Diagnosis of Plasmodium gallinaceum in Infected Mosquitoes by Multiplex PCR

Abstract

Abstract

The aim of this project was to use multiplex PCR as a detective reaction for Plasmodium gallinaceum in Aedes aegypti mosquitoes. Laboratory-reared female mosquitoes were divided into two groups–groups 1 and 2–and allowed to feed on the blood of parasitized and non-parasitized chickens, respectively. The engorged female mosquitoes were kept individually in vials at -20oC. The DNA was extracted from clotted blood from the mosquitoes’ stomachs using QIAamp Blood Mini Kit (Qiagen). Multiplex PCR was used to detect P. gallinaceum from DNA samples with two forward primers; FP1 (5’ ACT TGA CCG ATT GTT CCT CAT CGC CTT T 3’) and FP2 (5’ AGT TCG TGA ATA TGA TTT GTC TGG T 3’), together with reverse primer RP1 (5’ TTG TTG CCT TAA ACT TCC TTG TGT T 3’). From the experiment, the PCR condition 95^oC for 30 sec, 55^oC for 30 sec and 72^oC for 3 min, gave the most satisfactory result. Two distinct bands of 279 and 1,527 bp were only shown in P. gallinaceum-positive samples. This technique, therefore, could be used for mosquito surveillance for avian malaria.

Keywords : Plasmodium gallinaceum, mosquito, multiplex PCR