Abstract
BackgroundThe malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies. In the past, this has relied on morphological and cytogenetic methods but these have been largely superseded by a robust allele-specific PCR (AS-PCR). One disadvantage of AS-PCR is the requirement for post-PCR processing by gel electrophoresis of PCR products. In this study, three new high-throughput 'closed-tube' assays were developed and compared with the previously described AS-PCR technique.MethodsProtocols for three fluorescence-based assays based on Melt Curve Analysis (MCA), High Resolution Melt (HRM) and TaqMan SNP genotyping were developed to detect and discriminate Anopheles parensis, Anopheles leesoni, Anopheles vaneedeni, Anopheles rivulorum and An. funestus s.s. The sensitivity and specificity of these assays were compared with the widely used AS-PCR in a blind trial using DNA extracted from wild-caught mosquitoes.ResultsThe TaqMan assay proved to be the most sensitive and specific of the three new assays. The MCA and HRM assays initially gave promising results, but were more sensitive to both DNA quality and quantity and consequently showed a higher rate of incorrect identifications.ConclusionThe TaqMan assay proved to be the most robust of the three protocols tested in this study. This assay very effectively identified all five members of the An. funestus group using fluorescently-labeled probes with distinct emission and excitation spectra allowing their independent detection in a single reaction. This method is at least as sensitive and specific as the gold standard AS-PCR approach and because it has no requirement for post-PCR processing is simpler and more rapid to run. The one disadvantage of the TaqMan assay is the cost of this assay, both in terms of initial capital outlay and running cost per sample, which is higher than AS-PCR. However, the cost of both the real-time PCR machine and fluorescently labelled probes required is falling and in the future the cost of this assay is likely to become closer to that of standard PCR.
Highlights
The malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies
The Anopheles funestus group consists of five subgroups of mosquitoes namely Anopheles funestus, Anopheles rivulorum, Anopheles minimus, Anopheles aconitus and Anopheles culicifacies and includes one of the most important African vectors of malaria Anopheles funestus s.s. [1,2,3]
Anopheles funestus s.s., An. rivulorum and An. leesoni have a widespread distribution across sub-Saharan Africa, while An, parensis is found through eastern and southern Africa and An. vaneedeni is restricted to South Africa [1,2]
Summary
The malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies. The first of these was an allele-specific polymerase chain reaction (AS-PCR) assay, which identified An. leesoni, An. vaneedeni, An. rivulorum and An. funestus s.s. by exploiting species-specific polymorphisms in the ribosomal DNA gene (rDNA) [11]. This method is unable to distinguish An. parensis from An. vaneedeni and, an improved AS-PCR protocol was developed to detect and discriminate all five species and this has rapidly become the 'gold standard' for identification of members of the species group [12]. Disadvantages of the current PCR approach include the requirement for post-PCR processing (gel electrophoresis of PCR products) and manual scoring of test samples which can be prone to error due to the similar amplicon sizes generated by certain species
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