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目的设计马尔尼菲青霉菌(PM)种特异性引物,采用荧光定量PCR的方法,并结合传统的形态学检查,以达到可以在临床实验室进行快速诊断的目的。方法取患儿血液、骨髓进行真菌双相培养,观察真菌生长情况及菌落形态,显微镜下观察菌体特征,并将培养物涂片进行一系列染色。针对PM 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。结果 PM为双相性真菌,于25℃为青霉相,于37℃为酵母相,并均有典型的菌落形态特征。染色可见菌体根据染色方法不同、形态不同呈现不同特征。荧光定量PCR方法的特异性较好,与该菌属内的其他细菌间无交叉反应。结论 PM的特征性菌落形态对该菌有诊断价值,对该菌进行某些简单的染色可作为快速诊断与鉴别该菌的主要辅助手段,而实时荧光定量PCR方法在检测PM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 design specific primers for Penicillium marneffei (PM) using fluorescence quantitative PCR (PCR) in combination with traditional morphological examination to achieve rapid diagnosis in clinical laboratory. Methods The blood and bone marrow of children were used to carry out the biphasic culture of fungi. The growth and colony morphology of the fungi were observed. The characteristics of the cells were observed under a microscope and the cultures were smeared for a series of staining. Specific primers for PM 5.8S rRNA were designed and used to detect real-time fluorescence quantitative PCR using SYBR GreenⅠ fluorescent dye. Results PM was a biphasic fungus with a penicillium phase at 25 ° C and a yeast phase at 37 ° C with typical colony morphological characteristics. Staining bacteria can be seen according to different staining methods, different shapes show different characteristics. The specificity of the fluorescence quantitative PCR method is good, and no cross-reactivity with other bacteria within the genus. Conclusion The characteristic colony morphology of PM has diagnostic value to this bacterium, and some simple staining of this bacterium can be used as the main auxiliary method for rapid diagnosis and identification of this bacterium. The application of real-time fluorescence quantitative PCR in detecting PM can be greatly Shorten the clinical diagnosis time, improve the accuracy and efficiency of clinical diagnosis.