Nested RT-PCR method for the detection of European avian-like H1 swine inflenza A virus

doi:10.1016/S2095-3119(15)61092-9

Journal of Integrative Agriculture ›› 2016, Vol. 15 ›› Issue (05): 1095-1102.DOI: 10.1016/S2095-3119(15)61092-9

Nested RT-PCR method for the detection of European avian-like H1 swine inflenza A virus

WEI Yan-di¹, PEI Xing-yao¹, ZHANG Yuan², YU Chen-fang¹, SUN Hong-lei¹, LIU Jin-hua¹, PU Juan¹

¹ Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture/State Key Laboratory of Agrobiotechnology/College of Veterinary Medicine, China Agricultural University, Beijing 100193, P.R.China
² China Institute of Veterinary Drug Control, Beijing 100086, P.R.China

收稿日期:2015-05-04 出版日期:2016-05-03 发布日期:2016-05-03
通讯作者: PU Juan, Tel: +86-10-62732982, Fax: +86-10-62733837, E-mail: pujuanzgz@126.com
作者简介:WEI Yan-di, E-mail: wyd900@163.com
基金资助:
This work was supported by the National High-Tech R&D Program of China (2012AA101303).

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

WEI Yan-di¹, PEI Xing-yao¹, ZHANG Yuan², YU Chen-fang¹, SUN Hong-lei¹, LIU Jin-hua¹, PU Juan¹

¹ Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture/State Key Laboratory of Agrobiotechnology/College of Veterinary Medicine, China Agricultural University, Beijing 100193, P.R.China
² China Institute of Veterinary Drug Control, Beijing 100086, P.R.China

Received:2015-05-04 Online:2016-05-03 Published:2016-05-03
Contact: PU Juan, Tel: +86-10-62732982, Fax: +86-10-62733837, E-mail: pujuanzgz@126.com
About author:WEI Yan-di, E-mail: wyd900@163.com
Supported by:
This work was supported by the National High-Tech R&D Program of China (2012AA101303).

摘要/Abstract

Abstract: Swine influenza A virus (swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like (EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription (RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets (outer and inner) were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit (PFU) mL^–1 which was over 104 PFU mL^–1 for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Key words: nested RT-PCR , swine influenza A virus , European avian-like H1 , HA gene , molecular diagnosis

WEI Yan-di, PEI Xing-yao, ZHANG Yuan, YU Chen-fang, SUN Hong-lei, LIU Jin-hua, PU Juan. Nested RT-PCR method for the detection of European avian-like H1 swine inflenza A virus[J]. Journal of Integrative Agriculture, 2016, 15(05): 1095-1102.

WEI Yan-di, PEI Xing-yao, ZHANG Yuan, YU Chen-fang, SUN Hong-lei, LIU Jin-hua, PU Juan. Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus[J]. Journal of Integrative Agriculture, 2016, 15(05): 1095-1102.

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Nested RT-PCR method for the detection of European avian-like H1 swine inflenza A virus

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

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