16S rRNA基因在检测自发性腹膜炎腹水病原菌中的应用

Application of 16S rRNA gene in detection of pathogenic bacteria in ascites from patients with spontaneous peritonitis

目的应用聚合酶链反应(PCR)检测细菌16SrRNA基因,建立快速诊断自发性腹膜炎腹水病原菌的一种新方法。方法通过对已知病原菌16SrRNA基因保守区和变异区的序列分析,设计通用引物,对实验室已知的8种病原菌、人类基因组DNA、巨细胞病毒、白色假丝酵母菌和空白对照进行PCR扩增,检测其特异性;采用倍比稀释法和与细菌培养法比较进行敏感性检测。结果 8种已知病原菌被扩增后均获得1032bp的特异性产物,其与人类基因组DNA、巨细胞病毒、白色假丝酵母菌无交叉反应;通过稀释扩增,敏感性测试可达1pg大肠埃希菌DNA;52份标本PCR扩增15份有阳性产物,阳性率为28.8%,传统的培养法5份为阳性,阳性率为9.6%,两种方法比较差异有统计学意义(P<0.05)。结论 PCR方法能快速、特异、敏感地检出自发性腹膜炎腹水中的病原菌。

OBJECTIVE To establish a new method of rapid diagnosis of pathogenic bacteria in ascites from patients with spontaneous peritonitis through the detection of 16S rRNA with polymerase chain reation（PCR）. METHODS Universal primers were designed through a known pathogen of a conservative district and variation of 16S rRNA gene sequence analysis. The specificity was detected through PCR amplifying for 8 kinds of known pathogenic bacteria, human genomic DNA, cytomegalovirus, Candida albicans and blank-control. The sensitivity was detected through doubling dilution and bacterial culture. RESULTS The 8 known pathogens were amplified to obtain specific product of 1032 bp, which had no cross reaction with human genomic DNA, cytomegalovirus, and C. albicans. The sensitivity test could be improved to 1 pg Escherichia coli DNA after dilution amplifying. A total of 15 positive PCR amplification products of 52 specimens（28.8%） and 5 positive products by traditional culture method（9.6%）, the difference between the two method was statistically significant （P〈 0.05）. CONCLUSION The method of PCR is fast, specific and sensitive to detect pathogenic bacteria in ascites from patients with spontaneous bacterial peritonitis.