title: Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus.
abstract: In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry.
authors.affiliation: Laboratory of Avian Diseases, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Thanh Trung
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Hyuk-Joon
authors.initials: HJ
authors.lastname: Kwon
authors.forename: Il-Hwan
authors.initials: IH
authors.lastname: Kim
authors.forename: Seung-Min
authors.initials: SM
authors.lastname: Hong
authors.forename: Won-Jin
authors.initials: WJ
authors.lastname: Seong
authors.forename: Jin-Wook
authors.initials: JW
authors.lastname: Jang
authors.forename: Jae-Hong
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis* / Q000821:virology; D001717:Birds; D001351:Infectious bronchitis virus / Q000235:genetics / Q000302:isolation & purification*; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009522:Newcastle disease virus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods; D014773:Virology / Q000379:methods; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary