基于PEDV M基因RT-LAMP快速检测方法的建立与应用

Establishment and application of RT-LAMP rapid detection method for porcine epidemic diarrhea virus

为建立一种快速、灵敏、方便的猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）早期诊断方法,本研究针对PEDV的M基因片段设计了4条引物,对反应体系的时间、温度进行了优化,对试验的特异性和敏感性进行了评估,初步建立了逆转录环介导恒温扩增技术（RT-LAMP）。结果表明,建立的PEDV RT-LAMP方法在65℃恒温下扩增60min,可通过琼脂糖凝胶电泳或SYBR Green I染料肉眼判断结果,灵敏性较普通RT-PCR高100倍。该方法与猪传染性胃肠炎病毒（TGEV）、猪轮状病毒（PoRV）、猪繁殖与呼吸综合征病毒（PRRSV）和猪瘟病毒（CSFV）等无交叉反应,具有良好的特异性。采用该RT-LAMP对100份临床疑似发病粪便拭子、小肠组织、初乳样本进行检测,结果较RT-PCR更灵敏。因此,本研究初步建立了可用于临床PEDV检测的RT-LAMP检测方法,为PEDV的临床快速诊断和综合防控提供了一种新的手段。

The aim of this study is to establish a rapid,sensitive,convenient porcine epidemic diarrhea virus（PEDV）diagnostic methods.Four primers based PEDV M gene were designed,and initially established a reverse loop-mediated isothermal amplification（RT-LAMP）,and the specificity and sensitivity were assessed.The results showed that PEDV RT-LAMP method established at65℃isothermal amplification 60 min,can be judged by agarose gel electrophoresis or SYBR Green dye results by the naked eye,the sensitivity is 100 times higher than the ordinary RT-PCR.The method was also specific for PEDV and did not cross react to TGEV,PoRV,PRRSV and CSFV.The RT-LAMP was used to detect 36 clinical samples,and the results were in complete agreement with RT-PCR.Therefore,this study preliminarily established the RT-LAMP detection method which can be used for clinical PEDV detection,and provides a new method for rapid diagnosis and comprehensive prevention and control of PEDV.