Simultaneous detection of novel H7N9 and other influenza A viruses in poultry by multiplex real-time RT-PCR.
BACKGROUND
A novel reassortant H7N9 influenza A virus has crossed the species barrier from poultry to cause human infections in China in 2013 and 2014. Rapid detection of the novel H7N9 virus is important to detect this virus in poultry and reduce the risk of an epidemic in birds or humans.


RESULTS
In this study, a multiplex real-time RT-PCR (rRT-PCR) assay for rapid detection of H7N9 and other influenza A viruses was developed. To evaluate the assay, various influenza A viruses, other avian respiratory viruses, and 1,070 samples from poultry were tested. Fluorescence signals corresponding to H7 and N9 subtypes were detected only when H7 and N9 subtypes were present, while the fluorescence signal for the influenza A M gene was detected in all specimens with influenza A strains. The fluorescent signal can be detected in dilutions as low as 56 copies per reaction for the H7, N9 and M genes. Intra- and inter-assay variability tests showed a reliable assay. In poultry samples, a comparison of rRT-PCR with virus isolation showed a high level of agreement.


CONCLUSIONS
The multiplex rRT-PCR assay in this study has good specificity, sensitivity and reproducibility, and will be useful for laboratory surveillance and rapid diagnosis of H7N9 and other influenza A viruses.
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. qdxxl1989@163.com.
Xiaolong
X
Xu
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. baohongmei@caas.cn.
Hongmei
H
Bao
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. ganzhitg@163.com.
Yong
Y
Ma
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. conansun6812@163.com.
Jiashan
J
Sun
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. zyhzs1234@163.com.
Yuhui
Y
Zhao
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. wangyunhe1130@163.com.
Yunhe
Y
Wang
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. shijianzhong@caas.cn.
Jianzhong
J
Shi
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. zengxianying@caas.cn.
Xianying
X
Zeng
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. lyb@hvri.ac.cn.
Yanbing
Y
Li
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. wxr@hvri.ac.cn.
Xiurong
X
Wang
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People's Republic of China. chenhualan@caas.cn.
Hualan
H
Chen
D000818:Animals; D002681:China; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009439:Neuraminidase / Q000235:genetics; D011200:Poultry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods; D014763:Viral Matrix Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D019267:Hemagglutinin Glycoproteins, Influenza Virus; C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase