TaqMan探针实时荧光定量PCR快速检测土拉弗朗西斯菌

Rapid and quantitative identification of Francisella tularensis with TaqMan probe by real-time QF-PCR

目的建立TaqMan探针实时荧光定量PCR方法(QF-PCR),快速检测土拉弗朗西斯菌。方法针对土拉弗朗西斯菌的外膜蛋白fopA基因,利用Primer 5.0设计引物及TaqMan探针,人工合成fopA基因保守片段,克隆到载体作为阳性标准品,进行荧光定量检测,制作定量标准曲线,并以合成fopA基因为模板,PF、PR为引物,研究其灵敏度、特异性和准确性。结果构建了含fopA基因的重组质粒,以不同浓度的重组质粒制作标准曲线,在103～107拷贝数之间有较好的线性关系。灵敏度试验表明,该方法可检测到30.6个拷贝数的重组质粒,比普通PCR灵敏度高;特异性试验表明,能选择性检测土拉弗朗西斯菌,而与其他病原菌无交叉反应,与普通菌落PCR结果一致;重复性试验表明,拷贝数为3.06×106样品5次平行试验,标准差为0.201,变异系数为0.88%。结论本研究建立了TaqMan探针QF-PCR快速检测技术,具有灵敏度高、特异性强、重复性好等特点,可用于快速、实时、定量检测土拉弗朗西斯菌,为快速检测生物战剂级微生物建立了一种人工合成特异基因TaqMan探针实时荧光定量PCR新方法。

In order to develop a real-time quantitative fluorescent polymerase chain reaction（QF-PCR） method for rapid and quantitative identification of Francisella tularensis（F.tularensis） by using TaqMan probe,the F.tularensis outer membrane protein FopA gene fopA was synthesized and cloned into vector as the positive standards.The primers and TaqMan probe were designed by Primer 5.0.Results showed that TaqMan QF-PCR method targeting gene fopA was established,which produced a good standard curve.Linears ranged from 103 to 107 copies per microliter.The minimum detection limit was 30.6 copies of recombinant plasmids,which was more sensitive than conventional PCR.F.tularensis could be specifically detected from other similar microbes.Repeatability tests showed that the coefficient of variation of 5 parallel tests in same concentration（3.06×106 DNA copies） was 0.88% and the standard deviation was 0.201.In conclusion,the QF-PCR with TaqMan probe is sensitive,specific and repeatable,and could be used in safe,rapid,real-time and quantitative identification of F.tularensis.With the synthesis of template DNA,we could rapidly develop detection methods for biological agents and dangerous bacteria.