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Molecular Diagnosis of Rifampicin Antibiotic Resistance in Mycobacterium Avium Subspecies Paratuberculosis Isolated from Sheep Clinical Samples
Abstract
The present study was conducted to identify parts of the rpoB gene in MAP of Indian sheep isolates. The rpoB gene (Beta sub unit of RNA polymerase) is involved in conferring the rifampicin antibiotic resistance in MAP bacteria. The resistance is due to the presence of mutations in the 81 bp variable region of the rpoB gene including C1367T and T1442C mutations identified by several workers. The study was further extended to diagnose the prevalence rate of rifampicin antibiotic resistance in Indian MAP isolates using RFLP-PCR test. A total of ten MAP isolates (Six sheep post-mortem tissues and four fecal samples) were diagnosed by acid fast staining procedure. After confirmation of the positive samples, these were also diagnosed by the presence of the Insertion sequence (IS900 gene) using IS900 gene specific primers. Further, new primers were designed to amplify and characterize the portion of rpoB gene from India isolates. All ten samples were found for the presence of the IS900 gene which confirmed the presence of MAP infection in these clinical samples. Further, rpoB gene specific primers were used to diagnose the rpoB mutations at C1367T and T1442C in the gene. Out of ten, nine samples could amplify the product by using rpoB gene specific primers. Out of which two were heterozygous resistant (600 bp, 379 bp, and 221 bp band) and seven were homozygous susceptible (uncut 600 bp band) after KasI digestion. The products of rpoB gene were further sequenced from representative isolates. The results confirmed the presence of the similar sequence of the MAP of elsewhere sequence, however none of the mutation was detected at C1367T and T1442C in the gene of Indian isolates. In the present study, a new RFLP-PCR test was developed for diagnosis of the rifampicin antibiotic resistance in MAP isolates of sheep samples. Although, However, the sample size was too small to confirm the reliability of the Hence, further study is required to verify the DNA test on more number of the clinical isolates.
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References
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