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

Zhixun Xie,Zhiqin Xie,Qing Fan,Yaoshan Pang,Mazhar I Khan,Xianwen Deng,Jiabo Liu,Xie Liji

doi:10.1186/1746-6148-8-133

Abstract

BackgroundBovine 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.ResultsA 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.ConclusionsThe 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.

Highlights

Bovine rotavirus (BRV) infection is common in young calves
Restriction enzyme analysis performed with EcoRI and EcoRV on the reverse transcription loop-mediated isothermal amplification (LAMP) (RT-LAMP) product validated no nonspecific reaction in RT-LAMP assay
Specificity and Sensitivity of RT-LAMP assay The bovine rotavirus-specific RT-LAMP assay amplified strains neonatal calf diarrhea virus (NCDV)-014, and 8 Guangxi field bovine rotavirus strains, which have been isolated from the Guangxi dairy farms, and exhibits no cross-reactivity with other pathogens (Table 1)

Summary

Introduction

Bovine rotavirus (BRV) infection is common in young calves. This viral infection causes acute diarrhea leading to death. The recently described loop-mediated isothermal amplification (LAMP) can amplify specific target DNA sequences with high sensitivity and can be completed within 60 minutes under isothermal conditions without the need of a thermal cycler and specialized laboratory [11]. This technique eliminates the heat denaturation step for DNA synthesis used in conventional PCR, and relies instead on auto-cycling strand displacement DNA synthesis achieved by a DNA polymerase with high strand displacement activity and a set of two specially designed inner and two outer primers. The objective of the study reported here was to develop and optimize the reverse transcription LAMP (RT-LAMP) assay for the detection of group A BRV in calves

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Discussion

Conclusion