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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Edwin Kamau, Saba Alemayehu, Karla C Feghali, LaDonna S Tolbert, Bernard Ogutu, Christian F Ockenhouse
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-23) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

EK conceived and designed the study, acquired, analysed and interpreted the data; wrote the first draft of the manuscript. SA performed the sequencing experiments, SNP assay for the clinical samples and reviewed manuscript. KF analysed sequences generated and reviewed manuscript. LT processes cultured samples, run some experiments and reviewed manuscript. BO was the principles investigator the Phase IIb pediatric clinical trial. CFO authorized, supported, contributed intellectually, and reviewed the manuscript.

Abstract

Background

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. 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.

Methods

TaqMan 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.

Results

Data 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.

Conclusion

TaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.
Zusatzmaterial
Authors’ original file for figure 1
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Literatur
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