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Open Access Highly Accessed Methodology article

Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus

Zhixun Xie12*, Qing Fan12, Jiabo Liu12, Yaoshan Pang12, Xianwen Deng12, Zhiqin Xie12, Xie Liji12 and Mazhar I Khan3*

Author Affiliations

1 Department of Biotechnology, Guangxi Veterinary Research Institute, 51 You Ai Road, Nanning, Guangxi, 530001, China

2 Guangxi Key Laboratory of Animal Vaccines and Diagnostics, 51 You Ai Road, Nanning, Guangxi, 530001, China

3 Department of Pathobiology & Veterinary Science, University of Connecticut, Storrs, CT, 06260-3089, USA

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BMC Veterinary Research 2012, 8:133  doi:10.1186/1746-6148-8-133

Published: 15 August 2012

Abstract

Background

Bovine rotavirus (BRV) infection is common in young calves. This viral infection causes acute diarrhea leading to death. Rapid identification of infected calves is essential to control BRV successfully. Therefore development of simple, highly specific, and sensitive detection method for BRV is needed.

Results

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized for rapid detection of BRV. Specific primer sets were designed to target the sequences of the VP6 gene of the neonatal calf diarrhea virus (NCDV) strain of BRV. The RT-LAMP assay was performed in a water bath for 60 minutes at 63°C, and the amplification products were visualized either directly or under ultraviolet light. This BRV specific RT-LAMP assay could detect 3.32 copies of subtype A BRV. No cross-reactions were detected with other bovine pathogens. The ability of RT-LAMP to detect bovine rotavirus was further evaluated with 88 bovine rectal swab samples. Twenty-nine of these samples were found to be positive for BRV using RT-LAMP. The BRV-specific-RT-LAMP results were also confirmed by real-time RT-PCR assay.

Conclusions

The bovine rotavirus-specific RT-LAMP assay was highly sensitive and holds promise as a prompt and simple diagnostic method for the detection of group A bovine rotavirus infection in young calves.

Keywords:
BRV; VP6 gene; Reverse transcription loop-mediated isothermal amplification (RT-LAMP)