聚合酶链反应检测肝硬化患者腹水中细菌DNA

目的:探讨PCR方法检测肝硬化患者腹水中细菌DNA的可行性.方法:在细菌16S rRNA基因保守区设计一对通用引物,对7种对照菌株、人基因组DNA、HBV DNA、37份肝硬化患者腹水进行聚合酶链反应扩增.结果:7种对照菌株均获得530 bp DNA片段.而与人基因组DNA、HBV DNA无交叉阳性反应,敏感性试验可检测出1pg的细菌DNA.37份腹水中有9份获得53bp DNA片段,阳性率24.3%(9/37),而腹水细菌培养阳性率为5.4%(2/37),两者比较差异有显著性意义(P＜0.05).结论:将通用引物通过PCR方法扩增细菌16S rRNA基因,具有高度敏感性、特异性,可应用于肝硬化患者腹水中细菌DNA的检测及细菌移位的研究.

Detection of Bacterial DNA in Ascites of Patients with Cirrhosis by PCR

Objective:To detect bacterial DNA in ascites of patients with cirrhosis by PCR.Methods:A set of universal primers was designed based on the conservative regions in bacterial 16S ribosomal RNA genes. 7 control bacterial strains,human DNA,HBV DNA and 37 ascites of patients with cirrhosis were amplified with PCR.Results:530 bp DNA fragments were amplified in all 7 control bacterial strains. Human DNA and HBV DNA were not amplified by this method. The sensitivity of the test could be improved to lpg bacterial DNA. 530 bp DNA fragmens were detected from 9 of 37 ascites(24.3%).In 2 cases(5.4%),ascitic culture was positive. There was significant difference between the two methods for detecting the presence of bacteria( P <0.05)?Conclusion:PCR amplification of bacterial 16S rRNA genes using universal primers is highly sensitive and specific. The method can be used in the detection of bacterial DNA present in ascites of patients with cirrhosis and the study of bacterial translocation in cirrhosis.