Use of peptide nucleic acid fluorescent in situ hybridization technique in the detection of Mycobacterium avium paratuberculosis in fecal samples from dairy herds.

Novel peptide nucleic acid (PNA) probes were designed for direct detection of Mycobacterium avium paratuberculosis (MAP) by the Fluorescent in situ Hybridization technique from fecal samples. For this study, fecal samples (n = 218) were collected from cattle (n = 180) and buffaloes (n = 38) with a history or incidence of cases of chronic intermittent diarrhoea from organized dairy farms in the state of Punjab. Fluorescent in situ Hybridization was done on highly positive (acid-fast positive) fecal samples (n = 54) using a newly designed MAP probe. The designed probe was standardized for the standard culture of Mycobacterium avium subsp. paratuberculosis (ATCC K10) at hybridization conditions of 55 °C and 30% formamide concentration. The probe showed no cross-reactivity with the other Mycobacterial species, viz., M. intracellulare, M. smegmatis, M. fortuitum, and M. vaccae, except Mycobacterium kansasii. However, M. kansasii and Mycobacterium avium subsp. paratuberculosis were clearly differentiated by the probe morphologically. Out of 54 fecal samples, 7 samples tested positive by PNA- FISH which were also tested positive by conventional PCR using the IS900 gene, while the remaining fecal samples were negative by PCR.