Please wait a minute...
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
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
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:; PING Ji-hui, E-mail:   
About author:  SHI Lin, Mobile: +86-13624076068, E-mail:; * 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.

Chen H T, Zhang J, Sun D H, Ma L N, Liu X T, Cai X P, Liu Y S. 2008. Development of reverse transcription loop-mediated isothermal amplification for rapid detection of H9 avian influenza virus. Journal of Virological Methods, 151, 200–203.
Chen X, Smith G J, Zhou B, Qiu C, Wu W L, Li Y, Lu P, Duan L, Liu S, Yuan J, Yang G, Wang H, Cheng J, Jiang H, Peiris J S, Chen H, Yuen K Y, Zhong N, Guan Y. 2007. Avian influenza A (H5N1) infection in a patient in China, 2006. Influenza and Other Respiratory Viruses, 1, 207–213.
Dukes J P, King D P, Alexandersen S. 2006. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus. Archives of Virology, 151, 1093–1106.
Eiris J S, De Jong M D, Guan Y. 2007. Avian influenza virus (H5N1): A threat to human health. Clinical Microbiology Reviews, 20, 243–267.
Fouchier R A, Munster V, Wallensten A, Bestebroer T M, Herfst S, Smith D, Rimmelzwaan G F, Olsen B, Osterhaus A D. 2005. Characterization of a novel influenza A virus he­magglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology, 79, 2814–2822.
Gao M, Cui J, Ren Y, Suo S, Li G, Sun X, Su D, Opriessnig T, Ren X. 2012. Development and evaluation of a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus. Journal of Virological Methods, 185, 18–23.
Gou H, Deng J, Pei J, Wang J, Liu W, Zhao M, Chen J J. 2014. Rapid and sensitive detection of type II porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip. Journal of Virological Methods, 209, 86–94.
Mu J H E, McCarl B A, Hagerman A, Bessler D. 2015. Impacts of bovine spongiform encephalopathy and avian influenza on U.S. meat demand. Journal of Integrative Agriculture, 14, 1130–1141.
Notomi T, Okayama, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, E63.
Postel A, Letzel T, Frischmann S, Grund C, Beer M, Harder T. 2010. Evaluation of two commercial loop-mediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes. Journal of Veterinary Diagnostic Investigation, 22, 61–66.
Shi L, Sun J S, Yang Z P, Bao H M, Jiang Y P, Xiong Y Z, Cao D, Yu X W, Chen H L, Zheng S M, Wang X R. 2014. Development of a DNA microarray-based multiplex assay of avian influenza virus subtypes H5, H7, H9, N1, and N2. Acta Virologica, 58, 14–19.
Shivakoti S, Ito H, Murase T, Ono E, Takakuwa H, Yamashiro T, Otsuki K, Ito T. 2010. Development of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of avian influenza viruses in field specimens. Journal of Veterinary Medical Science. 72, 519–523.
Tong S, Li Y, Rivailler P, Conrardy C, Castillo D A, Chen L M, Recuenco S, Ellison J A, Davis C T, York I A, Turmelle A S, Moran D, Rogers S, Shi M, Tao Y, Weil M R, Tang K, Rowe L A, Sammons S, Xu X, et al. 2012. A distinct lineage of infl uenza A virus from bats. Proceedings of the National Academy of Sciences of the United States of America, 109, 4269–4274.
Tsai S M, Liu H J, Shien J H, Lee L H, Chang P C, Wang C Y. 2012. Rapid and sensitive detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. Journal of Virological Methods, 181, 117–124.
Wang X, Shi L, Tao Q, Bao H, Wu J, Cai D, Wang F, Zhao Y, Tian G, Li Y, Qao Q M, Chen H. 2010. A protein chip designed to differentiate visually antibodies in chickens which were infected by four different viruses. Journal of Virological Methods, 167, 119–124.
Wei Y D, Gao W H, Sun H L, Yu C F, Pei X Y, Sun Y P, Liu J H, Pu J. 2016. A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses. Journal of Integrative Agriculture, 15, 2105–2113.
Wu Y, Tefsen B, Shi Y, Gao G F. 2014. Bat-derived influenza-like viruses H17N10 and H18N11. Trends in Microbiology, 22, 183–191.
Xu X, Bao H, Ma Y, Sun J, Zhao Y, Wang Y, Shi J, Zeng X, Li Y, Wang X, Chen H. 2015. Simultaneous detection of novel H7N9 and other influenza A viruses in poultry by multiplex real-time RT-PCR. Virology Journal, 12, 69.
Yan L, Zhou J, Zheng Y, Gamson A S, Roembke B T, Nakayama S, Sintim H O. 2014. Isothermal amplified detection of DNA and RNA. Molecular Omics, 10, 970–1003.
Yoshikawa T, Ihira M, Akimoto S, Usui C, Miyake F, Suga S, Enomoto Y, Suzuki R, Nishiyama Y, Asano Y. 2010. Detection of human herpesvirus DNA by loopmediated isothermal amplification. Journal of Clinical Microbiology, 42, 1348–1352.
Yuan W, Wang J, Sun M, Zheng Y, Li L, Zhang X, Sun J. 2014. Rapid detection of encephalomyocarditis virus by one-step reverse transcription loop-mediated isothermal amplification method. Virus Research, 189, 75–78.
Zhang X, Lowe S B, Gooding J J. 2014. Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP). Biosensors and  Bioelectronics, 61, 491–499.
Zowalaty M E, Bustin S A, Husseiny M I, Ashour H M. 2013. Avian influenza: Virology,diagnosis and surveillance. Future Microbiology, 8, 1209–1227.
No related articles found!
No Suggested Reading articles found!