Tissue lysis buffer atl

In the case of bead-beating three different, commercially available bead mills (Fig. 1) were used such as (0.5 mm Glass) PowerBead Tubes; Qiagen, Hilden, Germany (L1.1, L1.2), LightCycler® SeptiFast Lysis kit; Roche Diagnostics, Risch-Rotkreuz Switzerland (L1.3, L1.4), and MagNa Lyser Green Beads; Roche Diagnostics (L1.5, L1.6). For grinding two sample agitators; (L1.1, L1.3, L1.5) a standard laboratory vortex (10 min at 1800 x rpm) and the MagNa Lyser Instrument (L1.2, L1.4, L1.6), Roche Applied Sciences; Penzberg, Germany (30 sec at 5000 x rpm) were used. In the case of bead-beating 800 µl PBS was added to the specimens. Performing chemical lysis a lysis mixture of 500 µl lysis buffer and 60 µl proteinase K was prepared. To perform chemical lysis, commercially available ATL - Tissue Lysis Buffer (Qiagen Hilden, Germany) and BLB - Bacterial Lysis Buffer (Roche Applied Sciences) lysis buffers were used according to manufacturer’s instructions (L2.1, L2.2, L2.5) or for overnight incubation at 56 °C (L2.3, L2.4).

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.

Analyses were performed at ZFMK and—for ZSM samples—at the Canadian Centre for DNA Barcoding (CCDB) in Guelph. Total genomic DNA was isolated from legs (ZSM) or nondestructively from complete specimens (ZFMK) to preserve undamaged vouchers. Entire specimens were incubated in 180µl Qiagen ATL tissue lysis buffer at 56°C for 16 hr by addition of 20µl Qiagen proteinase K. The following laboratory workflow was implemented at ZFMK: A Qiagen (Hilden, Germany) BioSprint96 magnetic bead extractor and corresponding kits were used, strictly following the manufacturer's specifications. We amplified 658 bp from the 5'‐end of the COI (cytochrome c oxidase subunit I) gene with primers HCO2198‐JJ and LCO1490‐JJ (Astrin & Stüben, 2008 (link)) or alternatively HCO2198‐JJ2 and LCO1490‐JJ2 (Astrin et al., 2016 (link)) using standard PCR conditions (see Astrin et al., 2016 (link)) in reaction volumes of 20 μl, including 2.0 μl of DNA template, and using the “Multiplex PCR Master Mix” (Qiagen). PCR products were subsequently sent for bidirectional Sanger sequencing to BGI (Hong Kong, China). CCDB laboratory protocols are available under https://ccdb.ca/resources/.