脑膜炎奈瑟菌实时荧光定量PCR的检测方法建立及评估

Establishment and evaluation of real-time fluorescence quantitative PCR for detection of Neisseria meningitidis

目的建立脑膜炎奈瑟菌实时荧光定量PCR的检测方法,为快速、准确检测脑膜炎奈瑟菌提供有效的手段。方法选用脑膜炎奈瑟菌夹膜转运基因ctrA作为靶基因,设计合成引物和探针,建立脑膜炎奈瑟菌实时荧光定量PCR的检测方法。并对该方法的特异性、敏感性、可重复性等进行方法学评价。结果采用该方法对流感嗜血杆菌、金黄色葡萄球菌、肺炎链球菌、淋病奈瑟菌、卡他莫兰汉菌、大肠埃希菌等进行检测,结果均为阴性。建立的该方法线性范围为1×10^(8)copies/μl~1×10^(1)copies/μl;检测灵敏度达到1.0×10^(0)copies/μl;根据Ct值和标准品拷贝数的对数值建立的标准曲线回归方程为y=-3.2596x+36.815,r=0.9991。以1×10^(2)copies/μl~1×10^(1)copies/μl的脑膜炎奈瑟菌菌株核酸为检测样品,分别进行8次重复性检验,标准差和变异系数分别为0.267、0.91%和0.356、1.09%。结论成功建立了一种基于实时荧光定量PCR的脑膜炎奈瑟菌的快速高灵敏的检测方法。

Objective To establish a real-time quantitative PCR method for the detection ofNeisseria meningitidis,and toprovide an effective means for the rapid and accurate detection ofNeisseria meningitidis.Methods The membrane transportgenectrA of Neisseria meningitidiswas selected as the target gene,and primers and probes were designed and synthesized to es-tablish a real-time quantitative PCR method for the detection ofNeisseria meningitidis.The specificity,sensitivity and repeat-ability of the method were evaluated.Results This method was used to detectHaemophilus influenzae,Staphylococcus aureus,Streptococcus pneumoniae,Neisseria gonorrhoeae,CataranghaniaandEscherichia coli,and the results were all negative.Thelinear range of the established method was 1×10^(8) copies/μl-1×10^(1) copies/μl.The detection sensitivity was 1.0×10^(0) copies/μl.The regression equation of the standard curve wasy=-3.2596 x+36.815,r=0.9991.The nucleic acid samples ofNeisseria meningitidis(1×10^(2) copies/μl-1×10^(1) copies/μl)were used for 8 times repeatability tests.The standard deviation and coef-ficient of variation were 0.267,0.91%,and 0.356,1.09%,respectively.Conclusion A rapid and sensitive method for thedetection ofNeisseria meningitidisbased on real-time quantitative PCR was established.