title: A multiplex RT-PCR assay for detection and differentiation of avian H3, H5, and H9 subtype influenza viruses and Newcastle disease viruses.
abstract: Avian influenza viruses (AIVs) and Newcastle disease viruses (NDVs) co-circulate in the poultry population in China. These viruses cause repeated disease outbreaks that exhibit similar clinical symptoms and epidemiological patterns. H5 and H9 influenza viruses are the major pathogens infecting poultry stocks. Recently, H3 AIV (one of the main subtypes in waterfowl) has become endemic in chickens. A multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was designed for simultaneous detection and differentiation of avian H3, H5, H9 subtype AIVs and NDVs. Four primer sets were evaluated, three of which specifically targeted the hemagglutinin genes of H3, H5 and H9 AIVs, while the other targeted the NDV fusion gene. The sensitivity and specificity of the mRT-PCR assay was determined. The assay detected the major clades or genotypes of all of the reference AIVs and NDVs currently circulating in China. In addition, the mRT-PCR results obtained from screening 380 clinical swabs and 12 experimental tracheal samples were consistent with those obtained using conventional virus isolation methods. The mRT-PCR assay was established successfully for the detection and differentiation of avian H3, H5, and H9 subtype AIVs and NDVs. The method should, therefore, provide a valuable diagnostic tool for these infections.
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Qingdong
authors.initials: Q
authors.lastname: Tang
authors.forename: Jinliang
authors.initials: J
authors.lastname: Wang
authors.forename: Jingnan
authors.initials: J
authors.lastname: Bao
authors.forename: Honglei
authors.initials: H
authors.lastname: Sun
authors.forename: Yipeng
authors.initials: Y
authors.lastname: Sun
authors.forename: Jinhua
authors.initials: J
authors.lastname: Liu
authors.forename: Juan
authors.initials: J
authors.lastname: Pu
mesh_terms: D000818:Animals; D002645:Chickens; D002681:China; D017931:DNA Primers / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009521:Newcastle Disease / Q000821:virology*; D009522:Newcastle disease virus / Q000235:genetics / Q000302:isolation & purification*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods
chemical_list: D017931:DNA Primers