真菌特异性通用引物的聚合酶链反应系统的实验与临床研究

Experimental and Clinical Study on Detection of Medically Important Fungi by PCR with A Universal Fungus-specific Primer System

目的 为了判断临床和实验室标本中是否存在病原性真菌。方法 设计了一个以热启动聚合酶链反应 (PCR)为基础的实验方法 ,以一段真菌特异性DNA序列作为通用引物进行检测。引物序列为 :①AACTTAAAGGAATTGACGGAAG ;②GCATCACAGACCTGTTATTGCCTC。结果 该法能在 3小时之内对所用的全部 2 3种共 42株重要病原性真菌及经培养证实有真菌的 2 2份临床标本成功地扩增出一条310bp的DNA片段 ,但对其它微生物和人体细胞均未扩增出类似片段。该法敏感性高 ,特异性强。结论 采用通用性强的引物系统配合特异性高的热启动PCR技术检测临床和实验室标本中是否存在病原性真菌的方法有重要的应用潜力。

Objective To detect pathologic fungi existed in experimental or clinical specimens. Methods A hot initiated polymerase chain reaction (PCR) based method with a set of universal fungus specific primers that are capable of detecting a wide range of medically important fungi is developed in this paper. Such primers allow specific amplification of fungal DNA but not other eukaryotes or prokaryotes. The gene sequences are:①AACTTAAAGGAATTGACGGAAG;②GCATCACAGACCTGTTATTGCCTC. Results A 310bp product was successfully amplified from all 42 strains of 23 fungal species studied, and from 22 culture proved clinical specimens within 3 hours, but not from any strains of other microbes and human cells. This detection system is of high sensitivity. Conclusion This highly universal primer system in combinaition with highly specific hot initiated PCR might be used in the detection of medically important fungi in experimental or clinical specimens.