建立16S rDNA的PCR-SBT法细菌鉴定及在败血症中的应用  

Establishment of 16S rDNA PCR-SBT for identification of bacteria in patients with sepsis

作　　者：蔡莹 魏建波[2] 鲍亚萍[2] 施新萍[2] 黄晶晶 

机构地区：[1]临安市中医院检验科,浙江临安311300 [2]浙江省立同德医院检验科,浙江杭州310012

出　　处：《中华医院感染学杂志》2015年第10期2161-2164,共4页Chinese Journal of Nosocomiology

基　　金：浙江省医药卫生科技计划基金资助项目(2010KYA045)

摘　　要：目的建立16SrDNA PCR测序(PCR-SBT)方法快速鉴定败血症患者体内的细菌菌种。方法对5株标准菌株及临床常见的20属27种细菌进行16SrDNA保守序列PCR扩增,扩增产物经纯化后直接测序,并利用Blast软件从GenBank数据库中搜索相关菌株的16SrDNA序列,采用Clustal-X软件进行多序列比对和同源性分析,以确定细菌的种属;同时对124例败血症患者进行血培养并与PCR-SBT法比较,探讨其在临床应用中的价值。结果所有细菌菌株通过PCR方法均获得约1 500bp的保守序列,经测序分析后5株标准菌株的序列与预期标准序列完全一致,27种临床分离株测序鉴定结果与生化鉴定结果一致;124例败血症患者中84例PCR法阳性,阳性率67.7%,显著高于血培养的30.6%(P<0.01)。结论建立16SrDNA的PCR-SBT法鉴定细菌快速可靠,诊断败血症优于传统的血培养法。OBJECTIVE To establish 16S rDNA PCR sequence‐based typing （PCR‐SBT ） for the rapid identification of bacteria in the patients with sepsis .METHODS The 16S rDNA conservative sequence PCR amplification was performed for 5 standard strains and 27 species of bacteria of 20 common clinical genera ,the amplified products were directly sequenced after the purification ,the 16S rDNA sequences of the relevant strains were searched from the GenBank database by using Blast software ,the multiple sequence alignment and homological analysis were per‐formed with the use of Clustal‐X software so as to determine the species of the bacteria ,the blood culture was con‐ducted for 124 sepsis patients and was compared with the PCR‐SBT method ,and its clinical value was evaluated . RESULTS Totally about 1 500 bp conservative sequences have been acquired from all the strains through the PCR method ,and the sequencing analysis showed that the sequences of the 5 standard strains were completely consist‐ent with the expected standard strains ,and the results of the sequencing in identification of the 27 clinical isolates were as same as the results of the biochemical identification .Of the 124 sepsis patients ,84 were tested positive for the PCR method with the positive rate of 67 .7% ,significantly higher than 30 .6% of the blood culture （P〈0 .01） .CONCLUSION The 16S rDNA PCR‐SBT is rapid and reliable for the identification of the bacteria and is su‐perior to the traditional blood culture method in the diagnosis of sepsis .

关 键 词：16S rDNA 多聚酶链反应一测序方法 败血症 

分 类 号：R446[医药卫生—诊断学]