多重PCR快速诊断甲真菌病

Direct detection of pathogenic fungi in onychomycosis based on multiplex PCR assay

目的探讨多重PCR技术从临床标本中检测甲真菌病病原真菌的方法。方法采用蛋白酶K消化法和煮沸法处理临床标本，从中提取致病菌DNA，然后用真菌通用引物NS，皮肤癣菌特异性引物CHS1和酵母特异性引物ACT1同时进行PCR扩增检测，并与直接镜检和培养法作对比。结果共收集104例临床标本，PCR检测、直接镜检和培养的敏感度分别为93．3％、100％和64．4％，特异度分别为100％、86．4％和100％，阳性预测值分别为100％、84．9％和100％，阴性预测值分别为95．2％、100％和78．7％。PCR方法在24小时内即可完成从标本处理至结果判读的全过程。结论多重PCR检测具有较好的特异性和准确性，可初步将甲真菌病的三大类病原菌区分开，且比培养缩短了近2周的时间，为临床快速诊断和及时、合理地治疗甲真菌病提供参考。

Objective To develop a rapid and reliable multiplex PCR procedure to detect fungi from specimens of onychomycosis. Methods Nail samples were taken from patients with clinically suspected onychomycosis. DNA was extracted from the specimens by digestion with proteinase K and boiling method. Pathogens were directly detected by triplex PCR assay based on universal fungus-, dermatophyte-, and yeast-specific primer pairs. The results were compared with those from microscopy and culture. Results One hundred and four patients were included in this study. Of them, forty-five （43.3%） were finally diagnosed as onychomycosis. For PCR, microscopy and culture, the sensitivity was 93.3%, 100% and 64.4%, respectively, the speciality was 100%, 86.4% and 100%, respectively, the positive predictive value was 100%, 84.9% and 100%, respectively, and the negative predictive value was 95.2%, 100% and 78.7%, respectively. This molecular diagnostic process needed no more than 24 h from specimen preparation to results interpretation. Conclusions The multiplex PCR assay could distinguish the three groups of mycose with a high specificity and sensitivity, shorten the detection time by approximately 2 weeks compared with culture, and provide evidence for rapid clinical diagnosis and timely mycosis treatment.