rDNA-ITS2 polymerase chain reaction assay for the sibling species of Anopheles fluviatilis

Species-specific differences in the nucleotide sequences of the second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) were used to develop a diagnostic polymerase chain reaction (PCR) assay for two of the sibling species of the Anopheles fluviatilis complex, members of which are major vectors of malaria in central and northern parts of India. This assay consisted of a three primer reaction, which could amplify the DNA of both the species producing fragments of two distinct sizes, 350 bp for species X and 450 bp for species Y, respectively. The assay was found to be highly specific and sensitive.