猪多杀性巴氏杆菌可视化LAMP检测方法的建立

该研究旨在建立一种快速检测猪多杀性巴氏杆菌的LAMP方法。根据GenBank中猪多杀性巴氏杆菌外膜蛋白H基因(ompH)序列,在其保守区域设计了多套LAMP引物,通过LAMP反应,评价其特异性和敏感性,并加入SYBR Green Ⅰ,对其进行肉眼判定。结果显示：建立的LAMP方法在56℃水浴中1 h可对猪多杀性巴氏杆菌核酸进行高效扩增,在反应结束后加入SYBR Green Ⅰ可肉眼进行判断；该方法具有很强的特异性,对其它相关猪病病原菌检测结果均为阴性；其DNA核酸的检测量为0.05pg,是PCR的100倍,显示出很高的敏感性；用建立的LAMP方法对5株猪多杀性巴氏杆菌阳性样品进行检测,结果表明LAMP法与PCR检测的结果是一致的。结果表明该研究建立了一种操作简便、快速、敏感、特异的可对猪多杀性巴氏杆菌进行快速检测并可肉眼判定结果的可视化LAMP方法,适合在基层使用。

Development of a loop-mediated isothermal amplification assay for visual detection of Pasteurella multocida

A rapid detection method of Pasteurella multocida (Pm) was established by loop-mediated isothermal amplification assay(LAMP) in this study. According to the published outer membrane proteins H(ompH) gene sequences of Pm in GenBank, multiple pairs of primers were designed targeting the conserved region of ompH gene. The specificity and sensitivity of the LAMP were evaluated. Meanwhile, the amplified products were colored by SYBR Green Ⅰ after completion of the reaction, so that the amplification can be observed with naked eyes. LAMP showed a highly efficient amplification for Pm genomic DNA which performed at 56℃ for 1 h. The results detected with naked eyes by the addition of SYBR Green Ⅰ at the end of reaction was observed. The LAMP showed a strong specificity that the results were all negative towards other swine pathogens detection; and it also showed a high sensitivity with a detectin limit of 0.05pg DNA, which was 100-fold higher than that of PCR. The results of 5 clinical positive samples detected by LAMP developped in this study were consistent with the ones tested before by PCR test. The results revealed a visualized LAMP method which can detect Pm was established and the results can be read with naked eyes. And the method was easy to operation, rapid, specific and sensitive, which was suitable for rapid detection of Pm.