Molecular analysis

All day-0 samples were subjected to analysis for distribution and spread of chloroquine and antifolate drug resistance markers. Filter papers were prepared and extracted for parasite genomic DNA using QIAamp DNA Blood Mini Kit (QIAGEN) according to manufacturer’s instruction. The target genes were amplified by using the specific pair of primers (Table 1). In this study, chloroquine resistance marker, ‘AAG’ insert in pvcrt-O; multi-drug resistance marker, mutations in pvmdr1; antifolate resistance makers, mutations in pvdhps and pvdhfr were amplified and analysed.

Pairs of primers used to amplify the target genes

PCR reactions were performed in a reaction mixture that contained 0.25 mM of each dNTP, 10 mM Tris–HCl (pH 9.0), 30 mM KCl, 1.5 mM MgCl₂, 1.0 units of Taq polymerase (Accupower^© premix, Bioneer, Seoul, Korea), 0.02 µM primers, and 2 µL of genomic DNA.

For target gene amplifications, initial denaturation at 95 °C for 10 min was followed by 35 cycles of 95 °C for 30 s, 58 °C (pvdhps) or 60 °C (pvcrt-O) or 62 °C (pvdhps and pvdhfr) for 45 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. Amplified products were checked by 1% agarose gel electrophoresis stained with Red safe^© (iNtRON, Seongnam, Republic of Korea). The PCR clean-up was proceeded by MEGAquick-spin DNA fragment purification Kit (iNtRON, Republic of Korea) and sequencing. The nucleotide and amino acid sequences were aligned and analysed by Lasergene^® software (DNASTAR, Madison, WI, USA) using the reference strain of Sal-1 retrieved from Plasmodium data base [5]. The nucleotide sequences were submitted to GenBank under accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"KX000945-KX000959","start_term":"KX000945","end_term":"KX000959","start_term_id":"1189015807","end_term_id":"1189015835"}}KX000945-KX000959.