title: Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum.
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.
authors.affiliation: Division of Epidemiology &Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi, 110077, India.
authors.forename: Madhvi
authors.initials: M
authors.lastname: Chahar
authors.affiliation: Division of Epidemiology &Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi, 110077, India.
authors.forename: Neelima
authors.initials: N
authors.lastname: Mishra
authors.affiliation: Division of Epidemiology &Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi, 110077, India.
authors.forename: Anup
authors.initials: A
authors.lastname: Anvikar
authors.affiliation: Division of Epidemiology &Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi, 110077, India.
authors.forename: Rajnikant
authors.initials: R
authors.lastname: Dixit
authors.affiliation: Division of Epidemiology &Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi, 110077, India.
authors.forename: Neena
authors.initials: N
authors.lastname: Valecha
mesh_terms: D000962:Antimalarials / Q000494:pharmacology / Q000627:therapeutic use; D002738:Chloroquine / Q000494:pharmacology / Q000627:therapeutic use; D017931:DNA Primers / Q000378:metabolism; D016054:DNA, Protozoan / Q000302:isolation & purification / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation; D015800:Protozoan Proteins / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D000962:Antimalarials; D017931:DNA Primers; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D002738:Chloroquine