蝎形引物PCR快速检测环境水样中沙门菌方法研究

目的]以蝎形引物PCR技术为基础建立一套环境水样沙门菌的快速检测方法。方法]选择沙门菌himA基因设计出以荧光素(FAM)和淬灭物(DABCYL)双标记探针的茎环尾状结构构成蝎形引物，对8种沙门菌和8种非沙门菌经增菌、DNA提取后，以蝎形引物PCR方法进行检测，探讨该检测方法的可行性、特异性和灵敏度，并与普通引物PCR法检测50份环境水样进行比较。结果]蝎形引物PCR方法检测沙门菌，细菌密度与荧光相对强度存在着量比关系，与普通引物PCR法比较，具有快速、特异、灵敏度高的特点，在50μl PCR及反应体系中最低可检出2cfu沙门菌。结论]沙门菌蝎形引物在PCR扩增中，探针可特异地与目的DNA片段杂交，使DABCYL不能有效地淬灭FAM产生的荧光，荧光可经仪器直读检出。所建立的快速检测方法可定性或定量的用于环境水样中沙门菌检测。

Studies on Rapid Detection of Salmonellae in Environmental Water Samples Using A Scorpion Primer PCR Assay

To establish a new method based on scorpion primer and PCR for rapid detection of the salmonellae in environmental water samples. FAM and DABCYL labeled scorpion primers were designed by a 122 bp section of himA gene of the salmonellae. Eight types of salmonella bacteria and eight types of non salmonella bacteria were delivered in the experiment to prove the feasibility,specificity,sensitivity for detecting salmonella by scorpion primer PCR assay and to explore the relationship between fluorescence intensity and salmonella density. The amplified product of salmonella by scorpion PCR could be detected by spectrofluorophotometer according to fluorescence. The results showed that the relative fluorescent intensity of the scorpion PCR amplified product correlates with the amount of salmonellae DNA. As less as 2 cfu per PCR reaction could be detected by the assay. It was also proved that scorpion primer PCR assay is more rapid,and more sensible for detecting salmonella than the general PCR assay. Conclusion] Scorpion PCR technology relies on energy transfer between FAM and DABCYL. The probe labeled with FAM and DABCYL on the tail of scorpion primer of salmonella could hybridize to an amplicon of target DNA,which resulted in that the fluorescence from FAM cannot be quenched effectively by ADBCYL and detected directly by the spectrofluorophotometer. This assay can be used to rapidly detect the salmonella in environmental water samples quantitatively.