A huge variety of different RT-PCR assays and protocols are used in public and private diagnostic laboratories throughout Europe. Only some of these assays have been
published, and validation data are in general not available. It is therefore of outermost importance, that research activities are launched with the focus to improve, validate, implement and standardizes the diagnostic procedures used in Europe and globally.
Recently, a ring trial to evaluate PRRSV real-time RT-PCR detection methods in several European laboratories showed, that none of the in-house or commercial assays tested, could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity (Wernike et al. 2012), and a combination of methods was recommended.
Furthermore, PRRSV is a single stranded RNA virus, which is prone to antigenic drift.
As described, studies have shown that some European countries such as Lithuania, Latvia, Belarus and Russia harbor exceptionally diverse EU-genotype PRRSV strains (Stadejek et al. 2008). Alignments of these strains with the primer and probe sequences of published PRRSV RT-PCR assays indicated that the majority of the assays would not be able to recognize these recent strains. Updated PRRSV sequence data from many parts of Europe is very sparse; therefore, it is not clear if various assays in diagnostic use will produce a substantial number of false negative results when field samples are tested. Thus, it is obvious that there is a need for surveillance programs with the aim to continuously monitor the drift of PRRSV by sequencing subsets of circulating strains and by building joint PRRSV databases with public accessibility. The sequencing should focus on, but not be limited to ORF5 and ORF7. Full genome sequence data are presently sparse in GenBank, but when more such sequences become available, it is the hope to identify a more conserved region than the widely used ORF7, which could be the target for sensitive and robust real-time RT PCR assays.
To maximize the diagnostic performance of RT-PCR assays, it is necessary to perform in-house validation of assays, including the RNA extraction procedures, for each diagnostic specimen. It is also very important to continuously check the applicability of primers and probes for their ability to detect new variants of PRRSV. This can be done by in silico analysis and by participation in ring trials.
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