Genolyse isolation kit

DNA from colonies was isolated using the Genolyse isolation kit (Hain Lifescience, Nehren, Germany).
The strains were classified as non-tuberculosis mycobacteria species using the GenoType Mycobacterium CM test (Hain Lifescience) based on the DNA-Strip technology. Briefly, the DNA was extracted and then subjected to multiplex amplification with biotinylated primers. Following this, reverse hybridisation was conducted.
MAP was detected by real-time PCR using the VetMax M. paratuberculosis 2.0 Kit (Thermofisher Scientific, Waltham, MA, USA). The test targets the insertion sequence IS900, part of the IS1110 family of insertion sequences. It was repeated between 14 and 18 times in MAP genome.
All tests were performed according to the manufacturers’ manuals.

Mycobacterial isolation was conducted as described previously [44 (link)]. Briefly, the tissues (i.e., lymph nodes and other organs) were removed from the animal and decontaminated with 5% oxalic acid. After homogenization, the sediment was plated on Stonebrink and Petragnani media in triplicate and incubated at 37 °C for 12 weeks, and was checked every seven days. If colonies appeared on the media, the DNA was isolated with the GenoLyse Isolation Kit (Hain Lifescience, Nehren, Germany) and classified to MTBC by using the GenoType Mycobacterium CM Test (Hain Lifescience, Nehren, Germany).

The culture was performed according to the standard procedure used at the National Veterinary Research Institute (NVRI) in Pulawy (Poland). Briefly, the collected material was decontaminated with a 5% solution of oxalic acid (Sigma-Aldrich, Saint Louis, MO, USA). Homogenization was performed in a stomacher (MiniMix, Interscience, France) for three minutes (1500 g, 10 min). The supernatant was removed and the pellet was washed twice with sterile physiological sodium chloride solution (Polfa, Lublin, Poland).
The obtained pellet was seeded on two solid media: Stonebrink (Becton Dickinson, Franklin Lakes, NJ, USA) and Löwenstein–Jensen (Becton Dickinson, Franklin Lakes, NJ, USA). The media were incubated at 37 °C. Growth was assessed every seven days for 12 weeks.
The DNA was isolated from the cultures and the genotype determined as described previously [1 (link)]. Briefly, DNA isolation was performed using a Genolyse isolation kit (Hain Lifescience, Nehren, Germany). Strains were determined using the GenoType^®MTBCassay (Hain Lifescience, Nehren, Germany).