血清凝集、基因测序联合检测群霍乱弧菌的应用

目的 避免霍乱弧菌检测假阳性,提高检测正确率。方法 随机选取2013年1～7月,全国各省市CDC送往中国CDC霍乱初筛阳性菌株14株; 用LB营养琼脂培养12 h,挑取单菌落进行霍乱弧菌血清凝集,同时水煮模板法提取菌株的DNA。针对弧菌属16SrDNA序列设计引物,进行弧菌16SrDNA PCR检测,电泳观察16SrDNA产物,将16SrDNA阳性产物送测序公司测序,测序结果在NCBI网站上进行Blast比对,分析比较血清凝集和Blast比对结果。结果 共选取14株菌进行实验,血清凝集阳性12株,2株未凝。经弧菌16SrDNA扩增,电泳观察14株菌均扩增出相应的片段,说明所选菌株均为弧菌属。将14株菌的16SrDNA阳性产物测序,并将测序结果进行Blast比对:2株血清未凝集菌均是哈维氏弧菌; 12株血清凝集阳性的菌中,1株是需钠弧菌,其余11株是霍乱弧菌。结论 血清凝集和基因测序联合检测群霍乱弧菌,可避免霍乱弧菌检测假阳性,提高检测正确率。

Application of DetectingVibrio Cholerae Combined with Serum Agglutination and Gene Sequencing

Objective To avoid false positive detection ofVibrio cholerae and improve the detection correct rate.Methods1～7 months of 2013 were randomly selected,the national various provinces and cities CDC to China cholera CDC positive screening 14 strains.LB nutrient agar 12 hours,take single colony to Vibrio cholera serum agglutination,extraction of strain DNA at the same time boiled template method.For Vibrio 16SrDNA sequence and design primers for PCR detection of Vibrio,16SrDNA,electrophoresis were used to observe the 16SrDNA products,16SrDNA positive products sent to sequencing company sequencing,sequencing results were Blast comparison on theNCBI website for the analysis and comparison of serum agglutination and Blast alignment.Results 12 strains was positive for agglutination and 2 strains of non agglutination in 14 strains.The Vibrio 16SrDNA amplification,electrophoresis were used to observe the 14 isolates that were amplified fragment corresponding,that the selected strains were vibrio.The 16SrDNA positive products were 14 strains,and the sequencing the Blast results:2 strainsof bacteria were Vibrio harveyi for non agglutination,in 12 positive strains ofserum agglutination; 1 strains was Vibrio natriegen and 11 strains were Vibrio cholerae.Conclusion Detection of Vibrio choleraecholerae combined with serum agglutination and gene sequencing can avoid falsepositive result of Vibrio cholerae,and improve correct rate of the detection.