The isolation and characterization of phages with lytic activity against Mycobacterium avium subspecies paratuberculosis, and their application using Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification assay for rapid detection

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully.