ABSTRACT
The significant strides made in reducing global malaria burden over the past decades are being threatened by the emergence of multi-drug resistant malaria. Mechanisms of resistance to several classes of antimalarial drugs have been linked to key mutations in the Plasmodium falciparum genes. Pyrimethamine targets the dihydrofolate reductase of the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS), and specific point mutations in the dhfr-ts gene have been assigned to resistant phenotypes. Several molecular methods are available to detect the mutant genotypes including DNA sequencing and PCR-based methods. In this study, we report the development of PfSNP-LAMP to detect nucleotide polymorphism in the dhfr gene associated with N51I mutation and antifolate resistance. The PfSNP-LAMP method was validated with genomic DNA samples and parasite lysates prepared from sensitive and pyrimethamine resistant strains of P. falciparum.
Subject(s)
Mutation , Nucleic Acid Amplification Techniques , Plasmodium falciparum/genetics , Polymorphism, Single Nucleotide , Tetrahydrofolate Dehydrogenase/genetics , DNA Primers , DNA, Protozoan/genetics , Drug Resistance/genetics , Folic Acid , Genome, Protozoan , Genotype , Malaria, Falciparum/diagnosis , Plasmodium falciparum/drug effects , Pyrimethamine/pharmacology , Sequence Analysis, DNA , Specimen Handling , Thymidylate Synthase/geneticsABSTRACT
Malaria is largely a preventable and curable disease. However, a delay or an inappropriate treatment can result in serious adverse outcomes for patient. Rapid, simple and cost-effective diagnostic tests that can be easily adapted and rapidly scaled-up at the field or community levels are needed. In this study, accelerated detection methods for the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) dihydrofolate reductase-thymidylate synthase were developed based on the loop-mediated isothermal amplification (LAMP) method. The developed methods included the use of species-specific biotinylated primers to amplify LAMP amplicons, which were then hybridized to specific FITC-labeled DNA probes and visualized on a chromatographic lateral flow dipstick (LFD). The total LAMP-LFD assay time was approximately 1.5h. The LAMP-LFD assays showed similar detection limit to conventional PCR assay when performed on plasmid DNA carrying the malaria dhfr-ts genes. The LAMP-LFD showed 10 folds higher detection limit than PCR when performed on genomic DNA samples from Pf and Pv parasites. The dhfr-ts LAMP-LFD assays also have the advantages of reduced assay time and easy format for interpretation of results.
Subject(s)
Malaria, Falciparum/parasitology , Malaria, Vivax/parasitology , Nucleic Acid Amplification Techniques/methods , Plasmodium falciparum/isolation & purification , Plasmodium vivax/isolation & purification , DNA Primers/genetics , DNA, Protozoan/genetics , Humans , Multienzyme Complexes/genetics , Plasmodium falciparum/genetics , Plasmodium vivax/genetics , Polymerase Chain Reaction/methods , Protozoan Proteins/genetics , Sensitivity and Specificity , Tetrahydrofolate Dehydrogenase/genetics , Thymidylate Synthase/geneticsABSTRACT
BACKGROUND: In recent years, Thai indigenous chickens have increasingly been bred as an alternative in Thailand poultry market. Due to their popularity, there is a clear need to improve the underlying quality and productivity of these chickens. Studying chicken genetic variation can improve the chicken meat quality as well as conserving rare chicken species. To begin with, a minimal set of molecular markers that can characterize the Thai indigenous chicken breeds is required. RESULTS: Using AFLP-PCR, 30 single nucleotide polymorphisms (SNPs) from Thai indigenous chickens were obtained by DNA sequencing. From these SNPs, we genotyped 465 chickens from 7 chicken breeds, comprising four Thai indigenous chicken breeds--Pradhuhangdum (PD), Luenghangkhao (LK), Dang (DA) and Chee (CH), one wild chicken--the red jungle fowls (RJF), and two commercial chicken breeds--the brown egg layer (BL) and commercial broiler (CB). The chicken genotypes reveal unique genetic structures of the four Thai indigenous chicken breeds. The average expected heterozygosities of PD=0.341, LK=0.357, DA=0.349 and CH=0.373, while the references RJF= 0.327, CB=0.324 and BL= 0.285. The F(ST) values among Thai indigenous chicken breeds vary from 0.051 to 0.096. The F(ST) values between the pairs of Thai indigenous chickens and RJF vary from 0.083 to 0.105 and the FST values between the Thai indigenous chickens and the two commercial chicken breeds vary from 0.116 to 0.221. A neighbour-joining tree of all individual chickens showed that the Thai indigenous chickens were clustered into four groups which were closely related to the wild RJF but far from the commercial breeds. Such commercial breeds were split into two closely groups. Using genetic admixture analysis, we observed that the Thai indigenous chicken breeds are likely to share common ancestors with the RJF, while both commercial chicken breeds share the same admixture pattern. CONCLUSION: These results indicated that the Thai indigenous chicken breeds may descend from the same ancestors. These indigenous chicken breeds were more closely related to red jungle fowls than those of the commercial breeds. These findings showed that the proposed SNP panel can effectively be used to characterize the four Thai indigenous chickens.
