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

Woo Jun Bang,Kwang Shik Choi,Myung Soon Kim,Heung Chul Kim,Jihun Ryu,So Youn Lee,Hyeon Seung Lee,Sung Tae Chong,Terry A Klein

doi:10.1186/s12936-021-03808-w

Woo Jun Bang, Kwang Shik Choi + Show 7 more

Open Access

https://doi.org/10.1186/s12936-021-03808-w

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Journal: Malaria journal	Publication Date: Jun 28, 2021
Citations: 10	License type: open-access

Affiliation: Kyungpook National University, 59th Medical Wing

Abstract

BackgroundGenus 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.MethodsAnopheles 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.ResultsDNA 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.ConclusionA 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.

Highlights

Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide
Sample collection Eight species of Anopheles mosquitoes used in the study were collected at six sites in/near the demilitarized zone (DMZ) where most malaria infections are contracted: 1) Neutral Nations Supervisory Commission (NNSC) camp adjacent to the Panmunjeom (37°57′17.19′′N; 126°40′47.91′′E); 2) Daeseongdong (village of approximately 200 residents inside the DMZ (37°56′28.31′′N; 126°40′37.38′′E)); 3) South Gate (South gate entrance to the DMZ) (37°56′03.53′′N; 126°43′15.46′′E)); 4) Camp Bonifas (US Army installation (37°55′55.25′′N; 126°43′21.73′′E)); 5) Warrior Base (US Army training sites approximately 3 km from the south gate of DMZ), (37°55′03.96′′N; 126°44′29.74′′E)); and, 6) Dagmar North training area (37°58′29.85′′N; 126°50′40.88"E)
Molecular species diagnosis A total of 165 DNA samples extracted from individual Anopheles species were used: An. sinensis (20), An

Summary

Introduction

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. 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. In the Republic of Korea (ROK), P. vivax, P. falciparum and P. malariae were eradicated in 1979 by the National Malaria Eradication Service (NMES) of the Korean Government [6], and the World Health Organization (WHO) declared the country malaria free [7]. Except for imported malaria cases, only P. vivax is present in ROK and, following its peak of > 4000 cases in 2010, continues to be responsible for 300–500 cases annually [9,10,11]

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