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

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.