One-step multiplex RT-PCR for detection and subtyping of swine influenza H1, H3, N1, N2 viruses in clinical samples using a dual priming oligonucleotide (DPO) system

• Published in: Journal of virological methods 2008-07, Vol.151 (1), p.30-34
• Main Authors: Lee, C.S., Kang, B.K., Lee, D.H., Lyou, S.H., Park, B.K., Ann, S.K., Jung, K., Song, D.S.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; DNA Primers - genetics; DPO; Fundamental and applied biological sciences. Psychology; Influenza A virus - classification; Influenza A virus - genetics; Influenza A virus - isolation & purification; Influenza A Virus, H1N1 Subtype - classification; Influenza A Virus, H1N1 Subtype - genetics; Influenza A Virus, H1N1 Subtype - isolation & purification; Influenza A Virus, H3N2 Subtype - classification; Influenza A Virus, H3N2 Subtype - genetics; Influenza A Virus, H3N2 Subtype - isolation & purification; Korea; Microbiology; Multiplex PCR; Orthomyxoviridae Infections - diagnosis; Orthomyxoviridae Infections - veterinary; Orthomyxoviridae Infections - virology; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - analysis; RNA, Viral - isolation & purification; Sensitivity and Specificity; Swine - virology; Swine Diseases - diagnosis; Swine Diseases - virology; swine influenza virus; Swine influenzavirus; Techniques used in virology; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2008.04.001

The swine influenza virus (SIV) H1N1, H1N2, and H3N2 subtypes circulate in Korean farm. A novel multiplex RT-PCR (m-RT-PCR) was developed to detect and subtype swine influenza viruses. This m-RT-PCR assay could identify H1, H3, N1 and N2 from clinical samples in single tube reaction using DPO system. Korean SIVs are closely related to the United States influenza viruses, and primers were developed for SIV from North American viruses and recently Korean isolates. The sensitivity of the m-RT-PCR was 10TCID 50/ml for H1N1, H1N2 or H3N2. The lowest viral concentrations detected by single PCR were 1TCID 50/ml for each subtype. Non-specific reactions were not observed when other viruses and bacteria were used to assess the m-RT-PCR. The results of m-RT-PCR were more effective than virus isolation or hemagglutination (HA) test. This assay using a DPO system provides a rapid, sensitive, and cost-effective laboratory diagnosis for detecting and subtyping of SIV in pigs.