青藏高原放牧家畜小肠结肠炎耶尔森氏菌LAMP检测方法的建立及应用

Establishment and Application of LAMP Detection Method for Yersinia enterocolitica in Grazing Livestock in Qinghai-Tibet Plateau

为了建立特异性、敏感性和适用性强的LAMP检测方法,采用分子生物学方法,根据GenBank数据库中该细菌的outL基因,进行了多序列比对和保守区域确定。根据其保守序列,在线设计和筛选了一套LAMP引物。温度优化及特异性和敏感性验证后建立了检测该病菌的LAMP检测方法,并对青藏高原放牧牛、羊临床粪便样品进行了检测。结果表明,LAMP检测方法的最适温度为63℃;检测其与大肠杆菌、沙门氏菌、阪崎肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌及蜡样芽胞杆菌无交叉反应;检测下限为100 fg目的基因;利用建立的方法检测了208份放牧牦牛、藏羊粪便DNA样品,共检测到26份阳性样品,阳性率为12.5%。这表明成功建立了一种检测该病的特异性、敏感性和适用性强的LAMP检测方法。

In order to establish a LAMP detection method with high sensitivity,specificity and applicability for Yersinia enterocolitica,outL gene sequences of Y.enterocolitica were downloaded from GenBank database to make multiple sequences blast by using molecular biology method.The conserved regions of the gene were determined.Based on the sequences of conserved gene,a set of LAMP primers were designed and screened out online.The LAMP method was established after temperature optimization,sensitivity and specificity verification.The clinical stool samples of grazing cattle and sheep were detected by the LAMP detection method.The results showed that the optimal temperature for LAMP detection method was 63℃.There was no cross-reactivity with Escherichia coli,Salmonella,Enterobacter sakazakii,Staphylococcus aureus,Pasteurella multocida and Bacillus cereus.The detection limit was 100 fg of the target gene.26 positive samples were detected among 208 fecal DNA samples of yak and Tibet sheep,the positive rate was 12.5%.These results demonstrated that a LAMP method for the detection of Y.enterocolitica with high sensitivity,specificity and applicability was established.