二聚体蝎型探针荧光定量PCR快速检测志贺菌方法的建立及初步应用  

Development and tentative application of fluorescent quantitative polymerase chain reaction by using duplex scorpion probe for detection of Shigella

作　　者：陈峰[1] 吴尔翔[1] 丁锁顺[1] 邵亚平[1] 章杰[1] 唐晓宇[1] 

机构地区：[1]南通出入境检验检疫局,南通226005

出　　处：《中国人兽共患病学报》2013年第9期886-890,共5页Chinese Journal of Zoonoses

基　　金：江苏出入境检验检疫局科研计划项目(No.2012KJ45)~~

摘　　要：目的利用二聚体蝎型荧光探针法,建立一种可广泛应用且敏感、特异的检测志贺菌的荧光定量PCR技术。方法根据编码志贺菌ipaH基因的核苷酸序列设计引物和荧光探针,采用基因重组技术构建用于志贺菌检测的定量标准品,通过对荧光定量PCR反应体系和反应条件的摸索,建立了检测志贺菌的二聚体蝎型探针定量PCR方法。结果成功构建了志贺菌重组质粒标准品和志贺菌实时荧光定量PCR方法;通过特异性、敏感性、稳定性和重复性验证,结果表明具有较好的特异性、敏感性、稳定性和重复性;对比分离培养标本,二者符合率为100%;对比TaqMan方法,本方法具有更敏感、省时、成本低的特点。结论建立了一种以二聚体蝎型荧光探针技术为基础的志贺菌荧光定量PCR检测法,为研制新型的检测试剂盒奠定了基础,可应用于食品卫生监管以及传染病诊断等。The present study aims to construct a variable and promising technology of fluorescent probe-based polymer- ase chain reaction （PCR） assay （the duplex scorpion probe） for detection of Shigella. The duplex scorpion probe was designed according to the gene sequence of ipaH gene, and then we recombined the vector to be used for the PCR reaction system, which was optimized and evaluated. Results showed that the plasmid containing the sequence of ipaH was constructed successfully; the assay for ipaH was developed with large detecting range, high sensitivity, specificity, stability, and accuracy. By compa- ring cultured specimens, the coincidence rates of results were 100%0. These results showed that a new real time PCR method for detecting Shigella has been successfully developed, which was based on duplex scorpion probe and fluorescent quantitative polymerase chain reaction, and laid a substantial foundation for the development of a new kit＇s competition in the market. Real- time PCR for Shigella based on duplex scorpion probe will be a novel and excellent method for the gene diagnosis of Shigella potentially.

关 键 词：二聚体蝎型探针 荧光定量PCR 志贺菌 分子探针 

分 类 号：R378.2[医药卫生—病原生物学]