口蹄疫病毒和水泡性口炎病毒二重RT-LAMP鉴别检测方法的建立

Development of duplex reverse transcription loop-mediated isothermal amplification assay for detection of foot-and-mouth disease virus and vesicular stomatitis virus

LAMP技术是一种快速新型的核酸检测技术,该技术利用4条引物在恒温下扩增目的 DNA,特异性好敏感性高。本试验旨在建立一种能同时鉴别诊断FMDV和VSV的二重RT-LAMP检测方法。根据口蹄疫病毒(FMDV)3D基因和水泡性口炎病毒(VSV)N基因的保守序列,设计了2套特异性引物,在每套引物的内引物中插入酶切位置EcoRⅠ,对反应条件进行了优化,建立了恒温快速的检测方法。结果显示:该方法特异性好,能检测到口蹄疫病毒的A,O,Asia1亚型和水泡性口炎的NJ和IND亚型,并与其他对照牛病原体不发生交叉反应;敏感性高,最低能够检测个100个FMDV病毒RNA和100个VSV病毒RNA;干扰性小,能同时检测两个模板的不同浓度组合。本试验建立的口蹄疫和水泡性口炎二重RT-LAMP方法具有简便、快速、特异、敏感等优点,可用于FMDV和VSV的临床检测和流行病学调查。

A loop-mediated isothermal amplification （LAMP） technique has been used as a novel nucleic acid detection method,whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. The purpose of this study was to developed the reverse transcription loop-mediated isothermal amplification （RT- LAMP） assay to detect foot-and-mouth disease virus and vesicular stomatitis vesicular simultane- ously. We designed two sets of the LAMP primers for conserved region of food-and-mouth disease 3D gene and stomatitis virus N gene respectively,in which a restriction enzyme cleavage site EcoR I was inserted into two pairs of inner primers to construct a multiplex LAMP （mLAMP） method for simultaneous detection of the FMDV and VSV. The results showed that the specificity of this assay was high, and able to detected FMDV and VSV without other any cross-reactions with other bovine pathogens. The detection limit of the duplex RT-LAMP assay was 100 copies of FMDV RNA and 100 copies of VSV viral RNA,indicating a good sensitivity of the assay. Different con- centration of FMDV and VSV could be identified when mixed together without any interference. These findings indicate that this duplex RT-LAMP assay is a simple, rapid, specific, and sensitive method for detection of FMDV and VSV, and is could applied in clinical detection and epidemiolog- ical investigation of FMDV and VSV.