Original Article: Real time reverse transcription (RRT)‐polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs

Abstract

Please cite this paper as: Slomka et al. (2010) Real time reverse transcription (RRT)‐polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza and Other Respiratory Viruses 4(5), 277–293. Background There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods. Objectives First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections. Methods RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v. Results The “perfect match” M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a “gold standard”, while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. “Perfect match” M gene RRT PCR had 100% sensitivity and 95·2% specificity for swabs, 93·6% and 98·6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99·1%, respectively, for the swabs, and 100% and 100% for the tissues. Conclusions Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.

Origin, distribution, and potential risk factors associated with influenza A virus in swine in two production systems in Guatemala

“…Influenza A virus RNA was detected by rRT‐PCR with matrix‐specific primers,12, 13 using the one‐step RT‐PCR or the Quantitec QuantiTect Probe RT‐PCR Kits (QIAGEN, Hilden, Germany) in the ABI 7300 Real‐Time PCR System (Applied Biosystems, Foster City, CA). Positive controls for matrix RNA detection included RNA extracts from inactivated virus (provided by the National Veterinary Services Laboratories, USDA, Ames, Iowa) or in vitro transcribed RNA from plasmid DNA containing the corresponding gene segment.…”

Genome plasticity of triple-reassortant H1N1 influenza A virus during infection of vaccinated pigs

“…Eleven 3-week-old specific-pathogen-free piglets were selected from a serologically IAV-negative swine herd and moved to the University of Minnesota animal research units. The IAV-negative status was confirmed by testing individual nasal swabs with RRT-PCR targeting the M gene (Slomka et al, 2010;Spackman & Suarez, 2008) and serum samples by ELISA (Influenza Ab Test kit; IDEXX Laboratories) for antibodies against NP (Ciacci-Zanella et al, 2010). Viral RNA was eluted using 50 ml of each sample into 50 ml elution buffer using an Ambion MagMax virus RNA isolation kit (Life Technologies).…”

“…PCR mix containing 5 ml RNA, 12.5 ml 2| buffer, 1.0 ml 25| enzyme mix, 1.67 ml detection enhancer, 5 pmol each primer and 1.5 pmol probe was run on a LightCycler 480 system (Hoffmann-La Roche) at 45 uC for 10 min, followed by 95 uC for 10 min, and 45 cycles at 94 uC for 1 s and 60 uC for 30 s. Fluorescence was recorded at 60 uC and a sample was considered positive if the C t was v40. This PCR protocol can detect IAVs in samples containing i200 copies of the target amplicon, and has 100 and 95 % diagnostic sensitivity and specificity, respectively (Slomka et al, 2010). (Spackman & Suarez, 2008) and ELISA, respectively.…”

Emergence of the pandemic H1N1 2009 influenza A virus in swine herds in Poland

“…The viral RNA extracted from the samples was used for partial amplification of the M gene of type A influenza virus in RRT-PCR using a QuantiTect Probe RT-PCR Kit (QIAGEN, Canada). Sequences of primers and a probe and RT-PCR protocol were acquired from Slomka et al (28).…”