RNA Extraction from Swine Samples and Detection of Influenza A Virus in Swine by Real-Time RT-PCR.

Abstract

Real-time reverse-transcription PCR (rRT-PCR) assays are currently the method of choice in many laboratories for the detection and subtyping of influenza A virus (IAV) in swine. Traditionally, nasal swabs and lung tissues (sometimes bronchoalveolar lavage and tracheal tissues) are the primary specimens for IAV testing. However, oral fluids are becoming more common for IAV prognostic profiling. In this chapter, we describe (1) procedures of RNA extraction from the common clinical specimens, (2) two rRT-PCR assays for detection of IAV in swine, and (3) an rRT-PCR assay for subtyping swine IAV. RNA extraction procedures include a magnetic bead method optimized for extraction from nasal swabs and tissue homogenates and a magnetic bead method optimized for extraction from oral fluids. Two rRT-PCR assays for detection of swine IAV include a USDA-validated IAV rRT-PCR targeting the matrix gene and the USDA-licensed VetMAX™-Gold Swine Influenza Virus Detection rRT-PCR kit (Thermo Fisher Scientific) targeting the nucleoprotein and matrix genes. The swine IAV subtyping assay described here is VetMAX™-Gold Swine Influenza Virus Subtyping rRT-PCR kit (Thermo Fisher Scientific) which distinguishes swine IAV H1 from H3 and N1 from N2.