环介导等温扩增技术快速检测猪流行性腹泻病毒

Development of loop-mediated isothermal amplification assays for detection of porcine epidemic diarrhea virus

为建立一种快速、灵敏的检测猪流行性腹泻病毒(PEDV)的环介导等温扩增(LAMP)检测方法,本研究针对PEDV的NP基因设计LAMP引物,对反应体系优化并进行特异性及敏感性等试验。结果表明,该方法在65℃下恒温扩增60 min,经SYBR Green I染色后仅肉眼观察即可判定结果。该方法特异性好,与传染性胃肠炎病毒、猪轮状病毒、猪瘟病毒及大肠杆菌等无交叉反应,对重组质粒最低检测限度为16拷贝/μL,灵敏度高于普通PCR。该LAMP检测方法特异性强,灵敏度高,操作方便,适于临床检测PEDV。

The objective of the present study was to develop a loop-mediated isothermal amplification （LAMP） assay for detection of porcine epidemic diarrhea virus （PEDV）.LAMP primers were designed based on the NP gene of PEDV.This assay was conducted under 65 ℃ in one hour and the results can be read by naked eye.This LAMP was specific that had no cross reaction with porcine transmissible gastroenteritis virus,porcine rotavirus,classical swine fever virus and E.coli.The sensitivity of the assay was 16 copies/μL recombinant plasmids,which was more sensitive than that of conventional PCR.The results showed that this LAMP assay was sensitive,specific,and easy to preform which could be applied to the rapid detection of PEDV infecton.