Use of real-time reverse transcriptase polymerase chain reaction assay and cell culture methods for detection of swine influenza A viruses

Abstract

To evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay performed on pooled nasal swab specimens, compared with virus isolation performed on individual nasal swab specimens by use of 2 cell culture lines for detection of swine influenza A viruses. 900 nasal swab specimens obtained from pigs at an abattoir and 62 nasal swab specimens submitted for diagnostic testing. Primers were chosen to amplify a conserved portion of the influenza virus matrix gene. Assay sensitivity was initially determined by testing serial dilutions of various subtypes of swine influenza viruses. Sensitivity and specificity were confirmed by use of nasal swab specimens with or without addition of known concentrations of influenza virus and further validated by testing nasal swab specimens obtained through an abattoir surveillance program or submitted for diagnostic testing. Aliquots of specimens were pooled in sets of 10, and results of real-time RT-PCR assays were compared with results of virus isolation of individual specimens in Madin Darby canine kidney (MDCK) and mink lung (Mv1Lu) cells. Real-time RT-PCR assay was highly specific (100%) and sensitive (88% to 100%). Among the 16 viruses isolated, 3 grew only in Mv1Lu cells and 3 grew only in MDCK cells. Results indicate that real-time RT-PCR assay is a fast and accurate test for screening numerous nasal swab specimens for swine influenza virus. Some viruses were isolated in only MDCK or Mv1Lu cells, indicating that use of >1 cell line may be required to isolate a broad range of influenza A viruses.