Rapid subtyping of H9N2 influenza virus by a triple reverse transcription polymerase chain reaction.
The aim of this study was to develop a rapid, cost-saving triple reverse transcription polymerase chain reaction (triple RT-PCR) for subtyping H9N2 avian influenza viruses (AIVs). The three primer pairs for amplification of target sequences of nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA) genes, respectively, were designed for subtyping the viruses in the triple RT-PCR. The sensitivity of triple RT-PCR was found to be 10(2) copies per reaction for each of NP, H9 and N2 gene. The specificity tests indicated that all of NP, HA and NA genes were positive for H9N2, only NP gene was positive for H5N1 and H1N1 AIVs, and the results were negative for the other avian viruses including Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, duck hepatitis virus and avian encephalomyelitis virus. A total of 112 clinical samples were evaluated by the assay and the results showed that the sensitivity and specificity of triple RT-PCR were in accordance with the virus isolation. In conclusion, this method is rapid and cost-effective making it feasible and attractive for large-scale epidemiological investigation of H9N2 influenza virus.
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Hao-Tai
HT
Chen
Jie
J
Zhang
Li-Na
LN
Ma
Yan-Ping
YP
Ma
Yao-Zhong
YZ
Ding
Meng
M
Wang
Xiang-Tao
XT
Liu
Yong-Guang
YG
Zhang
Yong-Sheng
YS
Liu
D000818:Animals; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D006389:Hemagglutinins, Viral / Q000235:genetics; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics; D053127:Influenza A Virus, H9N2 Subtype / Q000145:classification* / Q000235:genetics*; D009439:Neuraminidase / Q000235:genetics; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014758:Viral Core Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D006389:Hemagglutinins, Viral; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; C458881:hemagglutinin fusogenic peptide, influenza virus; C487630:NA protein, influenza A virus; D009439:Neuraminidase