Disclosed are compositions, assays, methods, diagnostic methods, kits and diagnostic kits for the specific and differential detection of a non-Mycobacterium avium subsp. paratuberculosis (non-MAP) organism, wherein a non-MAP organism is a Mycobacterium avium complex (MAC) organism that does not belong to the Mycobacterium avium subsp. paratuberculosis (MAP) organism, from samples including veterinary samples, clinical samples, food samples, forensic sample, an environmental sample (e.g., soil, dirt, garbage, sewage, air, or water), including food processing and manufacturing surfaces, or a biological sample. Exemplary non-MAP organisms including M. avium subsp. avium (MAA), M. avium subsp. hominissuis (MAH), and M. avium subsp. silvaticum (MAS) can be detected by the present compositions, kits and methods.