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Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay

Youling Wang12, Xiaoyuan Yuan2, Yufeng Li2, Kexiang Yu2, Jinxing Yang2, Huaiying Xu2, Yuxia Zhang2, Kangzhen Yu3, Ming Liao1* and Zhuoming Qin2*

Author Affiliations

1 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, PR China

2 Institute of Poultry Science, Shandong Academy of Agricultural Science, Jinan 250023, PR China

3 Ministry of Agriculture of the People's Republic of China, Beijing 100026, PR China

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Virology Journal 2011, 8:553  doi:10.1186/1743-422X-8-553

Published: 21 December 2011

Abstract

Background

From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus.

Results

The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation.

Conclusion

The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

Keywords:
Tembusu-related Flavivirus; newly-isolated virus; RT-PCR; RT-LAMP