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目的分析3起霍乱疫情病原O139霍乱弧菌分子分型特征和遗传相关性,探讨O139霍乱疫情流行特征。方法对2012年湖北省3起霍乱疫情分离鉴定的35株O139霍乱弧菌菌株用水煮法提取DNA,利用聚合酶链反应检测霍乱肠毒素CT基因;取新鲜培养的菌株,制备约4.3个麦氏单位的细菌悬浮液,经裂解、洗涤及限制性内切酶NotI和SfiI酶切后进行脉冲场凝胶电泳(PFGE)分子分型,用凝胶成像仪获取电泳图像,分析DNA片段并用BioNumerics V4.6软件UPGMA方法(复选Dice相关系数)进行聚类分析。结果 35株O139霍乱弧菌ctxA基因PCR扩增产物约为308bp,分离自聚餐食用的凉菜、病人、带菌者及厕所标本(对应病人家)O139霍乱弧菌均为产毒株,经NotI酶切分为9种PFGE带型,SfiI酶切分为6种PFGE带型;A市2株病人分离菌和4株带菌者分离菌PFGE带型为KZGN11O139.CN0077+KZGS12O139.CN0054,B市1株病人分离菌和1株厕所分离菌PFGE带型为KZGN11O139.CN0302+KZGS12O139.CN0058,C市和D市分离菌株优势型为KZGN11O139.CN0276+KZGS12O139.CN0007,包括3株食品分离菌、2株厕所分离菌及16株病人和带菌者分离菌,另有6株PFGE带型呈现多样性。结论 2012年湖北省霍乱疫情特点为散发以及聚餐导致的局限暴发,3起疫情的分离菌株带型各不相同,呈现多样性,其中2起为单一菌型感染,1起为混合菌型感染,传染来源复杂且不明确,提示应加强霍乱的病原学监测。
Objective To analyze the molecular typing characteristics and genetic correlation of three Vibrio cholerae O139 isolates from the cholera epidemic pathogen, and discuss the epidemiological characteristics of the cholera O139 epidemic. Methods 35 strains of Vibrio cholerae O139 isolates and identified from 3 cholera epidemic cases in Hubei province were extracted by boiling water method, and the CT gene of Cholera toxin was detected by polymerase chain reaction. Freshly cultured strains were used to prepare about 4.3 wheat The bacterial suspension was digested, washed and digested with restriction endonucleases NotI and SfiI. The PFGE molecular typing was performed. The gel electrophoresis image was used to obtain the electrophoresis images. The DNA fragments were analyzed and analyzed by BioNumerics V4 .6 software UPGMA method (check the Dice correlation coefficient) for cluster analysis. Results The PCR products of 35 strains of Vibrio cholerae O139 cholera toxin were about 308bp. Vibrio cholerae O139 isolates from cold dishes, patients, carriers and toilet specimens (corresponding to patients’ patients) Divided into 9 types of PFGE band, SfiI digested into 6 types of PFGE band; A city of 2 isolates and 4 strains of carriers of isolates PFGE band type KZGN11O139.CN0077 + KZGS12O139.CN0054, B City, a patient Isolation of bacteria and a toilet isolates PFGE band type KZGN11O139.CN0302 + KZGS12O139.CN0058, C and D city isolates strains predominance KZGN11O139.CN0276 + KZGS12O139.CN0007, including three strains of food isolates, two strains of toilet separation And 16 isolates from patients and carriers, and another 6 PFGE bands showed diversity. Conclusion The epidemic situation of cholera in Hubei province in 2012 was characterized by sporadic outbreaks and outbreaks caused by dinners. The strains isolated from the three outbreaks were different and showed diversity. Among them, two were single bacterial infection, one was mixed bacterial infection, The sources of infection are complex and unclear, suggesting that the etiology of cholera should be monitored.