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Background  Genus  Anopheles  mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria re-emerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight  Anopheles  species ( Anopheles sinensis ,  Anopheles pullus ,  Anopheles belenrae ,  Anopheles lesteri ,  Anopheles kleini ,  Anopheles sineroides ,  Anopheles koreicus, Anopheles lindesayi ) are distributed throughout Korea. Members of the  Anopheles  Hyrcanus group currently cannot be identified morphologically. The other species of  Anopheles  can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight  Anopheles  species present in the ROK.    Methods   Anopheles  spp. used in this study were collected near/in the demilitarized zone in ROK, where most malaria cases are reported. DNA from 165 of the  Anopheles  specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analysed for molecular identification.    Results  DNA from a total of 165  Anopheles  specimens was identified to species using a multiplex diagnostic system. These included: 20  An. sinensis , 21  An. koreicus , 17  An. lindesayi , 25  An. kleini , 11  An. lesteri , 22  An. sineroides , 23  An. belenrae , and 26  An. pullus . Each species was clearly distinguished by electrophoresis as follows: 1,112 bp for  An. sinensis ; 925 bp for  An. koreicus ; 650 bp for  An. lindesayi ; 527 bp for  An. kleini ; 436 bp for  An. lesteri ; 315 bp for  An. sineroides ; 260 bp for  An. belenrae ; and, 157 bp for  An. pullus .    Conclusion  A multiplex PCR assay was developed to identify  Anopheles  spp. distributed in ROK. This method can be used to accurately identify  Anopheles  species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

Multiplex PCR assay for the identification of eight Anopheles species belonging to the Hyrcanus, Barbirostris and Lindesayi groups