志贺氏菌可视浊度/荧光环介导等温扩增方法的建立

Establishment of the Visual Turbidity/Fluorescent LAMP Method for Detection of Shigella

为给实验室和现场检测提供一种快速且易于操作的志贺氏菌检测方法,通过将LAMP技术分别与荧光检测技术和浊度检测技术相结合,建立了灵敏、特异、准确的可视浊度/荧光环介导等温扩增(LAMP)检测方法。根据志贺氏菌基因保守序列,利用LAMP在线软件设计4条最佳引物,通过优化反应温度,分别建立了可视浊度环介导等温扩增检测方法(LAMP-浊度)以及可视荧光环介导等温扩增检测方法(LAMP-荧光),并分析所建立的两种方法的特异性和灵敏度。结果显示:所建立的LAMP-浊度方法最适反应温度是63℃,菌液灵敏度为13.6 cfu/mL,是普通PCR方法的10倍;所建立的LAMP-荧光方法最适反应温度是64℃,菌液灵敏度为1.36 cfu/mL,是普通PCR方法的100倍。利用4株志贺氏菌和10株非志贺氏菌,证实了建立的两种可视化LAMP方法具有良好的特异性。通过环介导等温扩增技术分别与浊度检测技术和荧光检测技术相结合,使其完成时间缩短至1 h内,整个扩增过程可实时监测,并可避免由于开盖加染料而导致的假阳性。

In order to provide laboratories and field tests with a rapid and easy-to-operate method to detect Shigella, the sensitive,specific and accurate visual turbidity/fluorescent loop-mediated isothermal amplification(LAMP) method was established by combing LAMP with fluorogenic assay and turbidity measurement respectively. Based on the conservative sequence of Shigella gene,2 pairs of primers were designed by use of LAMP online software. After optimizing the reaction temperature,and then the visual turbidity LAMP(LAMP-turbidity)and visual fluorescent LAMP(LAMP-fluorescent)methods were respectively established,and their sensitivity and specificity of the two methods were also analyzed. The results showed that optimum reaction temperature of LAMP-turbidity was 63℃ with the sensitivity of bacterial solution of 13.6 cfu/mL which was 10 times higher than that of traditional PCR;while for LAMP-fluorescence,the optimum reaction temperature was 64 ℃ with the sensitivity of bacterial solution of 1.36 cfu/mL which was 100 times higher than that of traditional PCR. Therefore,the two visual LAMPs were with good specificity as verified by 4 strains of Shigella and 10 strains of Non-Shigella. By the two methods,the completion time of the amplification process was shorten to less than one hour under real-time monitoring,so as to avoid any false positive that may caused by opening the cover or adding any dye.