A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax.

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380bp for An. minimus, 180bp for An. harrisoni, 150bp for An. aconitus, 310bp for An. varuna, 276bp for P. falciparum, and 300bp for P. vivax. The sensitivities were 0.5pg/μl (25sporozoites/μl) for P. falciparum DNA and between 0.5 and 5pg/μl (25-250sporozoites/μl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.