Opisthorchis-like eggs collected from PBS ethyl acetate sediments were used for DNA extraction. A 200 μl aliquot of each positive sample was treated with 1.4 mL ATL tissue lysis buffer (Qiagen), mixed continuously for 1 min or until the stool samples were thoroughly homogenized. The suspension was subjected to PBS incubation technique [28 (link)] and 5 cycles of freezing in liquid nitrogen followed by thawing at 98°C to 100°C [24 (link)] to break up the eggs. Subsequently, the suspension was heated at 70°C for 5 min before continuously mixing and centrifuging at 20,000 g for 1 min to sediment fecal pellets. Then 1.2 mL of supernatant was transferred into a new 2 mL microcentrifuge tube. The DNA was extracted from the supernatant using the QIAmp DNA stool mini kit (Qiagen) according to the manufacturer’s protocol. At the final step, DNA was eluted with 50 μl of elution buffer. At the final step, DNA was eluted with 50 μl of elution buffer. DNA extraction of the positive control (O. viverrini eggs) was also performed and doubled distilled water was used as negative control. Other negative fecal samples using wet smears, Kato thick smear and PBS ethyl acetate concentration technique were not used for the PCR assay.
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