长臂光敏生物素和光敏地高辛核酸探针检测医学真菌灵敏度的比较研究

The Sensitivity Comparing Study of DNA Probe Labelled with Long Arm Photobiotin and DNA Probe Labelled with Photodigoxin for the Detecting Medical Fungi

建立应用长臂光敏生物素核酸探针和光敏地高辛核酸探针杂交。检测真菌的方法 ,并比较二者检测临床常见医学真菌的敏感性。设计并合成医学真菌通用引物 ,用PCR扩增白色念珠菌标准株的核糖体RNA基因 ,用长臂光敏生物素或光敏地高辛标记其纯化的扩增产物制备成相应的核酸探针 ,然后用上述两种探针对标本中的PCR扩增产物进行Southern杂交 ,检测医学真菌。通过用白色念珠菌感染大鼠 ,建立念珠菌病的实验动物模型 ,验证血培养法、PCR法和PCR扩增结合Southern杂交 ,检测真菌的敏感性。结果表明 ,这两种核酸探针可分别与白色念珠菌、热带念珠菌、新型隐球菌、黄曲霉菌、烟曲霉菌等 9种真菌杂交呈阳性结果 ;与临床常见的细菌、病毒和哺乳动物组织细胞DNA杂交结果为阴性。利用实验动物模型比较了血培养法、PCR法和PCR扩增结合长臂光敏生物素核酸探针Southern杂交法检测真菌血症的灵敏度 ,实验证明后者的敏感性最高。总之 ,应用长臂光敏生物素核酸探针和光敏地高辛核酸探针与标本PCR扩增物杂交 ,检测上述真菌既特异和敏感 ,又不需放射性同位素。光敏地高辛核酸探针检测医学真菌的灵敏度略高于长臂光敏生物素核酸探针。

Two molecular methods for the detection of the medical fungi using DNA probe labelled with long arm photobiotin and DNA probe labelled with photodigoxin were developed and their sensitivities were compared.At first, a pair of universal primers of medically important fungi was designed and synthesized.Then the DNA of Candida albicans were amplified with the universal primers.The amplification products were then purified and labelled with the long arm photobiotin or photodigoxin as a probes to be used in the Southern blot analysis of the medically important fungi. Results showed that the medically important fungi including Candida albicans, Candida tropicalis, Candida kefyr, Candida parapsilosis, Candida glabrata, Candida lipolytica, Cryptococcus neoformans, Aspergillus fumigatus and Aspergillus flavus can be hybridized positively with the photobiotin or photodigoxin probes. All the bacteria , virus and human cells tested give negative results.Comparing with the routine blood culture method and PCR method, the Southern blot method using probes labelled with long arm photobiotin or photodigoxin is more sensitive. It is able to detect the presence of as few as 1-5 cells or 1-10 fg DNA of fungus.The two methods are specific, sensitive and safe and not involve any radioactivity.The method using photodigoxin probe is more sensitive than that using long arm photobiotin probe.