变性高效液相色谱技术快速检测下呼吸道致病性细菌的实验研究

Experimental study on rapid detection of lower respiratory tract pathogenic bacteria by denaturing high-performance liquid chromatography

目的运用变性高效液相色谱（DHPLC）技术检测5种下呼吸道致病性细菌，建立一种快速检测下呼吸道致病性细菌的分子生物学方法。方法以下呼吸道致病性细菌16SrRNA基因的保守区设计通用引物，并在引物前加上40-bpGC，特异性扩增该基因的保守区和可变区，运用DH—PLC技术对PCR产物进行分析。选取50株临床分离菌株验证该方法的有效性。结果通用引物可特异性扩增细菌16SrRNA，PCR产物经DHPLC分析后每种细菌均能得到特征性的洗脱峰。DHPLC检测临床分离菌株结果显示，与常规培养方法的符合率为100％。结论DHPLC技术具有准确、简便、快捷和高通量等特点，在临床检测下呼吸道致病性细菌中具有潜在的应用价值。

Objective To construct a new molecular biological method for the analysis of microbial species in lower respiratory tract infections based on 16S rRNA gene by denaturing high-performance liquid chromatograph（DHPLC）. Methods The universal primer set was analyzed basing on the highly conserved regions of 16S rRNA gene, DNA amplicons of lower respiratory tract were analyzed by DHPLC to generate peak profiles respectively. The incorporation of 40-bpGC clamp into the amplification primer was essential to effectively discriminate genetic differences identification. Results The primers could only amplify bacterial 16S rRNA. Bacterial of amplicons which incorporation of a 40-bpGC clamp were effectively discriminated genetic differences in DHPLC. The results of clinical isolates identification showed 100% according with the traditional method. Conclusion DHPLC has not only high accuracy, but also is a convenient, rapid and high-through technique for the discrimination bacteria. It has potential value in the detection of lower respiratory pathogenic bacteria.