Real-Time LAMP: a reliable tool for the surveillance of SNPs in chloroquine resistance Pfcrt gene

Resistance to chloroquine in Plasmodium falciparum is very common in India as compared to Plasmodium vivax . Mutation at K76T in Pfcrt gene is the major marker for chloroquine resistance in Plasmodium falciparum. Although various traditional methods are available for detecting these markers but they have certain limitations while their use in field settings due to requirement of trained personal, well-equipped labs and are time consuming. To overcome these challenges, we have designed Loop Mediated Isothermal Amplification Assay (LAMP) which is emerging as new widely used tool in field settings. This study optimized LAMP for the surveillance of SNPs in Pfcrt gene. LAMP primers were designed for K76T mutants and conditions for Real-Time LAMP assay were optimized. Clinical sensitivity and specificity for the assay were 100% and 80% respectively. Limit of detection was 1copy/µl and no cross-reactivity was observed with other parasitic strains. Study proved LAMP a reliable tool that can be used in field settings for the surveillance of markers of resistance against drugs which are currently in use and which have been withdrawn. These surveillance studies would help in providing data for resurgence of sensitivity for any drug that has been withdrawn and resistance for the drugs that are currently in use, thus ultimately taking part in modifying drug policies by the government accordingly.