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

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

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 2 8S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 ,AF42 5 5 94) ,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 3 76bp处有一共同条带外 ,分别在 2 94bp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。

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.