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A LAMP-SNP Assay Detecting C580Y Mutation in Pfkelch13 Gene from Clinically Dried Blood Spot Samples

Korean Journal of Parasitology 2021년 59권 1호 p.15 ~ 22
Khammanee Thunchanok, Sawangjaroen Nongyao, Buncherd Hansuk, Tun Aung Win, Thanapongpichat Supinya,
소속 상세정보
 ( Khammanee Thunchanok ) - Prince of Songkla University Faculty of Science Department of Microbiology
 ( Sawangjaroen Nongyao ) - Prince of Songkla University Faculty of Science Department of Microbiology
 ( Buncherd Hansuk ) - Prince of Songkla University Faculty of Medical Technology
 ( Tun Aung Win ) - Mahidol University Faculty of Graduate Studies
 ( Thanapongpichat Supinya ) - Prince of Songkla University Faculty of Medical Technology

Abstract


Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3’ end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.

키워드

Plasmodium falciparum; Pfkelch13 gene; artemisinin resistance; C580Y mutation; LAMP-SNP assay

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