中国媒介生物学及控制杂志

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核糖体28S-D3等位基因特异扩增鉴别微小按蚊亲缘种A和C

周水森;汤林华;夏明仪;薛海筹   

  1. 中国疾病预防控制中心寄生虫病预防控制所疟疾室 上海20005
  • 出版日期:2003-12-20 发布日期:2003-12-20

Allele-Specific Amplification (PCR-ASA) Method Based on 28S-D3 Region of Ribosomal DNA for Differentiation of Anopheles minimus A and C

ZHOU Shui-sen;TANG Lin-hua;XIA Ming-yi;et al   

  1. National Institute for Parasitic Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Shanghai 200025, China
  • Online:2003-12-20 Published:2003-12-20

摘要: 目的建立微小按蚊复合体不同亲缘种A和C的分子鉴别方法。方法用单蚊蚊腿消化提取基因组DNA,PCR特异扩增28S第3结构域(D3)基因,对PCR产物进行纯化、克隆、测序和序列分析;基于序列差异设计特异引物,进行等位基因特异扩增(PCR-ASA),根据扩增片段大小区分微小按蚊不同亲缘种。结果发现微小按蚊不同亲缘种A和C(GeneBank登录号:AF416782,AF425594),根据二者序列差异设计的特异引物能在普通琼脂糖凝胶上直观地区分两亲缘种,两者除在376bp处有一共同条带外,分别在294bp和112bp处出现各自的特异扩增带。结论我们建立的ASA分子鉴别方法能有效地将我国微小按蚊不同亲缘种A和C区分开来。

关键词: 微小按蚊, 亲缘种, 基因D3, 分子鉴别

Abstract: Objective To establish a molecular method for differentiation of two sibling species,Anopheles minimus A and An.minimus C,of An.minimus Complex:allele-specific amplification(PCR-ASA).Methods Genomic DNA was extracted from an individual mosquito'legs by the GNT-K method and the rDNA-28S-D3 gene was amplified by specific primers.PCR products were purified,cloned,sequenced and analyzed.Species-specific alleles were amplified in terms of specific primers based on sequence variation of 28S D3 gene between An.minimus A and An.minimus C.Results Two different D3 sequences(AF416782,AF415594),blasted as An.minimus A and An.minimus C,were detected in this study.An.minimus A and C can be visually distinguished according to the specific bands in 294 bp and 112 bp,respectively.Conclusion Two sibling species,Anopheles minimus A and C,found in southern China,can be distinguished by the PCR-ASA method established in this study.