摘要
目的:建立一种检测马尔尼菲青霉菌的实时荧光定量PCR的方法。方法:针对马尔尼菲青霉菌5.8S rRNA设计特异性PCR引物,采用核酸荧光染料SYBR GreenⅠ进行实时荧光定量PCR检测,探讨该方法的灵敏度和特异性,并进行临床样品检测验证。结果:该方法的特异性较好,与该菌属内的其他细菌间无交叉反应;灵敏度可检测出10个细胞/mL全血,在检测范围内线性良好,相关系数R2=0.981。临床样品检测和传统的培养方法结果完全相符。结论:该方法特异性好,灵敏度高,操作简单,检测时间短;临床样品检测具有很好的准确性,从本研究的结果显示实时荧光定量PCR方法在检测马尔尼菲青霉菌中的应用可以大大缩短临床的诊断时间,提高临床诊断的准确度和效率。
Objective： To develop a quantitative Real-Time PCR assay for detection ofpenicillium marneffei. Methods： A pair of PCR primers of penicillium marneffei 5.8S rRNA was designed, and Real-Time PCR assay with SYBR Green I fluorescent dye was performed. Specificity and sensitivity of the assay were verified. The accuracy of the assay was verified, and clinic samples were detected with the PCR assay. Results： The PCR assay was specific to penicillium mameffei, and it had not cross-react with other strains of this Genus. Its detection sensitivity reached 10 cells per ml of blood. It had good linearity in the detection range, and R2 is 0.981. The results of detecting clinic samples were consistent with that of traditional culture method. Conclusions： The quantitative Real-Time PCR assay has good specificity and sensitivity. The method is easy manipulation and quick. The good accuracy of the assay was shown in detecting clinic samples. The clinic application of the quantitative Real-Time PCR can accelerate the diagnosis of penicilliosis mamefei, and impr- ove the diagnostic accuracy and efficiency.
出处
《现代生物医学进展》
CAS
2014年第7期1247-1249,1387,共4页
Progress in Modern Biomedicine
基金
广州市医药卫生科技项目(201102A213023)
