| 基于SSUrRNA基因序列的间日疟原虫LAMP检测技术的建立 |
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| 引用本文： | 高世同,张仁利,黄达娜,张荣锦,耿艺介,李晓恒,吴少庭. 基于SSUrRNA基因序列的间日疟原虫LAMP检测技术的建立[J]. 中国病原生物学杂志, 2011, 0(3) |
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| 作者姓名： | 高世同 张仁利 黄达娜 张荣锦 耿艺介 李晓恒 吴少庭 |
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| 作者单位： | 深圳市疾病预防控制中心;广东药学院; |
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| 基金项目： | 深圳市科技计划资助项目(No.200902090) |
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| 摘 要： | 目的克隆分析间日疟原虫小亚基亚单位核糖体核糖核酸(SSUrRNA)特异性基因序列,建立间日疟原虫环介导等温扩增(LAMP)检测技术。方法针对间日疟原虫SSUrRNA种特异性基因序列设计1对引物,采用聚合酶链反应(PCR)从血样核酸提取物中扩增SSUrRNA基因片段,纯化后与pGEM-Teasy载体连接,构建重组质粒并转化大肠埃希菌JM109,PCR与双酶切鉴定筛选阳性克隆并测序,设计6条寡核苷酸片段,LAMP检测感染血样中间日疟原虫DNA,扩增产物作琼脂糖电泳分析或直接荧光染色肉眼观察。结果间日疟原虫SSUrRNA基因扩增片段大小约为235 bp;阳性克隆重组质粒插入的SSUrRNA基因扩增片段含有235个核苷酸,与GenBank中的Sal-1株、Belem株间日疟原虫相同序列进行比对,同源性为100%,与PV2008/TR/DEL株、PVK1294株、E1 Salvador株的同源性均为99%,与三日疟原虫、卵形疟原虫及恶性疟原虫的序列同源性均低于95%。将含有SSUrRNA靶基因的重组质粒以蒸馏水倍比稀释后做LAMP,试验的灵敏度为10 copy/μl;特异性检验显示间疟原虫患者血样DNA呈阳性反应,恶性...
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| 关 键 词： | 疟原虫 间日 核糖体核糖核酸 核酸扩增技术 |
Establishment of a loop-mediated isothermal amplification assay for detecting blood-stage Plasmodium vivax based on the specific sequence of an SSUrRNA-encoding gene |
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| GAO Shi-tong,ZHANG Ren-li,HUANG Da-na,ZHANG Rong-jin,GEN Yi-jie,LI Xiao-hen,WU Shao-ting. Establishment of a loop-mediated isothermal amplification assay for detecting blood-stage Plasmodium vivax based on the specific sequence of an SSUrRNA-encoding gene[J]. Journal of Pathogen Biology, 2011, 0(3) |
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| Authors: | GAO Shi-tong ZHANG Ren-li HUANG Da-na ZHANG Rong-jin GEN Yi-jie LI Xiao-hen WU Shao-ting |
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| Affiliation: | GAO Shi-tong1,ZHANG Ren-li1,HUANG Da-na1,ZHANG Rong-jin2,GEN Yi-jie1,LI Xiao-hen1,WU Shao-ting1 (1.Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China,2.Guangdong Pharmaceutical College,Guangzhou 510224,China) |
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| Abstract: | Objective To establish a loop-mediated isothermal amplification(LAMP) method for detecting Plasmodium vivax based on the cloning and analysis of a specific SSUrRNA-encoding gene(SSUrDNA) fragment.Methods A pair of primers was designed according to a specific P.vivax SSUrDNA sequence,and the SSUrDNA fragment was amplified by PCR from the DNA extracts of a P.vivax-infected blood sample.After purification,the gene fragment was ligated with plasmid pGEM-Teasy to construct a recombinant plasmid,which was transfo... |
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| Keywords: | Plasmodium vivax ribosomal RNA nucleic acid amplification techniques |
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