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

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• 作者：Liang Bai (8) (9)
  Guo-ding Zhu (10) (8)
  Hua-yun Zhou (8)
  Jian-xia Tang (8)
  Ju-lin Li (8)
  Sui Xu (8)
  Mei-hua Zhang (8)
  Li-nong Yao (11)
  Guang-quan Huang (12)
  Yong-bin Wang (13)
  Hong-wei Zhang (14)
  Si-bao Wang (9)
  Jun Cao (15) (8)
  Qi Gao (10) (8)
  
  8. 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鈥檚 Republic of China
  9. Institute of Plant Physiology & Ecology ; Shanghai Institute for Biological Sciences ; Chinese Academy of Sciences ; Shanghai ; People鈥檚 Republic of China
  10. Department of Parasitology ; Medical College of Soochow University ; Suzhou ; 215123 ; People鈥檚 Republic of China
  11. Zhejiang Center for Disease Control and Prevention ; Hangzhou ; People鈥檚 Republic of China
  12. Hubei Center for Disease Control and Prevention ; Wuhan ; People鈥檚 Republic of China
  13. Shandong Institute of Parasitic Diseases ; Jining ; People鈥檚 Republic of China
  14. Henan Center for Disease Control and Prevention ; Zhengzhou ; People鈥檚 Republic of China
  15. Public Health Research Center ; Jiangnan University ; Wuxi ; People鈥檚 Republic of China
  
• 关键词：Anopheles sinensis ; Knockdown resistance (kdr) ; AllGlo probe ; qPCR
• 刊名：Malaria Journal
• 出版年：2014
• 出版时间：December 2014
• 年：2014
• 卷：13
• 期：1
• 全文大小：1,145 KB
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• 刊物主题：Parasitology; Infectious Diseases; Tropical Medicine;
• 出版者：BioMed Central
• ISSN：1475-2875

文摘

Background Anopheles 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. Methods A 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. Results Six 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. Conclusion The 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.