The loop-mediated isothermal amplification coupled with lateral flow dipstick (PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparumdhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities (10 to 87,634 parasites per microliter of blood). The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.
Ultrasensitive single-nucleotide polymorphism detection using target-recycled ligation, strand displacement and enzymatic amplification
