聚合酶链反应检测病原真菌及新生隐球菌的实验研究

Experimental study on detection of pathogenic fungi and cryptococcus neoformans by polymerase chain reaction

目的使临床系统性真菌病的诊断提高到分子水平。方法首先应用一对外侧的基于ｒＤＮＡ的真菌通用引物进行ＰＣＲ扩增，此后应用新生隐球菌种特异性内侧引物对首轮扩增产物进行第二轮扩增鉴定。结果应用外侧引物对能对代表８属１７种的２５株医学重要真菌进行扩增，而对所有细菌和人ＤＮＡ均为扩增阴性，内侧引物对仅对新生隐球菌获得扩增。应用两轮ＰＣＲ扩增（通用ＰＣＲ和随后的种特异性ＰＣＲ）方法从获得临床标本到鉴定病原菌仅需８小时。

Objective The objective of the study was to develop the diagnosis of systemic mycoses to the sphere of molecular biology. Methods With the nested PCR methodology, a method for detecting fungal pathogens in clinical specimens was developed. The complete strategy consists of PCR amplification with the external fungal universal primers for rDNA and amplicon identification by the second cycle PCR amplification with the cryptococcus neoformans species specific primer pair. Results The external universal primers were capable of amplifying DNA from 25 strains representing 17 species (8 genera) of medically important fungi. Neither human nor a varity of pathogenic bacteria gave an amplified product. The internal species specific primers amplified DNA only from Cryptococcus neoformans. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species specific PCR) of a fungal pathogen within 8h from clinical specimens. Conclusion This PCR based detection and identification system provides a rapid laboratory method for the diagnosis of systemic mycoses and shows its potential for clinical applications.