Distinct swine sample matrices used for high throughput RNA purification and TaqMan~R RT-PCR amplification of North American and European PRRSV RNA

摘要

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an enveloped, ssRNA plus-sense virus, family Arteriviridae, order Nidovirales PRRSV. There are two main antigenic subtypes of PRRSV: the North American (NA) subtype and the European (EU) subtype. An integrated workflow has been developed that includes the isolation of viral RNA from diverse sample matrices followed by simultaneous amplification and subtyping of NA and EU PRRSV RNA. The workflow was validated using the Ambion~R MagMAX TM Viral-96 RNA Isolation Kit, Multiplex RT-PCR Reagents, primers/probes targeting PRRSV RNA, and PRRSV RNA synthetic transcripts as controls. As few as 50 copies of PRRSV RNA transcripts were consistently amplified using this workflow. An internal control,Xeno TM RNA, was used to monitor nucleic acid purification efficiency, detect the presence of reaction inhibitors, and assess functionality of reagents and reaction preparations, to minimize false negative results. Performance of this workflow was evaluated using more than 100 field samples of known PRRSV status. The reagents and instruments used provided fast, sensitive, and specific amplification of both NA and EU PRRSV subtypes from different types of swine samples.