用PCR技术直接检测致病真菌的实验研究

DETECTION AND IDENTIFICATION OF FUNGAL PATHOGENS IN HUMAN BLOOD SPECIMENS BY PCR ASSAY

目的 建立和评价从血标本中直接PCR检测病原真菌的方法。方法 用红、白细胞裂解液 ,基因释放剂等直接处理血标本 ,提取微量的靶DNA ,然后用真菌通用引物及种特异性引物进行PCR扩增。结果 在几种重要致病真菌制备的人血标本中检测出靶DNA ,其敏感性达 10个孢子 /ml以下 ,且从标本处理到报告结果仅需 6h。结论 用PCR方法可简便快速特异敏感地从血标本中直接检测病原真菌 ,提示可为临床快速诊断深部真菌病打下基础。

Aim To set up and evalute a method for detecting pathogenic fungi in human blood specimens Methods The blood specimens were directly disposed with erythrocyte lysis buffer,leukocyte lysis buffer and GeneReleaser,and then,a PCR was carried out with universal fungal and species-specific primers Results The target DNAs from all blood specimens,which contain 10 different strains tested,were amplified with PCR,and the theoretical lower limit of detection of the PCR assay is 10 CFU/ml The time from obtaining clinical specimens to reporting test result is about 6 hours Conclusions The described PCR assay allows for the highly sensitive,specific,simple and rapid detection and identification of fungal pathogens in human blood specimens,and has important value in the early diagnosis and monitoring of antifungal therapy