阪崎肠杆菌实时荧光TaqMan PCR 检测方法的研究
Study of real time TaqMan PCR for detction of Enterobacter sakazakii
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摘要: 目的 建立阪崎肠杆菌的TaqMan PCR检测方法。 方法 将阪崎肠杆菌的菌悬液连续10倍稀释后与健康人新鲜粪便混匀,制备成1.2100 ~ 1.2108 cfu/ml梯度浓度的粪便模拟标本并提取DNA,用以评价阪崎肠杆菌实时荧光TaqMan PCR法对临床模拟标本检测的灵敏度、特异性和稳定性。 结果 实时荧光TaqMan PCR法对阪崎肠杆菌粪便模拟标本的检测灵敏度为1.2103 cfu/ml; 其线性检测范围为:1.2103 ~ 1.2108 cfu/ml;在稳定性评价中,对含菌量为1.2108、1.2106和1.2104 cfu/ml的粪便模拟标本进行实时荧光TaqMan PCR法重复检测10次,结果显示扩增反应Ct 值的变异系数(CV)为0.65%~1.47%;在特异性评价中,除了阳性对照其余样本均未见特异性扩增曲线。 结论 本研究建立了阪崎肠杆菌实时荧光TaqMan PCR法。Abstract: Objective To establish the rapid detection assay of Enterobacter sakazakii and provide reliable technical basis for clinical application. Methods In order to evaluate the sensitivity, specificity and stability of the real time TaqMan PCR assay to detect Enterobacter sakazakii in feces specimens, we made the feces simulation specimens with the concentrations of Enterobacter sakazakii from 1.2100 to 1.2108 cfu/ml by 10 series dilutions of Enterobacter sakazakii strain and mixing well with healthy human feces. Results The sensitivity of real time TaqMan PCR assay of the feces simulation specimens was 1.2103 cfu/ml, and the range of the linearity was from 1.2103 to 1.2108 cfu/ml, the stability evaluation indicated that the coefficient of variation was from 0.65% to 1.47%, and the specificity was 100%. Conclusion The real time TaqMan PCR assay developed in our study could be used in the detection of Enterobacter sakazakii in feces specimens.
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