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)表注：DNA提取：采用Maxwell RSC全自动核酸提取仪（Maxwell RSC Instrument，Promega，美国），严格遵循厂商说明书并进行小幅优化操作。从滤纸片干血斑上获取3个直径为3.175mm（1/8英寸）的打孔圆片用于DNA提取，该样本量相当于15~20μL全血。将截取的滤纸片圆片置于30μL蛋白酶K与180μL试剂盒配套孵育缓冲液中，于70℃孵育90分钟，随后按照试剂盒提取说明书完成后续操作。提取得到的DNA用50μL洗脱缓冲液进行洗脱，于-30℃低温保存待用。 PCR扩增：诺氏疟原虫（Plasmodium knowlesi）鉴定：通过巢式PCR扩增诺氏疟原虫的部分细胞色素b基因（cytochrome b, cytb）与部分裂殖子表面蛋白1基因（merozoite surface protein-1, msp1）。其中细胞色素b基因的第一轮PCR采用实时荧光定量PCR，引物对为PCBF与PCBR；第二轮PCR采用常规PCR，引物对为PKCBF与PKCBR。裂殖子表面蛋白1基因的第一轮PCR采用实时荧光定量PCR，引物对为Pk_MSP1_F3与Pk_MSP1_R2；第二轮PCR采用常规PCR，引物对为Pk_MSP1_F2与Pk_MSP1_R2。 恶性疟原虫（Plasmodium falciparum）鉴定：采用实时荧光定量PCR，引物对为Pf F1与Pf R1。 间日疟原虫（Plasmodium vivax）鉴定：采用实时荧光定量PCR，引物对为PvF11与PvR7。 卵形疟原虫（Plasmodium ovale）鉴定：采用实时荧光定量PCR，引物对为POCBF与POCB_R1。 三日疟原虫（Plasmodium malariae）鉴定：采用实时荧光定量PCR，引物对为PMCBF与PMCBR。 缩写说明：cytb为细胞色素b基因；msp1为裂殖子表面蛋白1基因。（XLSX）