PCR primers, master mix, and condition of assay.

Table footnotes: DNA extraction: DNA was extracted from the dried blood spots on the filter papers with a Maxwell RSC Instrument (Promega, United States of America) in accordance with the manufacturer’s instructions with minor modification. Three punched-out circles of 3.175-mm (1/8-inch) diameter from the dried blood spot on the filter paper were used for DNA extraction, which was equivalent to 15–20 μL of whole blood. The punched-out filter paper circles were incubated in 30 μL of proteinase-K and 180 μL of incubation buffer from the kit at 70 °C for 90 minutes and then followed with the extraction instructions. The extracted DNA was eluted with 50 μL of elution buffer and preserved until use at −30 °C. PCR: For identification of P. knowlesi, partial cytb and partial msp1 of P. knowlesi were amplified by nested PCR. For primary PCR of the cytb, real-time PCR was performed using a primer set of PCBF and PCBR. For secondary PCR of the cytb, conventional PCR was performed using a primer set of PKCBF and PKCBR. For primary PCR of the msp1, real-time PCR was performed using a primer set of Pk_MSP1_F3 and Pk_MSP1_R2. For secondary PCR of the msp1, conventional PCR was performed using a primer set of Pk_MSP1_F2 and Pk_MSP1_R2. For identification of P. falciparum, real-time PCR was performed using a primer set of Pf F1 and Pf R1. For identification of P. vivax, real-time PCR was performed using a primer set of PvF11 and PvR7. For identification of P. ovale, real-time PCR was performed using a primer set of POCBF and POCB_R1. For identification of P. malariae, real-time PCR was performed using a primer set of PMCBF and PMCBR. Abbreviations: cytb, cytochrome b gene; msp1, merozoite surface protein-1 gene. (XLSX)