艾滋病相关支原体多重实时荧光聚合酶链反应检测方法的初步建立
Establishment of multiplex real-time PCR assay to detect AIDS-associated Mycoplasmas
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摘要: 目的 建立一种快速、灵敏和特异的艾滋病相关支原体(穿透支原体、发酵支原体和梨支原体)多重实时荧光聚合酶链反应(Multiplex real-time PCR)检测技术。 方法 使用Beacon Designer 7.0软件在穿透支原体和发酵支原体的ftsZ基因以及梨支原体的rpoB基因保守区域设计多重引物及荧光探针,建立并优化艾滋病相关支原体Multiplex real-time PCR检测体系。分别使用3种支原体阳性质粒标准品评价体系的灵敏度,使用8种其他支原体、14种常见致病菌和人类基因组核酸评价该体系的特异度,并与普通聚合酶链反应(PCR)检测方法进行比较。 结果 该Multiplex real-time PCR方法对穿透支原体和发酵支原体检测灵敏度为103拷贝,约为普通PCR的10倍,对梨支原体检测灵敏度为102拷贝,约为普通PCR 100倍。该体系对8种其他支原体、14种常见致病菌和人类基因组均不能扩增。 结论 本研究建立的Multiplex real-time PCR方法可同时快速、准确的检测穿透支原体、发酵支原体和梨支原体。有望用于临床标本检测,完善对艾滋病相关支原体的检测能力。Abstract: Objective To establish a rapid, sensitive and specific multiplex real-time PCR assay for the detection of AIDS-associated Mycoplasmas. Methods Three sets of primers and probes were designed based on the specific sequence of the ftsZ gene (M. penetrans and M. fermentans) and the rpoB gene (M. pirum) and the Multiplex real-time PCR assay was established. Eight other Mycoplasmas and 14 pathogens were used to evaluate the specificity of the assay. The sensitivity of the assay was evaluated and compared with conventional PCR assay by using standard concentration positive plasmids. Results No specific amplifications were presented by using 22 other pathogens. The sensitivity of this Multiplex real-time PCR assay was about 10 times higher than that of conventional PCR for M. penetrans and M. fermentans, and 100 times higher than M. pirum. Conclusion This Multiplex real-time PCR assay was sensitive and specific, which could be used for the detection of AIDS-associated mycoplasmas and might be used in the clinical detection.
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