One-step Multiplex RT-PCR Assay for the Detection of Peste des petits ruminants Virus in Clinical Samples

摘要

A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7–17 days post infection (p.i.). and in oral swabs 7–15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.