Fig 2: DNA gel electrophoresis of the qPCR products showing that the primers were specific in amplification. Panel a: L) DNA ladder; 1) GAL3ST2 (177 bp); 2) CYP7A1 (171 bp); 3) CALB1 (116 bp); 4) TYRO3 (186 bp); 5) CNG4 (243 bp); 6) POMC (115 bp); 7) RHOBTB3 (171 bp); 8) KCNH1 (160 bp); 9) MMP13 (128 bp); 10) KLB (224 bp); 11) GATA3 (157 bp); 12) SPP1 (134 bp). Panel b: L) DNA ladder; 1) YWHAZ (61 bp); 2) TBP (147 bp); 3) IBV T-as positive control (181 bp); 4) CA9 (80 bp); 5) OTOP2 (202 bp); 6) CGA (166 bp); 7) CLDN16 (186 bp); 8) GJA8 (186 bp); 9) SS2 (160 bp); 10) BPIFB3 (234 bp); 11) PPARGCIB-Positive control (82 bp); 12) TLR3-Positive control (203 bp). The upper (purple) and lower markers (green) act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The DNA gel in Agilent 2100 Bioanalyzer was performed as per the manufacturer’s instructions of Agilent DNA 1000 Kit. The size of the individual amplicons are consistent with the expected size

Fig 3: PCR-RFLP of the pfdhfr gene, amplified region of M3-F/primers.BsrI cleaved the 522 bp fragment into 190 and 332 bp indication of 108asn mutation (A). DraI detect 164leu mutations producing 28, 107, 171 and 245 bp fragments for wild type and 107, 143 and 245 bp for mutant (B). Digestion with NlaIII produce 53, 93 and 376 bp for wild type and 146 and 245 bp for mutant at codon 16 (C) and digestion with Tsp5091 yielded 55, 65, 120, and 153 bp fragments for wild type and 55, 65, 120 and 218 bp fragments for mutant for detection on polymorphism at codon 51 (D). Lane L: DNA ladder of Agilent DNA 1000 Kit (Agilent Technologies, Molecular Probes Inc, USA), The controls are in Lane 1 and 2: A: Lane 1: Plasmodium falciparum KI strain (mutant); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: field samples from Kalabakan; well 10: PCR negative control (no DNA was added to the PCR reaction). B and C: Lane 1: Plasmodium falciparum KI strain (wild type); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: field samples from Kalabakan; well 10: PCR negative control (no DNA was added to the PCR reaction). D: Lane 1: Plasmodium falciparum KI strain (wild type); Lane 2: P. falciparum W2 strain (mutant) Lane 3–9: field samples from Kalabakan; well 10: PCR negative control (no DNA was added to the PCR reaction).

Fig 4: PCR-RFLP of the pfdhfr gene, amplified region of M4-F primers. The 326 bp fragment was cut by Alu I into 180 and 118 bp fragments, indications for wild type ser108 and 299 bp for mutant (A). BstNI digested only mutant allele into 145 and 181 bp indicates mutation 108thr. All the tested samples showed wild type (B). Reaction with XmnI produced fragment 163 and 189 bp for wild type; and 26, 137 and 163 bps for 59arg mutation (C). Lane L: DNA ladder of Agilent DNA 1000 Kit (Agilent Technologies, Molecular Probes Inc, USA), The controls are in Lane 1 and 2: A: Lane 1: Plasmodium falciparum KI strain (mutant); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: samples from Kalabakan; Lane 10: PCR negative control (no DNA was added to the PCR reaction). B: Lane 1: Plasmodium falciparum KI strain (wild type); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: samples from Kalabakan; Lane 10: PCR negative control (no DNA was added to the PCR reaction). C: Lane 1: Plasmodium falciparum KI strain (mutant); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: samples from Kalabakan; Lane 10: PCR negative control (no DNA was added to the PCR reaction).

Fig 5: Amplification of the gene fragments from the eggshell gland tissue of laying hens to assess the specificities of the primers used in the current study.L, DNA ladder; 1. 18S rRNA (63 bp); 2. ACTB (139 bp); 3. ALB (197 bp); 4. B2M (194 bp); 5. CA2 (99 bp); 6. CST3 (130 bp); 7. GAPDH (113 bp); 8. HMBS (131 bp); 9. HPRT1 (245 bp); 10. RPL4 (235); 11. SLC25A38 (139 bp); 12. FECH (112 bp). All the amplified products were in accordance to the expected sizes. The amplified products were run on Agilent 2100 Bioanalyzer using Agilent DNA 1000 Kit as per the manufacturer’s instructions. The upper (purple) and lower (green) markers act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The standard curve (plotting migration time against DNA amplicon size), in conjunction with the markers, is then used to calculate DNA fragment sizes for each well from the migration times measured (for more detail see Agilent 2100 Bioanalyzer Users Guide for Molecular Assays).