牛乳房炎金黄色葡萄球菌LAMP检测方法的建立及初步应用

为建立灵敏、快速、可视化的牛乳房炎金黄色葡萄球菌(Staphylococcus aureus,SA)环介导等温扩增技术(LAMP),根据GenBank公布的金黄色葡萄球菌nuc基因核苷酸序列为靶序列,设计并合成一组特异性LAMP引物,分别优化反应条件和反应体系,进行特异性和灵敏度试验,并用优化后的方法对临床分离的金黄色葡萄球菌样品进行检测。结果表明,建立的方法可以在64℃反应60 min,得到清晰的梯状条带;反应产物经10×SYBR GreenⅠ染色后,肉眼直接观察或是紫外线照射之后均可判定结果;敏感性试验表明,与常规PCR相比,该方法检测到的金黄色葡萄球菌最低浓度为1×101 CFU/mL,灵敏度提高了100倍;该方法特异性良好,与大肠埃希菌、沙门菌、伪结核棒状杆菌均无交叉反应;31份临床分离的金黄色葡萄球菌样本检测的结果表明,该方法与传统的PCR检测结果一致。建立的LAMP检测方法特异性、灵敏度良好,可用于临床牛乳房炎金黄色葡萄球菌感染的检测。

Establishment and Preliminary Application of LAMP for Detecting Staphylococcus aureus from Bovine Mastitis

In order to establish a sensitive,rapid and visualized method for the detection of Staphylococcus aureus loop-mediated isothermal amplification(LAMP),the nucleotide sequence of the highly conserved region of the S.aureus nuc gene published in GenBank was as target sequencea group of S.aureus-specific LAMP primers were designed and synthesized,and the reaction conditions(time,temperature)and reaction system(internal and external primer concentration ratio,dNTP),were optimize and then specificity and sensitivity tests in turn were conducted.The optimized method was used to test clinically isolated S.aureus samples.The results showed that the established method could obtain clear ladder strips after reacting at 64℃for 60 min.The reaction products were stained with 10×SYBR GreenⅠ,and the results were judged directly by the naked eye or after ultraviolet irradiation.Sensitivity test showed that the minimum concentration of S.aureus which can be detected by this method is 1×101 CFU/mL,which is 100 times higher than that of the conventional PCR method.The method had good specificity,and no cross-reactions with E.coli,Salmonella and Corynebacterium pseudotuberculosis.The results of 31 clinical S.aureus samples showed that the method was consistent with the traditional PCR results.The established LAMP detection method in this experiment has good specificity and sensitivity,and can be used for the detection of clinical Staphylococcus aureus infection samples from bovine mastitis.