粪肠球菌TaqMan荧光定量PCR检测方法的建立

目的 建立用于检测粪肠球菌的TaqMan实时荧光定量-聚合酶链反应(real-time fluorescence quantitative-Polymerase Chain Reaction,FQ-PCR)方法。 方法 根据粪肠球菌的d-丙氨酸聚连接酶(ddl)基因设计TaqMan FQ-PCR的引物及探针,并使用39种常见致病菌和条件致病菌检测其特异性。将目的基因克隆到质粒中,制作标准曲线,确定方法的检测下限。制备血液模拟标本,直接提取核酸进行检测,探索临床应用的可能。 结果 在特异性评价实验中,除阳性对照外其余样本均未出现特异性扩增曲线。建立粪肠球菌TaqMan FQ-PCR方法的标准曲线,确定该方法的检测下限为20 copy/管。通过对粪肠球菌血液模拟标本的检测发现,本方法对模拟标本的检测灵敏度为3.9×103 cfu/ml。在稳定性评价中,对含菌量为3.9×108、3.9×106和3.9×104cfu/ml的血液模拟标本重复检测10次,结果显示扩增反应Ct值的变异系数(CV)为0.99%~1.84%。 结论 本研究建立了适用于临床标本检测的粪肠球菌TaqMan FQ-PCR方法。

Study of TaqMan real time PCR for detection of Enterococcus faecalis

Objective To establish a TaqMan real-time PCR assay to detect Enterococcus faecalis and provide reliable technical basis for its clinical application. Methods We designed the primers and probe for ddl gene of E. faecalis. The specificity of the assay was tested by using 39 common pathogens and opportunistic pathogens. In order to evaluate the sensitivity, the standard curve was built by using the target gene which was cloned into pMD18-T plasmid. We made the blood simulative specimens with the concentrations of E. faecalis from 3.9×100－3.9×108cfu/ml by 10 series dilutions of E. faecalis strain and mixing well with blood to evaluate the application in clinical sample detection. Results The target gene was positive in E.faecalis reference strains and clinical isolates but negative in other pathogens by this TaqMan real-time PCR assay. The standard curve showed that 20 copy E.faecalis per reaction could be detected by this assay. The sensitivity of this assay for blood simulative specimens was 3.9×103cfu/ml. The stability evaluation indicated that the coefficient of variation was from 0.99% to 1.84%. Conclusion The TaqMan real-time PCR assay established in our study could be used in the detection of E.faecalis in blood specimens.