应用Taq Man荧光PCR定性定量检测炭疽芽孢杆菌

目的　发展一种快速、敏感、特异的检测炭疽芽孢杆菌的方法。方法　应用基于TaqMan荧光探针的实时(real time)PCR技术,针对致病炭疽毒株的两个质粒pXO1、pXO2上的pagA ,cap基因和染色体上的ropB基因设计引物和探针定性、定量检测炭疽芽孢杆菌。构造并应用上述探针的外标对照及pagA的内标对照,采用多重荧光定量PCR技术建立荧光定量方法并发现假阴性;采用UDG抗污染和ROX矫正背景噪音,提高检验能力;用该方法检测炭疽芽孢杆菌疫苗株感染的动物脾脏标本,盲法评价该方法在实际工作的应用。结论　该方法能特异、灵敏、高效地检测炭疽芽孢杆菌,该方法的推广和应用对有效防范炭疽生物恐怖袭击、提高突发事件应对能力、快速诊断具有重要意义。

Establishment and application of Real-time fluorescence polymerase chain reaction based on the TaqMan probes for detection of Bacillus anthracis

To establish and develop a assay for quick,special and sensitive detection of Bacillus anthracis. By Using Real-time fluorescence polymerase chain reaction based on the TaqMan technology with anti-contamination UDG systems and ROX reference dye,the special probes and primers were designed from pag gene and cap gene on two plasmids, pXO1,pXO2,as well as a ropB gene on chromosome.The amplification efficiency of different company'sreagents were compared.The sensitivity were evaluated by serial dilution of gene colon, internal template and B.anthracis strains.The specifity was confirmed by amolifying real DNAs from bacteria,and the reprentitive strains were identified and assessed by dule-blind.An internal control was added to the reaction mixture in order to detect the presence of PCR inhibitors that were often found in biological samples.Applying the exterior and interior standards, we developed two ways to exactly quantify the samples,and we stimulated infective spleen samples to evaluate the ability for actual application.The results showed that the quantitative real-time PCR assay was sentitive and specific for rapid identification of B.anthracis. It is well suited to identify B.anthracis in case of emergency, bioterrorist attack and surveillance of epidemics.