Genotyping Plasmodium vivax Samples

Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Kit (QIAGEN, Valencia, CA, USA). Samples were genotyped using a set of 15 previously reported microsatellite markers [37 (link), 38 (link)]. Five microsatellites were used to genotype 27 samples in a test for accuracy (MS4, MS6, MS9, MS15 and MS20), and all microsatellites were used to measure genetic diversity using 271 P. vivax samples (primers are listed in Additional file 1). Five microliters of template DNA was used for PCR in a 50 μl total volume for each individual reaction. The PCR product was then separated on a QIAxcel Advanced System (QIAGEN). The QIAxcel Advanced System from Qiagen is commercially presented as a novel means to replace gel analysis of DNA, RNA, and proteins. It consists of 12 independent capillaries placed in a cartridge. Each cartridge allows processing about 100 samples per capillary and 96 samples per PCR plate. This system is marketed to have a resolution of 3–5 bp. The PCR product sizes were estimated using the QIAxcel ScreenGel Software.