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Journal of Integrative Agriculture  2019, Vol. 18 Issue (7): 1428-1435    DOI: 10.1016/S2095-3119(19)62700-0
Special Focus: Animal influenza virus Advanced Online Publication | Current Issue | Archive | Adv Search |
Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens
SHI Lin1, 2*, YU Xue-wu3*, YAO Wei4, YU Ben-liang4, HE Li-kun4, GAO Yuan4, ZHANG Yun-xian5, TIAN Guo-bin1, PING Ji-hui2, WANG Xiu-rong
1 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin150069, P.R.China          
2 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P.R.China
3 VICA Group, Shenyang Animal Husbandry Technology Co., Ltd., Shenyang 110027, P.R.China
4 Animal Epidemic Diseases Prevention and Control Center of Liaoning Province, Shenyang 110164, P.R.China
5 Qilu Animal Health Products Co., Ltd., Jinan 250110, P.R.China
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Abstract  
In recent years, the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus (AIV) gene recombination and reassortment.  Until now, traditional RT-PCR, fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV, but these methods require high-level instruments and experimental conditions, not suitable for the rapid detection in field and farms.  In order to develop a rapid, sensitive and practical method to detect and identify AIV subtypes, 4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification (RT-LAMP) method was established.  Using this method, the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein, without cross reaction with other susceptible avian viruses.  In addition, the detection limit of the common H1, H5, H7, and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU (plaque-forming unit), which was 10 times more sensitive than that using the routine RT-PCR.  Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18% (14/335) comparing with 3.58% (12/335) from real-time RT-PCR.  All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast, convenient and practical method for the clinic test and epidemiological investigation of AIV.
 
Keywords:  avian influenza virus (AIV)        RT-LAMP        diagnostic method        clinical specimens  
Received: 13 August 2018   Online: 13 December 2018   Accepted:
Fund: This study was supported by the Special Foundation for State Basic Research Program of China (2013FY113300-8) and the National Key R&D Program of China (2016YFD0500800).
Corresponding Authors:  Correspondence WANG Xiu-rong, E-mail: wangxiurong@caas.cn; PING Ji-hui, E-mail: jihui.ping@njau.edu.cn   
About author:  SHI Lin, Mobile: +86-13624076068, E-mail: hsyshilin@126.com; * These authors contributed equally to this study.

Cite this article: 

SHI Lin, YU Xue-wu, YAO Wei, YU Ben-liang, HE Li-kun, GAO Yuan, ZHANG Yun-xian, TIAN Guo-bin, PING Ji-hui, WANG Xiu-rong. 2019. Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens. Journal of Integrative Agriculture, 18(7): 1428-1435.

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