沙门菌PCR-dHPLC基因分型方法建立  

Genotyping of Salmonella spp. by 16S-23S rRNA assay

作　　者：张东方[1,2] 袁飞[1] 王娉[1] 杨海荣[1] 胡玥[1] 赵勇胜[1] 陈颖[1] 葛毅强[2,3] 

机构地区：[1]中国检验检疫科学研究院,北京100123 [2]中国农业大学食品科学与营养工程学院 [3]中国农村技术开发中心

出　　处：《中国公共卫生》2012年第11期1515-1518,共4页Chinese Journal of Public Health

基　　金：国家科技支撑计划项目(2009BAD91306);国家质检总局科研计划项目(2009IK312;2010IK178)

摘　　要：目的建立沙门菌聚合酶链反应-变性高效液相色谱(PCR-dHPLC)基因分型方法。方法采用16S～23S rRNA内转录间隔(ITS)作为沙门菌分型目的基因,确定特异性扩增引物,进行PCR扩增,扩增产物经dHPLC分离,根据dHPLC图谱峰型差异进行分型,并与血清学和生化分型结果比较。结果 89株沙门菌共分为12个dHPLC型(D型);所有沙门菌均有1个相同色谱峰,克隆测序结果表明,其片断大小为600 bp,其他8种食源性致病菌对照株无此色谱峰,应为沙门菌16S～23S rRNA基因序列的特征性条带,分型结果与血清学差异较大,与生化分型结果有一定一致性。结论所建立的沙门菌PCR-dHPLC基因分型方法具快速、操作方便、重复性好、成本低廉、高通量和自动化的特点。Objective To develop a polymerase chain reaction-denaturing high performance liquid chromatography（PCR-dHPLC）genotyping method for genotyping of Salmonella spp.Methods Specific primers of 16S-23S rRNA intergenic spacer sequence（ITS） region were used to subtype Salmonella spp.The PCR amplification products of experimental strains were separated by dHPLC.The results of genotyping achieved through the differences between dHPLC peaks and were comparaed to the results obtained by serological typing and biochemical typing.Results Totally 89 Salmonella spp.strains were successfully genotyped into 12 dHPLC types（D type）.All the Salmonella spp.strains had one same chromatographic peak,while other food-born pathogens showed no similar peak.DNA sequence analysis showed that the sequence was 600bp.The results indicate that the chromatographic peak is specific to Salmonella spp.Comparing the dHPLC genotyping results with those of serological typing and biochemical typing,we found dHPLC genotyping results were significantly differ from the serological typing results,while were consistent with that of the biochemical typing.Conclusion DHPLC is a novel,rapid,highly accurate,and cost-effective genotyping method for genotyping of Salmonella spp.

关 键 词：聚合酶链反应-变性高效液相色谱(PCR-DHPLC) 沙门菌 16S^23SrRNA基因区间 分子分型 

分 类 号：R446.5[医药卫生—诊断学]