实时荧光定量PCR检测巴尔通体方法的研究

发布时间：2018-10-21 15:24

【摘要】：目的：越来越多的巴尔通体物种感染人类疾病,严重的影响人类健康,给患者、家庭和国家造成重大的医疗卫生经济负担。本课题应用HRM技术建立快速检测巴尔通体属通用的方法。在确保检测出所有巴尔通体物种的情况下,应用双重HRM方法和双重实时荧光定量PCR探针法建立快速鉴定区分引起心内膜炎常见的两种病原体(Bq和Bh),为临床疾病的早期诊断、监测和流行病学调查等研究提供有效的手段。方法：查阅文献查找巴尔通体属的特异引物,应用生物信息学方法查找Bh和Bq特异基因,利用Primer Express 3和Beacon Designer7设计引物和探针。随后进行常规PCR扩增,将产物连接到pEASY(?)-T5 Zero克隆载体上制备标准品。优化扩增反应的退火温度、引物探针的浓度和熔解速率；评估各方法的特异性、敏感性及重复性,分析扩增效率和线性关系。结果：优化的退火温度均为60℃,引物浓度均为300 nmoL/L,探针浓度均为200nmoL/L和熔解速率为0.2℃/s。(1)HRM方法检测巴尔通体属方法结果显示特异性很高,只有巴尔通体物种扩增出荧光信号,熔解温度为81.05℃+0.31,其他阴性对照菌株均未见荧光信号；敏感性实验结果显示在20μL的反应体系中,Bh最低检出限为3.82×101个拷贝,相关系数R2为0.999,扩增效率E值为98.4%。重复性实验结果显示组内和组间的CV值分别为0.30%-0.62%和0.29%-0.36%。(2)双重HRM方法检测Bh和Bq结果显示特异性很高；敏感性实验结果显示在20μL的反应体系中,Bh和Bq最低检出限分别为3.64×101个拷贝和5.62×101个拷贝,此外也显示出良好的线性关系和扩增效率,其相关系数R2分别为0.998和0.997,E值分别为102.8%和104.7%。重复性实验结果显示组内和组间的CV值为0.22%-0.50%和0.50%-1.20%。(3)双重实时探针法检测Bh和Bq结果显示特异性良好；敏感性实验结果显示在20μL的反应体系中,Bh和Bq最低检出限分别为3.64x101个拷贝和3.28×101个拷贝,相关系数R2分别为0.994和0.998,扩增效率E值分别为100.0%和103.4%；重复性实验结果显示组内和组间的CV值分别为0.19%-0.53%和0.49%-1.66%。建立的方法敏感性均比常规PCR提高了100倍。结论：本研究建立了HRM方法特异性强、灵敏度高、稳定性好,可快速地检测出所有的巴尔通体物种,为巴尔通体所引起的CSD、战壕热、心内膜炎、BA和卡瑞恩病等一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。在检测出所有巴尔通体物种的基础上,同时也建立了双重HM技术和双重实时探针法可快速地检测区分引起心内膜炎常见的病原体Bq和Bh。
[Abstract]:Objective: more and more Bartonella species are infected with human diseases, which seriously affect human health. In this paper, HRM technique is used to establish a universal method for rapid detection of Bartonella. To ensure the detection of all Bartonella species, double HRM and double real-time fluorescence quantitative PCR probes were used to establish a rapid identification of two common pathogens causing endocarditis (Bq and Bh), as early diagnosis of clinical diseases). Studies such as surveillance and epidemiological investigation provide an effective means. Methods: the specific primers of Bartonella were searched and the specific genes of Bh and Bq were searched by bioinformatics. Primers and probes were designed by Primer Express 3 and Beacon Designer7. The product was then amplified by conventional PCR and ligated to pEASY (?)-T _ 5 Zero clone vector to prepare the standard product. The annealing temperature of amplification reaction, the concentration of primer probe and melting rate were optimized, the specificity, sensitivity and repeatability of each method were evaluated, and the relationship between amplification efficiency and linearity was analyzed. Results: the optimized annealing temperature was 60 鈩，

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