PCR-RFLP方法检测医学重要真菌的临床应用研究

Application of PCR-RFLP for detection of medically important fungi from both experimental animals and clinical samples

目的 :建立并应用PCR_RFLP技术区分临床重要真菌的方法 ,进行实验研究和临床应用。方法 :首先根据医学真菌的 18SrRNA基因合成了白色念珠菌、热带念珠菌、假热带念珠菌、近平滑念珠菌、光滑念珠菌、解脂念珠菌、新型隐球菌、烟曲霉菌和黄曲霉菌等 9种真菌的通用引物 ;建立鉴定医学真菌的PCR_RFLP方法。通过用白色念珠菌感染大鼠 ,建立了白色念珠菌感染的实验动物模型。将建立的方法用于临床 ,并与血培养法和协同凝集试验法进行了比较。结果 :PCR_RFLP法的灵敏度高于协同凝集试验和血培养法。经过一年多的临床应用 ,我们分别用PCR结合限制性内切酶鉴定技术、协同凝集试验法和血培养法检测了 118例血液病、肿瘤和其他发热患者的血液标本。应用协同凝集试验 (CoA)法检出真菌血症 19例 ,阳性率为 16 .1%。应用PCR和限制性内切酶技术共有 4 0例检出真菌血症 ,阳性率为 33.9% ;其中白色念珠菌血症占真菌血症的 6 5 .0 % ,热带念珠菌感染占 17.5 % ,假热带念珠菌感染占 10 % ,光滑念珠菌感染占 5 .0 % ,近平滑念珠菌感染占 2 .5 % ,为临床不明原因发热患者寻找病因、进行抗真菌治疗提供了参考依据。结论 :该技术具有快速、敏感、不需放射性同位素等优点 ,而且准确性极高 ,有着较好的临床应用前景。

Objective: To evaluate PCR_RFLP(polymerase chain reaction and restriction fragment length polymorphism) for detecting medically important fungi from both experimental animal and clinical samples.Methods:The previously established PCR_RFLP method was employed to detect 9 species of medically important fungi ( Candida albicans,C.tropicalis,C.pseudotropicalis,C.parapsilosis,C.glabrata,C.lipolytica,Cryptococcus neoformans,Aspergillus fumigatus,A.flavus ) from blood samples, and the routine blood culture method and CoA (coagglutination assay) method were used as control techniques.Results:Compared with the CoA method, the PCR_RFLP method is more sensitive and accurate in the experimental animal model and clinical application.Conclusions: The PCR_RFLP method is a rapid and sensitive technique with promising applications in clinical situation.