16S rDNA快速鉴定血液制品中污染细菌的测序方法的建立  

Establishment of PCR sequence-based typing for identifying bacteria based on 16S rDNA in the contaminate blood products

作　　者：应燕玲[1] 许先国[1] 朱发明[1] 吕杭军[1] 严力行[1] 

机构地区：[1]浙江省血液中心输血研究所,卫生部血液安全研究重点实验室,杭州310006

出　　处：《中华微生物学和免疫学杂志》2009年第10期880-883,共4页Chinese Journal of Microbiology and Immunology

基　　金：浙江省科技厅重大疾病防治项目（2006C13111）

摘　　要：目的建立基于16SrDNA快速鉴定细菌的PCR测序方法（PCR-SBT）。方法通过一组16SrDNA通用引物扩增得到基因组全长，PCR产物经纯化后直接测序分析。利用BLAST软件从GenBank数据库中搜索相关菌株的16SrDNA全序列，采用Clustal X软件进行多序列比对和同源性分析，确定细菌的种属，并与常规生化鉴定结果比较。利用大肠杆菌基因组一系列稀释度标本进行PCR扩增，检测方法的敏感性。结果实验利用多对引物建立了16SrDNA全长序列分析方法，13个标准菌株通过PCR．SBT方法获得约1400bp的全长序列。比对分析13个标准菌株测序结果与预期标准序列完全一致，证实建立的PCR—SBT方法结果可靠。利用建立的PCR—SBT法，对实验室从血小板制品和脐带血中分离得到不同未知菌株进行鉴定，成功确定了这些菌株的种属。以大肠杆菌DNA为模板，方法的最低检测限为反应体系DNA含量0．2ng。结论建立的基于16SrDNA的PCR—SBT方法是可行的，可快速准确地检测及鉴定细菌种类，尽早发现细菌污染血制品，对污染血制品输注后的针对性治疗方面具有潜在的应用价值。Objective To establish a PCR sequence-based typing（PCR-SBT） method for identifying the bacteria accurately and rapidly. Methods The full-length of 16S rDNA was amplified with a set of universal primers. The PCR products were purified and sequenced directly. The related sequences were obtained from the GenBank database using BLAST search software and the species of the bacteria were identified according to the multiple sequence alignment and homology comparison of the 16S rDNA sequences by CLUSTAL X software. Results The results showed that it has been established a PCR-SBT method for analysis 16S rDNA nucleotide sequences by multi-primer pairs and 13 standard bacteria strains were obtained about 1400 bp of the 16S rDNA sequences. The sequences of these bacteria were 100% concordance with the known standard nucleotide sequences, which confirmed the accuracy of the method. The different unknown strains that isolated from platelet and cord blood in the laboratory were successfully identified using the 16S rDNA PCR-SBT method. Estimation of the minimal levels of the genomic DNA detection was 0.2 ng in the PCR reaction. Conclusion It suggested that the PCR-SBT based on 16S rDNA was reliable and could identify the bacteria rapidly and accuracy. It can detect the bacterial contamination of blood products as early as possible and has a potential application value in the direct treatment of transfusion related bacterial contamination.

关 键 词：血液细菌污染 16S RDNA 多聚酶链反应-测序方法 

分 类 号：R927.2[医药卫生—药学]