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A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance (kdr) mutations in Anopheles gambiae from the Republic of the Congo.

Choi KS, Spillings BL, Coetzee M, Hunt RH, Koekemoer LL - Malar. J. (2010)

Bottom Line: Knockdown resistance (kdr) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa.Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid.Therefore, performing routine assay protocols blindly can result in the misinterpretation of results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vector Control Reference Unit, National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa.

ABSTRACT

Background: Knockdown resistance (kdr) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the kdr mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the kdr mutations in An. gambiae s.s. from the Republic of the Congo were compared with DNA sequence analysis.

Methods: A total of 52 pyrethroid and DDT resistant An. gambiae from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two kdr mutations (kdr-e and kdr-w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population.

Results: Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the kdr-e and kdr-w resistance mutations, seven specimens (13.5%) were homozygous for the kdr-e mutation, and 14 specimens (26.9%) were homozygous for the kdr-w mutation. A single individual was genotyped as heterozygous kdr-e mutation (1.9%) only and another as heterozygous kdr-w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous kdr-w, six specimens (11.5%) homozygous for the kdr-e mutation, one specimen (1.9%) heterozygous for the kdr-w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous kdr-e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis.

Conclusion: Both the kdr-e and kdr-w mutations were abundant in An. gambiae S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both kdr-e and kdr-w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of kdr is still held as the gold standard assay, this paper highlights the importance of kdr mutation confirmation via sequencing especially in regions where kdr frequency has never been reported before or where both the kdr-e and kdr-w mutations are present simultaneously.

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Scatter plot analysis of hydrolysis probe analysis fluorescence results for the kdr-e and kdr-w assays before and after adjustment of the allelic discrimination axes. A) The default allelic discrimination axes for the kdr-e mutation assay. B) After adjusting the allelic discrimination axes for the kdr-e mutation assay were shown. C) The default allelic discrimination axes for the kdr-w mutation assay. D) After adjusting the allelic discrimination axis for the FAM only.
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Figure 1: Scatter plot analysis of hydrolysis probe analysis fluorescence results for the kdr-e and kdr-w assays before and after adjustment of the allelic discrimination axes. A) The default allelic discrimination axes for the kdr-e mutation assay. B) After adjusting the allelic discrimination axes for the kdr-e mutation assay were shown. C) The default allelic discrimination axes for the kdr-w mutation assay. D) After adjusting the allelic discrimination axis for the FAM only.

Mentions: The first specific probe for the wild type allele was labelled with VIC and the second specific probe for the kdr-e or kdr-w alleles was labelled with FAM. It was essential to determine the standard values of fluorescence for scoring the genotypes in order to produce accurate results. The results from the hydrolysis probe analysis in this study were only consistent with the results from the DNA sequence when the allelic discrimination axes were adjusted (10% and 65% for the kdr-e allele susceptible and resistant probes with VIC and FAM respectively and 35% for the kdr-w resistant probe with FAM) from the original default axes as determined by the hydrolysis probe analysis programme were performed (Figure 1). No adjustment was needed for the wild type susceptible probe when it was run in the kdr-w reaction. One sample was identified as RR using kdr-w probe, while this specimen gave a failed reaction when using kdr-e analysis upon sequencing. This sample was shown to be homozygous for both kdr-e and kdr-w mutations. A second sample failed in kdr-w analysis, but was identified as RR using the kdr-e analysis. This specimen was also sequenced and shown as homozygous for both kdr-e and kdr-w. Therefore, 2/52 or 4% of samples resulted in discrepancies between the sequence and hydrolysis probe results. Adjustment of the allelic discrimination axes did not alter this result.


A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance (kdr) mutations in Anopheles gambiae from the Republic of the Congo.

Choi KS, Spillings BL, Coetzee M, Hunt RH, Koekemoer LL - Malar. J. (2010)

Scatter plot analysis of hydrolysis probe analysis fluorescence results for the kdr-e and kdr-w assays before and after adjustment of the allelic discrimination axes. A) The default allelic discrimination axes for the kdr-e mutation assay. B) After adjusting the allelic discrimination axes for the kdr-e mutation assay were shown. C) The default allelic discrimination axes for the kdr-w mutation assay. D) After adjusting the allelic discrimination axis for the FAM only.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959077&req=5

Figure 1: Scatter plot analysis of hydrolysis probe analysis fluorescence results for the kdr-e and kdr-w assays before and after adjustment of the allelic discrimination axes. A) The default allelic discrimination axes for the kdr-e mutation assay. B) After adjusting the allelic discrimination axes for the kdr-e mutation assay were shown. C) The default allelic discrimination axes for the kdr-w mutation assay. D) After adjusting the allelic discrimination axis for the FAM only.
Mentions: The first specific probe for the wild type allele was labelled with VIC and the second specific probe for the kdr-e or kdr-w alleles was labelled with FAM. It was essential to determine the standard values of fluorescence for scoring the genotypes in order to produce accurate results. The results from the hydrolysis probe analysis in this study were only consistent with the results from the DNA sequence when the allelic discrimination axes were adjusted (10% and 65% for the kdr-e allele susceptible and resistant probes with VIC and FAM respectively and 35% for the kdr-w resistant probe with FAM) from the original default axes as determined by the hydrolysis probe analysis programme were performed (Figure 1). No adjustment was needed for the wild type susceptible probe when it was run in the kdr-w reaction. One sample was identified as RR using kdr-w probe, while this specimen gave a failed reaction when using kdr-e analysis upon sequencing. This sample was shown to be homozygous for both kdr-e and kdr-w mutations. A second sample failed in kdr-w analysis, but was identified as RR using the kdr-e analysis. This specimen was also sequenced and shown as homozygous for both kdr-e and kdr-w. Therefore, 2/52 or 4% of samples resulted in discrepancies between the sequence and hydrolysis probe results. Adjustment of the allelic discrimination axes did not alter this result.

Bottom Line: Knockdown resistance (kdr) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa.Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid.Therefore, performing routine assay protocols blindly can result in the misinterpretation of results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vector Control Reference Unit, National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa.

ABSTRACT

Background: Knockdown resistance (kdr) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the kdr mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the kdr mutations in An. gambiae s.s. from the Republic of the Congo were compared with DNA sequence analysis.

Methods: A total of 52 pyrethroid and DDT resistant An. gambiae from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two kdr mutations (kdr-e and kdr-w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population.

Results: Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the kdr-e and kdr-w resistance mutations, seven specimens (13.5%) were homozygous for the kdr-e mutation, and 14 specimens (26.9%) were homozygous for the kdr-w mutation. A single individual was genotyped as heterozygous kdr-e mutation (1.9%) only and another as heterozygous kdr-w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous kdr-w, six specimens (11.5%) homozygous for the kdr-e mutation, one specimen (1.9%) heterozygous for the kdr-w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous kdr-e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis.

Conclusion: Both the kdr-e and kdr-w mutations were abundant in An. gambiae S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both kdr-e and kdr-w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of kdr is still held as the gold standard assay, this paper highlights the importance of kdr mutation confirmation via sequencing especially in regions where kdr frequency has never been reported before or where both the kdr-e and kdr-w mutations are present simultaneously.

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