Abstract
BackgroundAnti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods.MethodsTaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD) for each assay.ResultsData from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples.ConclusionTaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.
Highlights
Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria
single nucleotide polymorphism (SNP) assays performance To analyse the performance of the selected SNP assays, three P. falciparum strains, 3D7, 7G8 and K1 that differ
Data from genetic profiles of the P. falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the SNP assays were compared to
Summary
Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Advances that have been made far will only be sustained if monitoring and reporting of anti-malarial drug resistance becomes an in particular has positioned analysis and monitoring of genetic markers as an important alternative method for detection of drug resistance [7,8]. Plasmodium falciparum manifests its genetic diversity in several forms: single nucleotide polymorphisms (SNPs), microsatellite repeats, small insertions or deletions (indels) and gene duplication. Studies identifying SNPs and gene duplications associated with anti-malarial drug resistance have been exploited in evaluation of resistance to treatment [9]. Chloroquine resistance is attributed to SNPs in chloroquine resistance transporter (crt) and/or multidrug resistance gene 1 (mdr1) genes [15,16,17,18]
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