High Throughput Multiplex Assay for Species Identification of Papua New Guinea Malaria Vectors Members of the Anopheles punctulatus (Diptera Culicidae) Species Group

Center for Global Health and Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
The American journal of tropical medicine and hygiene (Impact Factor: 2.7). 01/2011; 84(1):166-73. DOI: 10.4269/ajtmh.2011.10-0438
Source: PubMed


Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An. punctulatus, An. koliensis, the An. farauti complex 1-8 (includes An. hinesorum [An. farauti 2], An. torresiensis [An. farauti 3]). Distinguishing these species from each other requires molecular diagnostic methods. We developed a multiplex polymerase chain reaction (PCR)-based assay specific for known species-specific nucleotide differences in the internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease (An. punctulatus, An. koliensis, An. farauti 1, An. hinesorum, and An. farauti 4). A set of 340 individual mosquitoes obtained from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay. Concordance between molecular and morphological diagnosis was 56.4% for An. punctulatus, 85.3% for An. koliensis, and 88.9% for An. farauti. Among 158 mosquitoes morphologically designated as An. farauti, 33 were re-classified by PCR as An. punctulatus, 4 as An. koliensis, 26 as An. farauti 1, 49 as An. hinesorum, and 46 as An. farauti 4. Misclassification results from variable coloration of the proboscis and overlap of An. punctulatus, An. koliensis, the An. farauti 4. This multiplex technology enables further mosquito strain identification and simultaneous detection of microbial pathogens.

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    • "Genomic DNA from individual mosquitoes was extracted using the Qiagen DNeasy® blood and tissue kit according to the supplementary protocol for purification of insect DNA. The species of each AP mosquito was determined using a PCR-based assay targeting the ITS2 locus [31]. "
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