Abstract
Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria.
Highlights
Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene
We developed a field based loop mediated isothermal amplification (LAMP) method for the detection of CQ resistance in P. falciparum caused by K76T point mutation of the P. falciparum chloroquine resistance transporter (Pfcrt) gene as a molecular marker
LAMP primers were designed for Pfcrt gene encoding K76T point mutation to detect CQ resistance, five primers set (Supplementary Table 1) was designed by using the software Primer explorer V4 program
Summary
Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. Other molecular methods are valuable for anti-malarial drug resistance detection but are, costlier, time-consuming and require highly sophisticated laboratory facilities with trained personnel including DNA sequencing[7] Since these methods cannot cope with the current demand of field based assays; there is an urgent need to evaluate a new simple, easy to use, rapid, less time consuming and cost effective field based method for detecting anti-malarial drug resistance. In comparison to other available isothermal amplification methods, LAMP is easy to use as one step method with high sensitivity and specificity[11] This method employs strand displacement synthesis primed by specially designed five set of primers, two outer (F3, B3) and two inner
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
