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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.

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