Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis

Malaria Journal

Methodology

Hong-wei Zhang¹  Guang-quan Huang²  Si-bao Wang³  Mei-hua Zhang⁴  Hua-yun Zhou⁴  Ju-lin Li⁴  Sui Xu⁴  Jian-xia Tang⁴  Qi Gao⁵  Guo-ding Zhu⁵  Liang Bai⁶  Jun Cao⁷  Yong-bin Wang⁸  Li-nong Yao⁹ 

[1] Henan Center for Disease Control and Prevention, Zhengzhou, People’s Republic of China;Hubei Center for Disease Control and Prevention, Wuhan, People’s Republic of China;Institute of Plant Physiology & Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China;Key Laboratory of Parasitic Disease Control and Prevention (Ministry of Health), and Jiangsu Provincial Key Laboratory of Parasite Molecular Biology, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province, People’s Republic of China;Key Laboratory of Parasitic Disease Control and Prevention (Ministry of Health), and Jiangsu Provincial Key Laboratory of Parasite Molecular Biology, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province, People’s Republic of China;Department of Parasitology, Medical College of Soochow University, 215123, Suzhou, People’s Republic of China;Key Laboratory of Parasitic Disease Control and Prevention (Ministry of Health), and Jiangsu Provincial Key Laboratory of Parasite Molecular Biology, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province, People’s Republic of China;Institute of Plant Physiology & Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China;Key Laboratory of Parasitic Disease Control and Prevention (Ministry of Health), and Jiangsu Provincial Key Laboratory of Parasite Molecular Biology, Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province, People’s Republic of China;Public Health Research Center, Jiangnan University, Wuxi, People’s Republic of China;Shandong Institute of Parasitic Diseases, Jining, People’s Republic of China;Zhejiang Center for Disease Control and Prevention, Hangzhou, People’s Republic of China;

关键词: Anopheles sinensis;    kdr;    AllGlo probe;    qPCR;   

DOI  :  10.1186/1475-2875-13-379

 received in 2014-07-16, accepted in 2014-09-20,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundAnopheles sinensis is one of the most important malaria vectors in China and other Southeast Asian countries. High levels of resistance have been reported in this species due to the long-term use of insecticides, especially pyrethroids, for public health and agricultural purposes. Knockdown resistance (kdr) caused by a single base pair mutation in the gene encoding the sodium channel is strongly associated with pyrethroid insecticide resistance in many Anopheles mosquitoes. There are few methods currently available for detecting kdr mutations in An. sinensis.MethodsA novel AllGlo probe-based qPCR (AllGlo-qPCR) method was developed to screen for the predominant kdr mutations in An. sinensis mosquitoes from the Jiangsu Province. The results from AllGlo-qPCR, allele-specific PCR (AS-PCR), and TaqMan-MGB probe-based qPCR (TaqMan-qPCR) were compared. A comparative analysis of the equipment required, ease of use and cost of the available methods was also performed. Finally, the AllGlo-qPCR method was used to detect the frequencies of kdr mutations from the other four provinces in central China.ResultsSix kdr genotypes were detected in An. sinensis from the Jiangsu Province by DNA sequencing. The AllGlo-qPCR method detected all of the kdr genotypes with a high level of accuracy (97% sensitivity and 98% specificity). AllGlo-qPCR correctly determined the kdr genotypes of 98.73% of 158 An. sinensis samples, whereas TaqMan-qPCR and AS-PCR correctly identified 96.84% and 88.61% of mutations, respectively. Furthermore, the AllGlo-qPCR method is simpler to perform, requires less equipment, and exhibits a moderate expense cost comparing with the other tested methods of kdr mutation detection. Samples collected from four of the other provinces in central China showed a high frequency of kdr mutation in An. sinensis, as detected by the established AllGlo-qPCR method.ConclusionThe novel AllGlo-qPCR method developed for kdr mutation detection in An. sinensis exhibits greater specificity and sensitivity than currently available methods and is more cost-effective; therefore, it represents a useful tool for entomological surveillance.

【 授权许可】

Unknown   
© Bai et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.