Redesigning Primer of ITS2 (Internal Transcribed Spacer 2) for Specific Molecular Characterization of Malaria Vectors Anopheles Species

Abstract

Anopheles mosquitoes are vectors of malaria, which is a serious health issue in Indonesia. Thus, vector control is an important approach taken to overcome this disease. The first and most important step in vector control is vector identification. As some Anopheles species share similar morphological features, molecular identification helps make the process more accurate by using specific DNA sequences as molecular markers such as Internal Transcribed Spacer 2 (ITS2). Many of the available ITS2 primers are universally designed for insects and, as such, are typically less specific for identifying certain genera, such as Anopheles sp. Therefore, redesigning a specific ITS2 primer is needed for specific Anopheles identification. Objective: Our objective was to redesign a specific PCR primer for Anopheles species. Methods: The redesigned primer, named sma-ITS2, was then tested using mosquito samples from the Anopheles genus and other genera. Each mosquito was identified morphologically and their genomes were extracted. DNA samples were then amplified using the redesigned primer. Results: The sma-ITS2 primer pair was capable of amplifying ITS2 sequences from all of the Anopheles samples and unable to amplify any of the non-Anopheles samples, suggesting that it is specific to Anopheles only. ll Anopheles samples were also able to be identified, only An. indefinitus were not able to be separated from its complex species, An. vagus. Conclusion: The sma-ITS2 primer pair was able to identify intra-species of Anopheles, but its efficiency in making differentiations within a species complex should be evaluated further