Mytaq mix

Nested PCR (nPCR) was performed as described previously [13 (link)] in a two-step procedure. In the first PCR round, amplification was performed using rPLU1 and rPLU5 primers for Plasmodium genus determination. The PCR mixture was prepared in a total volume of 20 μl, containing 10 μl of MyTaq™ mix (Bioline, UK), 0.4 μM of each primer and 2.5 μl of extracted DNA. PCR was performed under the following conditions: 94 °C for 5 min as the initial denaturation step; 25 cycles at 94 °C for 45 s, 58 °C for 45 s and 72 °C for 1 min; and a final extension step at 72 °C for 5 min. The amplified PCR product (1 μl) was used as a template for the second PCR round for Plasmodium species identification using (rFAL1 and rFAL2, rOVA-1 and rOVA2, rVIV1 and rVIV2, rMAL1 and rMAL2) primers [13 (link)]. The reaction mix contained 10 μl of MyTaq™ mix (Bioline, UK) and 0.4 μM of each primer, and the final reaction volume was made up to 20 μl by adding double distilled water. Amplification was performed under the following conditions: 95 °C for 5 min; 30 cycles of 94 °C for 1 min, 58 °C for 2 min and 72 °C for 5 min; and final extension at 72 °C for 2 min. A known Plasmodium positive samples and a negative control sample without DNA template was used in all the reactions as positive and negative control respectively.

AMOS-PCR was done as described previously [25 (link), 26 (link)]. The PCR mixture contained 1X MyTaq mix (Bioline), a combination of five primer sets specific for B. abortus, B. melitensis, B. ovis, B. suis (0.2 μM) and IS711 (1 μM), respectively, and 10 ng DNA per 25 μl reaction. The PCR conditions consisted of an initial denaturation at 95°C for three minutes followed by 35 cycles of 95°C for one minute, 55.5°C for two minutes and 72°C for two minutes.
Bruce-ladder PCR was also done as described previously [27 (link)]. PCR reactions (25 μl) composed of 1X MyTaq mix (Bioline), 0.4μM of each primer of the eight primer pairs and 10ng template DNA. PCR conditions consisted of initial denaturation at 95°C for three minutes, followed by 25 cycles at 95°C for 30 sec, 64°C for 45 sec and 72°C for three minutes and a final extension of 72°C for five minutes on an ABI 2720 Thermal Cycler (Applied Biosystems).
To confirm the identity of strains identified as B. suis and B. canis with Bruce-ladder, the previously described Suis-ladder multiplex PCR assay [46 (link)] was used.
PCR products were separated by gel electrophoresis on a 1.5% agarose gel subsequently stained with ethidium bromide and photographed under UV light.

Two pairs of primer were used to amplify the regions of interest. One pair recognized a sequence in which CpG sites were methylated (unmodified by bisulfite treatment).
Other pair recognized a sequence in which CpG sites were unmethylated (modified to UpG treatment). The primer sequences and X °C for each annealing temperatures were noted in Table 1.
MSP assay was carried out in a total of 15 μl containing 3 μl bisulfite-modified template DNA, 0.75 unit iTaq DNA polymerase (Biorad), 0.5 μM each primer, 7.5 μl MyTaq^TM Mix (Bioline). Thermal cycling was initiated at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 sec, annealing at the X °C for 30 sec, extension at 72 °C for 30 sec, and a final extension at 72 °C for 10 min (Note: X °C was the specific annealing temperature for each specific methylated or unmethylated primer). The PCR products were run on 2% agarose gel with visualized by ethidium bromide staining. Then, MSP products were sequenced to confirm the specificity of primers, examine the efficiency of bisulfite modification and the hypermethylation status of target gene.