双位点保守基因特异性PCR-CE-RFLP快速鉴定病原菌的实验与临床研究

目的]初步建立一个临床常见病原菌的标准PCR-CE-RFLP数据库,为临床快速鉴定病原菌奠定基础。方法]选取临床分离的病原菌共167株,筛选16S rRNA基因和16S-23S rRNA间区基因的合适引物,分别用FAM和JOE两种荧光素标记,调整PCR反应条件,让两种引物能同时在同一条件下反应,所得产物先用普通琼脂糖凝胶电泳,再分别用限制性内切酶HaeIII进行不完全酶切,酶切产物50倍稀释后进行毛细管电泳(PCR-CE-RFLP),测定酶切片段长度差异。结果]针对16S rRNA基因的RFLP谱数据只能将病原菌鉴定到"科"的程度,而单纯应用16S-23S rRNA间区基因的RFLP谱数据虽能区分绝大多数细菌,但个别种属内仍然出现相似的谱型。经过双位点PCR-CE-RFLP分析后,则可将所有细菌鉴定到"种"的程度。结论]利用16S rRNA基因和16S-23S rRNA间区基因PCR-CE-RFLP进行细菌鉴定简便快捷,能将传统方法需要十几个小时甚至几十小时的鉴定工作缩短到几个小时,是一种极有临床应用价值的快速诊断方法。

THE EXPERIMENTAL AND CLINICAL STUDY ON RAPID IDENTIFICATION OF PATHOGENIC BACTERIA BY MEANS OF TWO CONSERVATIVE GENE LOCI SPECIFIC PCR-CE-RFLP

Objective]To primarily construct a PCR-CE-RFLP database of common clinical pathogenic bacteria,and to lay foundation for rapid identification of pathogenic bacteria.Methods]167 clinical isolates were collected,and two pairs of primers aiming at 16S rRNA and 16S-23S rRNA spacer region were chosen appropriately,which were labeled with two kinds of dihydroxyfluorane FAM and JOE,respectively.The PCR reaction conditions were aslo modified to be with shorter annealing time and extention time,which allowed two kinds of primers to react simultaneously.The PCR product was given agarose gel electrophoresis,and then was digested by restriction endonuclease HaeIII incompletely.The mixture solutions were diluted for 50 times before given capillary electrophoresis,and the restriction fragment length polymorphism(RFLP)were detected.Results]Based on the analysis of PCR-CE-RFLP of 16S rRNA gene,the data could only be used to classify the bacteria into some families.While according to the information of two conservative gene loci specific PCR-CE-RFLP which came from 16S rRNA gene and 16S-23S rRNA spacer region gene,the pathogen bacterium could be identified to be species.Conclusions]PCR-CE-RFLP of 16S rRNA gene and 16S-23S rRNA spacer region gene is a convenient and rapid method to identify pathogenic bacteria,and is also a quick diagnosis measure which is of great clinical application value.