Proteinase k

mCherry10-expressing Mycobacterium tuberculosis (Mtb) was generated as described elsewhere [13 (link)]. Mtb H37Rv was obtained from American Type Culture Collection ((ATCC) 27294, Manassas, VA, USA). Strains were cultured as described in detail elsewhere [14 (link)]. In order to generate frozen stocks, mid-log-phase bacterial suspensions (OD₆₀₀, 0.2 to 0.4) were stored at −80°C. After four weeks of storage, the number of bacteria (colony forming units, CFU) was determined by plating serial tenfold dilutions on 7H10 plates (0.05% Tween 80 / 10% heat-inactivated bovine serum in phosphate buffered saline (PBS)) and counting colonies after 3–4 weeks of incubation at 37°C.
For spiking experiments, frozen stocks of Mtb H37Rv were thawed, centrifuged (4000xg), bacteria re-suspended in PBS and a defined amount of this solution added to buffer or saliva samples. To address the impact of proteinase K plus DTT treatment on bacterial viability, Mtb H37Rv spiked PBS samples were incubated in the presence or absence of proteinase K (18U/ml, Carl Roth, Karlsruhe, Germany) and Dithiothreitol (DTT, 2mM, Sigma-Aldrich, St. Louis, USA) for 60 minutes at 37°C and subsequently subjected to CFU analysis as described.

Direct DNA extraction from 5 g of muscle tissue (DE) was performed by using a slightly modified protocol published by the European Reference Laboratory for Parasites (Instituto Superiore di Sanità (ISS), Rome, MI-12) [36 ]. After thawing, pure muscle samples (5 g, Section 2.1) were vortexed at maximum speed with 2.5 g (±0.3 g) of sterile glass beads (Ø 4 mm) and 10 mL of lysis buffer, including 40 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS and 0.4 mg/mL proteinase K (30 mAnson-U/mg; Carl Roth, Karlsruhe, Germany), with proteinase K added separately after vortexing. After incubation at 56 °C (±3 °C) for 16–18 h in a hybridization oven under rotation, DNA extraction of 200 µL of the resulting lysate was carried out by using the “tissue protocol” of the QIAamp DNA Minikit (QIAGEN, Hilden, Germany) starting with the addition 200 µL of AL buffer. A total of 100 μL of 56 °C warm molecular biology grade water was used in two consecutive steps for elution of DNA by incubation for 5 min, followed by centrifugation at 6.000× g for one minute. DNA was stored at 4 °C. In each analysis, 5 g of minced pork spiked with 10⁶ in vitro cultivated tachyzoites (T. gondii strain RH) or water were used as positive and negative extraction control, respectively.

Tobacco leaves transiently expressing DMP1-eGFP, eGFP-DMP1 or eGFP were homogenized in extraction buffer without detergent. The resulting microsomes were pelleted by ultracentrifugation and resuspended in buffer without detergent, followed by Proteinase K (Carl Roth, Karlsruhe, Germany) treatment using 0.1 mg/ml Proteinase K for 15 min at 37°C. Detection by Western blotting was performed using anti-GFP and anti-DMP1 antibodies.