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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 the detection of Shigella dysenteriae fluorescence quantitative PCR in environmental water. Methods Based on the ipa H gene of Shigella dysenteriae, the ipa H gene was amplified by ordinary PCR and ligated with T vector to obtain ipa HT recombinant plasmid. The recombinant plasmids were subjected to gradient dilution to carry out quantitative PCR, and the standard curve and the minimum detection limit were observed. Seventeen strains of pathogenic bacteria that could exist in environmental water samples were extracted and the same reaction system was used for quantitative PCR. Specificity of the method; Shigella dysenteriae spiked in surface water samples to test the method of detection of environmental samples. Results The ipa H gene of Shigella dysenteriae was successfully amplified and ligated with T vector. The Ct values of recombinant plasmids were similar to those of the recombinant plasmids. The detection limits were as low as 10 copies. The detection results of 17 strains of pathogenic bacteria Was negative, indicating that the specificity of the square; surface water in the total number of 3.5 × 103 CFU / ml, Shigella dysenteriae spiked concentration as low as 5 CFU / ml, the method can still be detected, the method Anti-interference ability, can be used for direct detection of environmental water samples. Conclusion The rapid detection of Shigella dysenteriae in water can be achieved by real-time fluorescence quantitative PCR. The detection time is within 3 hours and the detection concentration is 5 CFU / ml. The specificity and anti-interference ability are good and can be used for rapid and direct detection of contaminated water samples.