การตรวจหาการกลายพันธุ์ที่ตำแหน่ง C580Y ของยีน Kelch13 Propeller ที่มีความสัมพันธ์กับการดื้อยาอาร์ทิมิซินินในเชื้อ Plasmodium falciparum โดยวิธี Loop - Mediated Isothermal Amplification

Abstract

Plasmodium falciparum resistance to artemisinin based combination therapies in Southeast Asia become a major obstacle for malaria control and elimination efforts. This could be more critical if it spreading to Africa, where plenty of people are infected with malaria. The important factor is limit the spreading of drug resistance malaria. The artemisinin resistance is related to the mutations of the Pfkelch13 gene, especially at the C580Y position. This point mutation is the most widespread in Southeast Asia, including Thailand. Accordingly, the employment of effective molecular biology techniques is one method that can be used for surveillance and monitoring of drug resistance. This study therefore developed SNP-loop-mediated isothermal amplification (SNP-LAMP) to detect C580Y mutation. A set of primers specific for Pfkelch13 C580Y gene were designed and the LAMP reaction conditions and concentration of reagent were optimized by using a plasmid containing the Pfkelch13 C580Y gene as positive control. It was found that the best LAMP reaction conditions were at 56°C 45 minutes and a concentration of 5 mM MgSO4, 0.8 mM dNTPs, 8 U Bst DNA polymerase and 1 M betaine. A positive reaction is designated when the color of hydroxynapthol blue (HNB) change from purple to sky blue when view with naked eye. The products confirmed by gel electrophoresis showed ladder like pattern bands. The LAMP primers were specifically distinguished only the C580Y mutation. No cross reactivity was observed with other Plasmodium spp., human DNA or Pfkelch13 wild type and Pfkelch13 P574L gene. LAMP reaction was able to detect as low as 50 copies of plasmid per reaction, The sensitivity is 10-fold higher than conventional PCR method. A total of 120 dried blood spots from patients were analyzed. The LAMP assay successfully differentiate positive samples containing Pfkelch13 C580Y gene from wild type. The detection limit of LAMP assay is 335 parasites/μl. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) showed all 100% along with good repeatability and reproducibility to detect C580Y mutation. Therefore, this technique has the advantages of rapidity, simplicity, sensitivity, specificity and could be used as a clinical diagnosis tool in field testing as well as surveillance of artemisinin resistance.