环境水体中痢疾志贺菌荧光定量PCR检测方法研究

Fluorescent quantitative PCR detection method of Shigella dysenteriae in environmental waters

目的对环境水体中痢疾志贺菌荧光定量PCR检测方法进行研究和评价。方法根据痢疾志贺菌侵袭性质粒抗原H(invasion plasmid antigen H,ipa H)基因设计引物,利用普通PCR方法扩增其ipa H基因并将其与T载体进行连接以获得ipa H-T重组质粒;对重组质粒进行梯度稀释后进行荧光定量PCR,观察其标准曲线及最低检出限;提取17株可在环境水样中存在的多种致病菌DNA并以同样反应体系进行荧光定量PCR,验证该方法的特异性;将痢疾志贺菌加标于地表水水样中,测试该方法的环境样品检测能力。结果成功扩增痢疾志贺菌ipa H基因并连接T载体;通过荧光定量PCR检测各梯度浓度重组质粒的Ct值间隔相近,检出限低至10 copies;17株致病菌DNA的检测结果均为阴性,显示该方特异性好;在环境地表水菌落总数为3.5×103 CFU/ml、痢疾志贺菌加标浓度低至5 CFU/ml时,仍可以通过该方法检出,显示该方法的抗干扰能力强,可用于环境水样的直接检测。结论通过荧光定量PCR方法可实现对水中痢疾志贺菌进行快速检测,检测时限3 h以内,检测浓度5 CFU/ml,特异性及抗干扰性好,可用于污染水样的快速直接检测。

Objective To study and evaluate one detection method of Shigella dysenteriae in environmental waters by fluorescence quantitative PCR. Methods Primers was designed based on the invasion plasmid antigen H（ipa H） gene, ipa H gene was amplified by PCR and jointed with p MD 18-T vector. The recombinant plasmid was treated by gradient dilute and determined by fluorescent quantitative PCR, the standard carve and its minimum limit of detection were observed. DNAs of 17 different pathogenic bacteria existed in environmental water samples were extract. The reliability and specificity of the detection method were evaluate by running fluorescence quantitative PCR with same reaction system. Shigella dysenteriae was marked into surface water samples and fluorescence quantitative PCR was performed with same reaction system to evaluate the capability of environmental samples detection. Results ipa H gene was successfully amplified and jointed with T vector. The differences between Ct values of each graded concentration of the recombinant plasmid were minimal. The specificity of the amplification of this detection method was good and the minimum limit of detection was under 10 copies. The results of all 17 pathogenic bacteria detection were negative, which showed good specificity. It can also be detected as marked surface water with total bacterial count of 3.5 ×10^3CFU/ml, Shigella dysenteriae concentration of 5 CFU/ml, which showed a good anti-jamming capability. Conclusion Fluorescence quantitative PCR is a rapid detection method of Shigella dysenteriae with great specificity and anti-jamming capability. The detection limit is lower than the traditional method, its detection period is within three hours and its minimum concentration limit is 5 CFU/ml. This method can be used for rapid detection of polluted water samples.