基于Luminex悬浮芯片的鼠疫耶尔森菌SNP分型方法研究  

Development of a multiplex SNP typing assay for Yersinia pestis Orientalis strains based on Luminex technology

作　　者：朱鹏[1,2] 张青雯[3] 祁芝珍[3] 崔玉军[2] 肖潇[2] 杨瑞馥[2] 谭周进[1,4] 宋亚军[2] 

机构地区：[1]湖南农业大学生物安全科学技术学院,长沙410128 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [3]青海省地方病预防控制所,西宁811602 [4]湖南中医药大学微生物学教研室,长沙410208

出　　处：《军事医学》2012年第7期502-507,共6页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31071111)

摘　　要：目的利用Luminex悬浮芯片技术,基于单核苷酸多态性(single nucleotide polymorphism,SNP)建立我国鼠疫耶尔森菌东方型菌株(以下简称东方型鼠疫菌)的基因分型方法,为进一步研究东方型鼠疫菌多态性并分析其系统发育关系奠定基础。方法利用多重PCR,同时扩增东方型鼠疫菌中18个具有分型意义的SNP位点,扩增产物进行多重等位基因特异性引物延伸(allele specific primer extension,ASPE)反应以及Luminex悬浮芯片技术分析,随后利用MasterPlex GT V2.3软件计算平均荧光强度(median fluorescence intensity,MFI)比值,判断各SNP位点的碱基状态;并对SNP分型方法的重现性进行评价。结果 Luminex多重SNP技术能够快速、高通量地检测出各SNP位点的MFI值,从而在8 h内一次性成功确定东方型鼠疫菌中18个SNP的碱基状态;36株东方型鼠疫菌基于18个SNP可以分为7个基因型。结论本文建立的Luminex多重SNP检测方法作为一种高通量检测SNP的技术平台,为后期的东方型鼠疫菌SNP分型及系统发育分析奠定了基础。Objective To develop a Luminex-based single nucleotide polymorphism （SNP） typing assay for the Chinese Yersinia pestis orientalis strains. Method 18 oriental strains specific SNPs were selected and amplified simultaneously by a multiplex PCR assay. The amplicons were subjected to allele specific primer extension （ASPE） and Luminex analysis. MasterPlex GT V2.3 software was used to calculate the median fluorescence intensity （MFI） ratios and call SNPs. Assays were repeated to evaluate their reproducibility. Results This assay yielded unambiguous SNP calls for all the 18 targeted SNPs simultaneously with reasonable reproducibility in 8 hours. Thirty-six Y. pestis Orientalis strains were grouped into 7 genotypes based on the SNP profiles. Conclusion The Luminex-assay presented here provides a reliable platform to screen the SNP of Y. pestis Orientalis strains in a high-throughput way, which will be of great help in phylogenetic analysis of this deadly bacterium.

关 键 词：Luminex技术 耶尔森菌 鼠疫 多态性 单核苷酸 基因分型 

分 类 号：R378.6[医药卫生—病原生物学]