山羊伪结核棒状杆菌PMA-PCR检测方法的建立

Establishment of PMA-PCR detection method for Corynebacterium pseudotuberculosis in Goat

本试验旨在建立伪结核棒状杆菌的活菌检测方法,为临床检测及疫病防控提供理论依据。利用PMA可选择性穿透死菌细胞膜,在强光作用下与死菌DNA共价交联,阻止以其DNA为模板的PCR扩增这一原理,将PMA与PCR鉴定相结合,并对PMA最佳曝光时间、最适工作浓度进行优化,建立伪结核棒状杆菌PMA-PCR活菌检测方法,并对其特异性进行分析。结果表明最佳曝光时间为15 min,PMA能够完全抑制死菌PCR扩增的最低浓度为20μmol/L,不抑制活菌PCR扩增的最大PMA浓度为30μmol/L。特异性试验结果显示,仅伪结核棒状杆菌可扩增出目的条带,而在链球菌、多杀性巴氏杆菌中未扩增出目的条带。对不同比例的死/活菌检测发现,该方法可以有效针对活菌进行检测,活菌的最低检测浓度为1×104 cfu/mL。本研究为伪结核棒状杆菌的活菌检测提供了一定的理论依据,也为由伪结核棒状杆菌诱发的山羊疫病防控奠定了基础。

The aim of this study was to establish a method for detecting live bacteria of Cory‐nebacterium pseudotuberculosis, and provide a theoretical basis for clinical detection and dis‐ease prevention and control. PMA can selectively penetrate the dead cell membrane, covalently cross-link with the dead DNA under strong light, and prevent the principle of PCR amplification using its DNA as a template, and combine PMA with PCR identification. The optimal exposure time and optimum working concentration of PMA were optimized, and the detection method of PMA-PCR for Corynebacterium pseudotuberculosis was established and its specificity was analyzed. The results showed that the optimal exposure time was 15 min. The minimum concentration of PMA which could completely suppress the PCR amplification of death bacteria was 20 μmol/L. And the maximum PMA concentration that did not inhibit PCR amplification of live bacteria was 30 μmol/L. The specif‐ic experimental results showed that only Pseudotuberculosis coryneform could amplify the target band, and the target band was not appeared in the amplification of Streptococci and Pasteurella multocida. Sensitivity testing found that the lowest detectable concentration of live bacteria was 1×10^4 cfu/mL. This study provided a theoretical basis for the detection of live bacteria in Corynebacterium pseudotuberculosis, and also laid a foundation for the prevention and control of goat disease induced by Corynebacterium pseudotuberculosis.