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Journal of Integrative Agriculture  2016, Vol. 15 Issue (9): 2105-2113    DOI: 10.1016/S2095-3119(15)61316-8
Animal Science · Veterinary Science Advanced Online Publication | Current Issue | Archive | Adv Search |
A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses
WEI Yan-di*, GAO Wei-hua*, SUN Hong-lei, YU Chen-fang, PEI Xing-yao, Sun Yi-peng, LIU Jin-hua, PU Juan
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture/College of Veterinary Medicine/State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, P.R.China
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Abstract  H9 subtype avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction (RT-PCR) assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR (dRT-PCR) was established. Two primer sets target the hemagglutinin (HA) gene of H9 AIV and the nucleocapsid (N) gene of IBV, respectively. Specific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other in·fectious avian viruses. The sensitivity of the dRT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×101, 1.5×101 and 1.5×101 50% egg infective doses (EID50) mL–1, respectively. The concordance rates between the dRT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the dRT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and surveillance of H9 AIVs and IBVs.
Keywords:  avian influenza viruses        H9 subtype       infectious bronchitis viruses        duplex RT-PCR  
Received: 04 November 2015   Accepted:
Fund: 

This work was supported by the National High-Tech R&D Program of China (2012AA101303).

Corresponding Authors:  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; GAO Wei-hua, E-mail: gwh212@163.com;

Cite this article: 

WEI Yan-di, GAO Wei-hua, SUN Hong-lei, YU Chen-fang, PEI Xing-yao, Sun Yi-peng, LIU Jin-hua, PU Juan. 2016. A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses. Journal of Integrative Agriculture, 15(9): 2105-2113.

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