rpoB作为蜡样芽胞杆菌群快速检测的标志基因的实验研究  

Using real-time quantitative PCR for the detection of rpoB gene of Bacillus cereus group

作　　者：朱诗应[1] 何胜菲[1] 王文博[1] 赵平[1] 任浩[1] 戚中田[1] 

机构地区：[1]第二军医大学微生物学教研室,上海市医学生物防护重点实验室,上海200433

出　　处：《热带医学杂志》2012年第10期1171-1174,共4页Journal of Tropical Medicine

基　　金：全军十二五科技重大项目(AWS11C001);第二军医大学军事医学专项(2010JS02);上海市重点学科建设项目(B901)

摘　　要：目的探讨常规PCR和实时荧光定量PCR(Q-PCR)方法检测蜡样芽胞杆菌群rpoB基因的特异性和敏感性。方法提取蜡样芽胞杆菌群和其他各种对照细菌的基因组DNA,合成蜡样芽胞杆菌群rpoB基因扩增引物,采用常规PCR和SYBRgreen实时定量PCR两种方法扩增rpoB基因片段,并将PCR产物克隆到pMD18-T载体后进行DNA测序。结果常规PCR和Q-PCR均能扩增出蜡样芽胞杆菌群rpoB基因的174bpDNA片段,而各种对照菌株均未见扩增。序列比对发现蜡样芽胞杆菌群细菌在该片段中存在5处核苷酸的不同,差异率为2.88%。以炭疽芽胞杆菌基因组DNA系列稀释作为扩增模板显示常规PCR最小检出量为3.42pg,Q-PCR的敏感性达到171fg,3次重复实验显示Q-PCR检测rpoB基因的灵敏度为(3.32×101±7.45×100)拷贝。结论以rpoB基因为检测靶基因的Q-PCR方法具有高度的特异性和良好的敏感性,能实现对蜡样芽胞杆菌群快速而准确的检测。Objective To develop a routine PCR and a real-time quantitative PCR（Q-PCR） assay for the detection of Bacillus cereus. Methods Genomic DNAs of various groups of Bacillus cereus were extracted. Specific primers for rpoB gene of Bacillus cereus were designed. The target rpoB gene fragment was amplified by a routine PCR and a SYBR green Q-PCR. The PCR products were cloned into pMD18-T plasmid vector and then sequenced. Results The rpoB gene fragment with a length of 174 bp was amplified from 4 species of Bacillus cereus. There was no non-specific amplification from the control bacterial strains by routine PCR or Q-PCR. Results from the sequencing of the amplified DNA fragment showed that there was 5 nucleotides diversity, and the sequence diversity was 2.88%. The sensitivity of routine PCR assay was 3.42 pg, while the sensitivity of the Q-PCR was 171 fg corresponding to （3.32×101±7.45×100） rpoB gene copies. Conclusions The developed Q-PCR method showed a good specificity and sensitivity for the detection of target rpoB gene. It may be used for rapid diagnosis of Bacillus cereus spp.

关 键 词：蜡样芽胞杆菌群 RPOB基因 实时定量PCR 

分 类 号：R378[医药卫生—病原生物学]