a family-wide rt-pcr assay for detection of paramyxoviruses and application to a large-scale surveillance study地利亚的family-wide rt - pcr试验检测和应用大规模监测研究.pdfVIP

A Family-Wide RT-PCR Assay for Detection of Paramyxoviruses and Application to a Large-Scale Surveillance Study Sander van Boheemen, Theo M. Bestebroer, Josanne H. Verhagen, Albert D. M. E. Osterhaus, Suzan D. Pas, Sander Herfst, Ron A. M. Fouchier* Department of Virology, Erasmus Medical Center, Rotterdam, The Netherlands Abstract Family-wide molecular diagnostic assays are valuable tools for initial identification of viruses during outbreaks and to limit costs of surveillance studies. Recent discoveries of paramyxoviruses have called for such assay that is able to detect all known and unknown paramyxoviruses in one round of PCR amplification. We have developed a RT-PCR assay consisting of a single degenerate primer set, able to detect all members of the Paramyxoviridae family including all virus genera within the subfamilies Paramyxovirinae and Pneumovirinae. Primers anneal to domain III of the polymerase gene, with the 39 end of the reverse primer annealing to the conserved motif GDNQ, which is proposed to be the active site for nucleotide polymerization. The assay was fully optimized and was shown to indeed detect all available paramyxoviruses tested. Clinical specimens from hospitalized patients that tested positive for known paramyxoviruses in conventional assays were also detected with the novel family-wide test. A high-throughput fluorescence-based RT-PCR version of the assay was developed for screening large numbers of specimens. A large number of samples collected from wild birds was tested, resulting in the detection of avian paramyxoviruses type 1 in both barnacle and white-fronted geese, and type 8 in barnacle geese. Avian metapneumovirus type C was found for the first time in Europe in mallards, greylag geese and common gulls. The single round family-wide RT-PCR assay described here is a useful tool for the detecti