title: Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Liang
authors.initials: L
authors.lastname: Bai
authors.forename: Guo-ding
authors.initials: GD
authors.lastname: Zhu
authors.forename: Hua-yun
authors.initials: HY
authors.lastname: Zhou
authors.forename: Jian-xia
authors.initials: JX
authors.lastname: Tang
authors.forename: Ju-lin
authors.initials: JL
authors.lastname: Li
authors.forename: Sui
authors.initials: S
authors.lastname: Xu
authors.forename: Mei-hua
authors.initials: MH
authors.lastname: Zhang
authors.forename: Li-nong
authors.initials: LN
authors.lastname: Yao
authors.forename: Guang-quan
authors.initials: GQ
authors.lastname: Huang
authors.forename: Yong-bin
authors.initials: YB
authors.lastname: Wang
authors.forename: Hong-wei
authors.initials: HW
authors.lastname: Zhang
authors.forename: Si-bao
authors.initials: SB
authors.lastname: Wang
authors.affiliation: 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. caojuncn@hotmail.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Cao
authors.forename: Qi
authors.initials: Q
authors.lastname: Gao
mesh_terms: D000818:Animals; D000852:Anopheles / Q000187:drug effects* / Q000235:genetics*; D001483:Base Sequence; D002681:China; D005838:Genotype; D007305:Insecticide Resistance / Q000235:genetics*; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity