Open AccessEstablishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum
Author(s) -
Madhvi Chahar,
Neelima Mishra,
Anup Anvikar,
Rajnikant Dixit,
Neena Valecha
Publication year - 2017
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep41119
Subject(s) - loop mediated isothermal amplification , plasmodium falciparum , chloroquine , multiplex , biology , detection limit , malaria , drug resistance , microbiology and biotechnology , virology , chemistry , genetics , immunology , chromatography , dna
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.