艰难梭菌数字化PCR-核糖体基因分型方法的建立与应用  

Establishment and Application of Digital PCR-ribosome Genotyping for Clostridium Difficile

作　　者：束振华[1] 陈思达[1] 范回生 古聪慧 黄震[1] Shu Zhenhua;Chen Sida;Fan Huisheng;Gu Conghui;Huang Zhen(Central Hospital of Longgang District,Shenzhen,Shenzhen 518115 China;The Fifth People s Hospital of Longgang District,Shenzhen,Shenzhen 518100 China;The People's Hospital of Luohu District,Shenzhen,Shenzhen 518001 China)

机构地区：[1]深圳市龙岗中心医院,广东深圳518115 [2]深圳市龙岗区第五人民医院,广东深圳518100 [3]深圳市罗湖区人民医院,广东深圳518001

出　　处：《锦州医科大学学报》2019年第3期27-32,I0005,I0006,共8页Journal of Jinzhou Medical University

基　　金：深圳市龙岗区科技发展资金项目,项目编号:20160606173802570;20160606173542416

摘　　要：目的建立艰难梭菌数字化PCR-核糖体基因分型方法并用于临床菌株的基因分型。方法利用荧光毛细管电泳技术代替琼脂糖凝胶电泳检测艰难梭菌PCR-核糖体分型产物,建立数字化PCR-核糖体分型方法;利用40株分属于10种核糖体型(RT)的艰难梭菌参考菌株构建常见基因型的数字化参考图谱,利用71株艰难梭菌临床菌株初步验证该方法的临床实用性。结果荧光毛细管电泳技术准确地检测出40株艰难梭菌参考菌株的PCR-核糖体分型片段,并以数字化的形式呈现出来,10种核糖体型艰难梭菌的PCR-核糖体分型数字化图谱差异明显。同为RT027的21株菌株间、RT002的3株菌株间和RT015的2株菌株间的数字化分型结果相似性都非常高,而7株RT001菌株则可分为4种亚型,2株RT014分为2种亚型。49株深圳地区儿童中分离出的艰难梭菌共分为22种核糖体型,其中RT017共7株,BA03和BA16各5株,BA13共4株。22株悉尼地区莫西沙星耐药的艰难梭菌均为高毒力核糖体型027(RT027)。结论成功建立艰难梭菌数字化PCR-核糖体分型方法,同时构建了艰难梭菌10种核糖体型的数字化PCR-核糖体分型参考数据库;深圳地区婴幼儿中分离出的艰难梭菌基因型以RT017为主,与国内成人中的分离株相似。悉尼地区莫西沙星耐药的艰难梭菌均为高毒力RT027。Objective To establish a digital PCR-ribosome genotyping method of Clostridium difficile and to apply it to genotyping on clinical strains. Methods Fluorescence capillary electrophoresis was used to detect the PCR-ribosome typing products of clostridium difficile instead of agarose gel electrophoresis and the digital PCR -ribosome typing method was established. The digital reference map of common genotypes was constructed by using 40 reference strains of clostridium difficile which belonged to 10 ribosomal body types (RT),and 71 clinical strains of clostridium difficile were used to verify the clinical practicability of this method preliminarily. The PCR- ribosomal typing digital profiles of 10 ribosomal Clostridium harringens were significantly different. Results Fluorescence capillary electrophoresis detected the PCR-ribosome typing fragments of 40 reference strains of clostridium difficile accurately and presented them in the form of digitalization. The PCR -ribosomal typing digital mappings of 10 ribosomal Clostridium difficiles were significantly different. The digital typing results of 21 strains of RT027,3 strains of RT002 and 2 strains of RT015 were very similar,while 7 strains of RT001 could be divided into 4 subtypes and 2 strains of RT014 could be divided into two subtypes. There were 22 ribosomal types,including 7 strains of RT017,5 strains of BA03 and 5 strains of BA16,and 4 strains of BA13 among the 49 strains of clostridium difficiles isolated from children in Shenzhen area. They were type 027 (RT027) with high virulence ribosome among all of the 22 strains of clostridium difficile resistant to moxifloxacin in Sydney. Conclusion The digital PCR-ribosome typing method for clostridium difficile has been established successfully and the digital PCR-ribosome typing reference database of 10 ribosome types of clostridium has been constructed. The genotype of clostridium difficile isolated from infants and young children in Shenzhen area is mainly RT017,which is similar to that isolated from adults in Chi

关 键 词：艰难梭菌 PCR-核糖体分型 荧光毛细管电泳 数字化 参考数据库 

分 类 号：R378.61[医药卫生—病原生物学]