Genotype mycobacterium cm test

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).

Mycobacterium avium species were identified and differentiated using a commercial GenoType Mycobacterium CM test (Hain Lifescience, Germany). The isolated DNA was selectively replicated by polymerase chain reaction (PCR). The resulting amplicons were transferred onto DNA strips covered with highly-specific probes which were complementary to the amplified DNA sequences. The amplicons were bound to their complementary sites, while the unbound fragments were removed during washing. Following this, a conjugate labeled with alkaline phosphatase was added, and the mycobacterial species were identified based on the hybridization pattern of specific probes placed on the strips with the product of the multiplex PCR reaction. The result was read using a dedicated template attached by the manufacturer.