ESTABLISHMENT OF PCR-RFLP METHOD BASED ON 28S-D3 REGION OF RIBOSOIBOSOMAL 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 Minimus Complex: polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). Methods Genomic DNA was extracted from the individual mosquito’s legs by the GNT-K method and the rDNA-28S-D3 gene was amplified by specific primers and PCR products were purified, cloned, sequenced and analyzed. MboII restriction enzyme was chosen based on the restriction maps of 28S D3 gene of An. minimus A and C to cut PCR products. Results Two different D3 sequences (GenBank: AF416782, AF415594), blasted as An. minimus A and C, were detected in this study. An. minimus A and C can be visually distinguished according to the electrophoresis pattern: the three fragments in 376 bp, 268 bp and 108 bp were observed in the lanes of An. minimus A while only a band in 376 bp for An. minimus C because of mutation in 96 bp, 97 bp. Conclusion Two sibling species, An. minimus A and C, were found in southern China and they can be distinguished by the PCR-RFLP method established in this study.