聚合酶链反应技术对真菌性角膜炎快速诊断的实验研究及临床应用

Experimental study and clinical application of fast diagnosis of mycotic keratitis using polymerase chain reaction

目的探讨聚合酶链反应（ＰＣＲ）技术与真菌性角膜炎快速诊断的可行性和优越性及其在临床应用中的价值。方法以一段真菌特异性通用引物并配合热启动聚合酶链反应技术来检测临床上常见的病原性真菌。结果８种真菌均扩增出了一条 ３１０ ｂｐ的阳性条带，而其它细菌、病毒和人体角膜组织均为阴性。在动物造型中，ＰＣＲ技术的敏感性为８６．７％，特异性为１００％；真菌培养的敏感性为５６．７％，特异性为７２．７％。在对临床标本的研究中，ＰＣＲ技术的敏感性为６６．６％，真菌培养的敏感性为３３．３％，角膜刮片的敏感性为４０％。结论采用通用性特异引物并配合热启动ＰＣＲ技术来检测实验室和临床标本中是否有真菌存在有重要的应用价值。

Objective To discuss the feasibility and superiority of fast diagnosis of mycotic keratitis using Polymerase Chain Reaction (PCR) and its application value in clinical diagnosis. Methods We used a hot-initiated PCR-based method with a set of universal fungus-specific primers that were capable of detecting a wide range of medically important fungi. ResultsA 310bp product was successfully amplified from 8 fungal species, but not from Staphylococcus aures, Psuedomonas aeruginosa ,HSV-I or human cells .In model of mycotic keratitis, the sensitivity of PCR was 86.7%,the specificity of PCR was 100%;the sensitivity of culture was 56.7%,the specificity of culture was 72.7%.The sensitivity of PCR, culture and scrapes were 66.7%,33.3% and 40% in 15 clinical specimens, respectively .Conclusion The highly universal primer system in combination with highly specific hot-initiated PCR is of great importance in the detection of medically important fungi in experimental and clinical specimens.