? Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses
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    2017, Vol. 16 Issue (09): 2055-2061     DOI: 10.1016/S2095-3119(17)61655-1
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Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses
WANG Xiu-rong*, GU Lin-lin*, SHI Jian-zhong, XU Hai-feng, ZHANG Ying, ZENG Xian-ying, DENG Guo-hua, LI Cheng-jun, CHEN Hua-lan
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, P.R.China
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Abstract    In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China.  Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus.  The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza.  Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus.  One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established.  Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity.  The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs.  Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples.  Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.
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Key wordsH7N9     highly pathogenic influenza virus     real-time RT-PCR     
Received: 2017-05-16; Published: 2017-05-18

This study was supported by the National Key R&D Program of China (2016YFD0500800) and the International Science & Technology Cooperation Program of China (2014DFR31260).

Corresponding Authors: Correspondence LI Cheng-jun, Tel: +86-451-51051688, E-mail: lichengjun@caas.cn; CHEN Hua-lan, Tel: +86-451-51997168, E-mail: chenhualan@caas.cn   
Cite this article:   
WANG Xiu-rong, GU Lin-lin, SHI Jian-zhong, XU Hai-feng, ZHANG Ying, ZENG Xian-ying, DENG Guo-hua, LI Cheng-jun, CHEN Hua-lan. Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses[J]. Journal of Integrative Agriculture, 2017, 16(09): 2055-2061.
http://www.chinaagrisci.com/Jwk_zgnykxen/EN/ 10.1016/S2095-3119(17)61655-1      or     http://www.chinaagrisci.com/Jwk_zgnykxen/EN/Y2017/V16/I09/2055
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