Development of a reverse-transcription loop-mediated isothermal amplification method for detection of rabbit hemorrhagic disease virus

• Published in: Journal of virological methods 2013-02, Vol.187 (2), p.274-277
• Main Authors: Yuan, Dongwei, Guo, Dongchun, Liu, Jiasen, Si, Changde, Jiang, Qian, Lin, Huan, Yang, Tiankuo, Qu, Liandong
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Caliciviridae Infections - diagnosis; Caliciviridae Infections - veterinary; Caliciviridae Infections - virology; China; detection limit; DNA Primers - genetics; electrophoresis; fluorescence; Fundamental and applied biological sciences. Psychology; genes; hemagglutination; Hemorrhagic Disease Virus, Rabbit - genetics; Hemorrhagic Disease Virus, Rabbit - isolation & purification; liver; Loop-mediated isothermal amplification; Microbiology; monitoring; mortality; Nucleic Acid Amplification Techniques - methods; Rabbit hemorrhagic disease virus; Rabbits; Reliability; reverse transcriptase polymerase chain reaction; Reverse transcription; reverse transcription loop-mediated isothermal amplification; RNA; Rodent Diseases - diagnosis; Rodent Diseases - virology; Sensitivity; Sensitivity and Specificity; Techniques used in virology; Temperature; Time Factors; ultraviolet radiation; Veterinary Medicine - methods; Virology; Virology - methods
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.11.020

► A novel method of one-step RT-LAMP detection of RHDV was described. ► Four specific primers were designed according to 27 isolates of RHDV-VP60. ► Specific amplification reaction for RHDV-VP60 different from other caliciviruses. ► A rapid, simple, sensitive and visually diagnostic method on RHDV was constructed. Rabbit hemorrhagic disease virus (RHDV) causes haemagglutination and severe liver damage, with a high mortality rate. To develop a rapid and sensitive method for the surveillance of RHDV, a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of four primers specific for the VP60 gene segment of RHDV. The established assay was performed at 64°C for 40min under isothermal conditions, and the results were visualized directly by electrophoresis or as fluorescent signals under ultraviolet light. The detection limit of the RT-LAMP assay was 10 copies of viral RNA per reaction, which was comparable to quantitative real-time RT-PCR, and 100-fold more sensitive than standard RT-PCR. Furthermore, seven viral RNAs of field isolates in China could be detected successfully using this assay. Overall, the newly established RT-LAMP assay indicates the potential usefulness of the technique as a simple, rapid and sensitive procedure, and can visually detect RHDV infection without the need for any specialized equipment.