected from any one patient population at a particular time may give only limited indication to the performance of that assay in another patient population, or even on the same patient population at a different time. Essentially this is due to differences and mutation of circulating viral strains in those populations, and highlights the need for assay validation on virus populations that represent local virus types and subtypes. Following validation there is then a clear need for ongoing quality control of assay performance.
Many of the limitations due to sequence variation can often be complicated by a lack of sequence information for local viral subtypes during assay development. This may be overcome by laboratories conducting their own sequencing studies, but this increases assay development costs considerably, as a broad range of viral strains need to be sequenced to provide adequate information. In general, we use sequence information on public databases as a tentative guide only and develop two PCR assays, targeting different genes, which are then evaluated in parallel.
Although these issues highlight some notable pitfalls concerning the application of molecular assays in viral diagnostics, they detract from the enormous potential of PCR over traditional techniques, such as DFA and virus isolation by cell culture. The major benefit of PCR is its potential for greater sensitivity and specificity compared to the traditional methods. We believe there is a need for acceptance that some viral strains may be missed by PCR in the same manner that the traditional methods may fail to detect low viral loads or non-viable organisms. Nonetheless, the problems concerning strain variation may be overcome by using two PCR assays with different gene targets in parallel, although this would inevitably increase the cost of testing.
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