摘要
目的应用16S rRNA宽范围聚合酶链反应(PCR)快速检测临床无菌体液感染,并与传统培养方法进行比较分析。方法收集94份临床无菌体液(血液、脑脊液、胸腹水、关节液)标本,提取细菌基因组DNA,应用16S rRNA宽范围PCR扩增,并将PCR阳性产物测序,然后将测序结果在美国国家生物技术信息中心(NCBI)上进行BLAST比对,从而确定菌种。同时,应用常规培养方法检测94份无菌体液标本,并对2种方法的结果进行比较。结果 16S rRNA宽范围PCR敏感性高,最低扩增浓度为103 CFU/mL,且该技术特异性高,其阳性扩增产物经测序比对后结果和培养结果完全吻合。另外,94例临床标本中应用培养方法培养出阳性例数为9例,阳性率为9.6%,而应用宽范围PCR,除培养阳性的9例均为阳性外,另外扩增出6例阳性标本,阳性率为15.9%,而此6例经测序证实分别为4株铜绿假单胞菌、1株黏质沙雷菌、1株肺炎链球菌。结论 16S rRNA宽范围PCR与培养技术比较起来,敏感性高,特异性强,有望成为临床无菌体液病原体感染快速筛查的方法。
Objective To investigate the application of 16S rRNA broad range polymerase chain reaction（PCR） to detect bacterial infection in clinical body fluid,and compare the results with traditional culture method.Methods 94 samples of clinical body fluid（blood,cerebrospinal fluid,pleural and peritoneal fluid and synovial fluid） were collected.Bacterial genome DNA was extracted,and 16S rRNA broad PCR amplification was administrated.The positive products of PCR were sequenced to determine bacterial species,and the sequence results were compared by National Center for Biotechnology Information（NCBI） BLAST.Meanwhile,94 samples were also detected by culture assay,and the results were compared.Results 16S rRNA broad range PCR was highly sensitive,and the lowest amplification concentration was 103 CFU/mL.The broad range PCR was also highly specific,and the coincidence rates between these 2 assays were 100%.In addition,9 samples were positive by culture assay,and the positive rate was 9.6%.However,15 samples were positive by broad range PCR assay,and the positive rate was 15.9%.Except 9 samples had the same results with culture assay,the other 6 samples were positive by broad range PCR,including 4 strains of Pseudomonas aeruginosa,1 strain of Bacillus prodigiosus and 1 strain of Streptococcus pneumoniae.Conclusions Compared with culture assay,16S rRNA broad range PCR is highly sensitive and specific.It is hopeful to be a new method for the rapid screening of clinical body fluid infection.
出处
《检验医学》
CAS
北大核心
2011年第12期865-868,共4页
Laboratory Medicine
基金
国家科技重大专项"十一五"资助课题(2009ZX10004-107)
