Use of the polymerase chain reaction to identify mosquito species of the Anopheles gambiae complex

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Abstract
A nonradiometric method has been developed for distinguishing between the sibling species Anopheles gambiae Giles and An.arabiensis Patton, two important Afrotropical vectors of malaria. DNA fragments of species diagnostic length are amplified by polymerase chain reaction (PCR) from a small amount of unknown DNA and three different PCR primers. All three PCR primers are based on ribosomal DNA (rDNA) sequences. A universal plus‐strand primer (A0) is derived from a conserved region at the 3' end of the 28S rDNA coding region. Two species‐specific minus‐strand primers (Aa05 and Ag1 3) are derived from sequences in the intergenic spacers. The Ag13 sequence is approximately 1.3 kb downstream of A0; the Aa05 sequence is about 0.5 kb downstream of A0. When mosquito DNA is amplified in the presence of all three primers, a 1.3 kb fragment is produced if An.gambiae DNA is used as template, and a 0.5 kb fragment is produced if An.arabiensis DNA is used. Amplification of DNA from An. gambiae I An.arabiensis hybrids produces both the 1.3 kb and the 0.5 kb fragments. Neither diagnostic fragment is produced when DNA from other species in the An.gambiae complex is used as template.