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. 2013 Jun 12:6:173.
doi: 10.1186/1756-3305-6-173.

Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay)

Kwang Shik Choi  1 , Maureen Coetzee, Lizette L Koekemoer
Affiliations

Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay)

Kwang Shik Choi et al. Parasit Vectors. .

Abstract

Background: Recent studies presented two clades (clades I and II) within the major malaria vector, Anopheles funestus s.s. on the mitochondrial DNA. We describe a hydrolysis probe analysis (TaqMan assay) method for the rapid identification of these two clades.

Findings: A total of 53 An. funestus s.s. from Malawi and Mozambique were tested for detection of clade types using the hydrolysis probe analysis. Results were compared to DNA sequence analysis to verify the accuracy of the probes TaqMan assay for this vector species. Analysis using the hydrolysis probe revealed that there were 21 individuals from Malawi and 13 individuals from Mozambique for clade I, and 19 individuals from Mozambique for clade II. The results were consistent with the results of DNA sequences. A field sample from northern Zambia revealed the presence of both clade types.

Conclusion: A diagnostic method using the hydrolysis probe analysis was developed to identify clade types within An. funestus s.s. This assay will be useful for screening clade types of field-collected An. funestus specimens accurately and efficiently in malaria vector research and control studies.

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Figures

Figure 1
Figure 1
Alignment from 3′ to 5′ end of the COI target region for probes. The alignment of An. funestus for the comparison were originated from GenBank (access No. AY423059). Mal and Moz represent Malawi and Mozambique respectively. Dots represent identity with respect to the An. funestus sequences. AG, CT and AT indicate polymorphic positions. The square indicates the sequences of the probes for clades I and II.
Figure 2
Figure 2
Hydrolysis probe analysis fluorescence results for clades I and II of An. funestus s.s. from Malawi and Mozambique. Scatter plot analysis (a) of A) clade I; B) clade II; C) negative control and quantitative peaks (b). Blue peaks are clade I and yellow peaks are clade II.
Figure 3
Figure 3
Hydrolysis probe analysis fluorescence results for clades I and II of An. funestus s.s. from Zambia. Scatter plot analysis (a) and quantitative peaks (b) of A) clade I; B) clade II; C) negative control. Blue peaks are clade I and yellow peaks are clade II. Positive controls (arrows) from the Figure 2 experiment are D) clade I and E) clade II.
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