16SrRNA基因PCR加反相杂交技术检测细菌DNA

目的探讨聚合酶链反应（ＰＣＲ）加反相杂交技术在细菌ＤＮＡ检测中的应用。方法以１６ＳｒＲＮＡ基因为靶序列，设计引物及寡核苷酸探针，采用ＰＣＲ法加反相杂交检测标准菌株及临床标本细菌ＤＮＡ。结果对２０株不同标准菌株进行ＰＣＲ扩增，均出现３７１ｂｐ长度的ＤＮＡ片段，敏感性试验可检测出１０－１２ｇ的细菌ＤＮＡ，与人基因组及病毒无交叉反应；２２份血培养阳性标本及４份脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交区分革兰阳性／阴性细菌与培养结果相符。结论１６ＳｒＲＮＡ基因ＰＣＲ加反相杂交技术检测细菌ＤＮＡ，具有敏感、快速、准确的特点，为细菌感染的临床诊断提供了科学的依据。

Detection of bacterial DNA with polymerase chain reaction and reverse hybridization of 16SrRNA gene

Objective To develop a molecular biologic technique for detection of bacterial DNA in all bacteria with polymerase chain reaction (PCR) and reverse hybridization.Methods A pair of primers were designed according to the gene encoding 16SrRNA. DNA fragments of different species and digested from clinical samples were detected with PCR, reverse hybridization of a universal bacterial probe, and a gram positive probe as well as a gram negative probe.Results 371bp DNA fragments were amplified from 20 different species. The sensitivity could be improved to 10 -12 g. No signal was observed when human DNA and viruses were used as templates. 22 blood samples and 4 cerebrospinal fluid samples, being positive with culture, were positive by using PCR. The gram negative and gram positive probes hybridized to clinic samples and different species, as predicted by Gram stain characteristics.Conclusions The method not only is sensitive, rapid and specific, but also provides an etiological basis for diagnosis of sepsis.