Abstract
Real-time RT-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 broncho-alveolar 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 (Life Technologies) targeting the nucleoprotein and matrix genes. The swine IAV subtyping assays described here are multiplex SIV HA (H1 and H3) and NA (N1 and N2) subtyping rRT-PCR reagents from Life Technologies.
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References
Detmer SE, Patnayak DP, Jiang Y, Gramer MR, Goyal SM (2011) Detection of influenza A virus in porcine oral fluid samples. J Vet Diagn Invest 23:241–247
Romagosa A, Gramer M, Joo HS, Torremorell M (2012) Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non-vaccinated pigs. Influenza Other Respi Viruses 6:110–118
Ramirez A, Wang C, Prickett JR, Pogranichniy R, Yoon KJ, Main R, Johnson JK, Rademacher C, Hoogland M, Hoffmann P, Kurtz A, Kurtz E, Zimmerman J (2012) Efficient surveillance of pig populations using oral fluids. Prev Vet Med 104:292–300
Goodell CK, Prickett J, Kittawornrat A, Zhou F, Rauh R, Nelson W, O’Connell C, Burrell A, Wang C, Yoon KJ, Zimmerman JJ (2013) Probability of detecting influenza A virus subtypes H1N1 and H3N2 in individual pig nasal swabs and pen-based oral fluid specimens over time. Vet Microbiol 166:450–460
Mackay IM, Arden KE, Nitsche A (2002) Real-time PCR in virology. Nucleic Acids Res 30:1292–1305
Watzinger F, Ebner K, Lion T (2006) Detection and monitoring of virus infections by real-time PCR. Mol Aspects Med 27:254–298
Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL (2002) Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 40: 3256–3260
Spackman E, Suarez DL (2008) Type A influenza virus detection and quantitation by real-time RT-PCR. Methods Mol Biol 436:19–26
Vincent AL, Ma W, Lager KM, Janke BH, Richt JA (2008) Swine influenza viruses a North American perspective. Adv Virus Res 72:127–154
Lorusso A, Vincent AL, Gramer ME, Lager KM, Ciacci-Zanella JR (2013) Contemporary epidemiology of North American lineage triple reassortant influenza A viruses in pigs. Curr Top Microbiol Immunol 370:113–132
Karasin AI, Brown IH, Carman S, Olsen CW (2000) Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J Virol 74:9322–9327
Karasin AI, Olsen CW, Brown IH, Carman S, Stalker M, Josephson G (2000) H4N6 influenza virus isolated from pigs in Ontario. Can Vet J 41:938–939
Lekcharoensuk P, Lager KM, Vemulapalli R, Woodruff M, Vincent AL, Richt JA (2006) Novel swine influenza virus subtype H3N1, United States. Emerg Infect Dis 12:787–794
Ma W, Gramer M, Rossow K, Yoon KJ (2006) Isolation and genetic characterization of new reassortant H3N1 swine influenza virus from pigs in the midwestern United States. J Virol 80:5092–5096
Ma W, Vincent AL, Gramer MR, Brockwell CB, Lager KM, Janke BH, Gauger PC, Patnayak DP, Webby RJ, Richt JA (2007) Identification of H2N3 influenza A viruses from swine in the United States. Proc Natl Acad Sci U S A 104:20949–20954
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Zhang, J., Harmon, K.M. (2014). RNA Extraction from Swine Samples and Detection of Influenza A Virus in Swine by Real-Time RT-PCR. In: Spackman, E. (eds) Animal Influenza Virus. Methods in Molecular Biology, vol 1161. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0758-8_23
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DOI: https://doi.org/10.1007/978-1-4939-0758-8_23
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Publisher Name: Humana Press, New York, NY
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