【作者】 袁春； 黄长形； 连建奇；

【Author】 YUAN Chun HUANG chang-xing LIAN Jian-qi. The Center of Diagnosis and Treatment for Infectious Diseases of PLA,Tangdu Hospital,Fourth Military Medical University,Xi’an 710038,China

【机构】 第四军医大学唐都医院全军感染病诊疗中心；

【摘要】 目的:探讨用 PCR 方法检测肝硬化患者腹水中细菌 DNA 的可行性,了解肝硬化患者发生细菌移位的高危因素。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 PCR 扩增。腹水中细菌 DNA 阳性者纯化后经核苷酸测序鉴别细菌种类, 同时收集患者临床资料,运用 SPSS 11.5软件对患者基本临床特征及主要检验结果进行统计分析。结果: 7种对照菌株均获得530 bpDNA 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 Ipg 的细菌 DNA。37份腹水中有9份获得530bp DNA 片段(24.3%),而腹水细菌培养阳性率为5.4%(2/37), 二者比较有显著性差异(P<0.05)。腹水中细菌 DNA 阳性和阴性的两组肝硬化患者在上消化道出血的发生率、血清总胆红素水平、凝血酶原活动度、Child-Pugh 积分、腹水总蛋白浓度、外周血白细胞总数等比较, 有显著性差异(P<0.05)。结论:用通用引物通过 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。肝功能损害、腹水调理活性低下和上消化道出血是肝硬化患者发生细菌移位的高危因素。更多还原

【Abstract】 Objective:To detect bacterial DNA in ascites of patients with cirrhosis by PCR and to understand the risk factors for bacterial translocation in patients with cirrhosis.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 samples of ascites of patients with cirrhosis were amplified with PCR.The corresponding bacteria were identified by nucleotide sequencing of purified PCR products.The clinical data of patients was also collected.The main clinical and analytical characteristics of 37 patients were statistically analysed using SPSS software 11.5.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 cultures was positive.The two methods for detecting the presence of bacteria were significant difference(p<0.05).There were significant differences in incidence of upper gastrointestinal hemorrhage,serum total bilirubin level,Child-Pugh score ,ascitic total protein and peripheral blood leucocyte population between group with bacteria DNA and group without bacteria DNA in ascites(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.Cirrhotic patients with severe hepatic impairment,deficient ascitic fluid opsonic activity or upper gastrointestinal hemorrhage are predisposed to bacterial translocation.更多还原