Direct DNA extraction to detect Mycobacterium bovis from the lungs of buffaloes positive to intradermal tuberculin testing

Abstract

Bovine tuberculosis (bovine TB), caused by Mycobacterium bovis, is an important significant zoonotic disease. The infection course is usually chronic, which directly affects animal health and production. Bacterial culture, which is time-consuming, is the gold standard of diagnosis. Nowadays, rapid molecular techniques such as polymerase chain reaction (PCR) are used for the rapid identification of bovine TB. However, a convenient and rapid DNA extraction method to detect mycobacterial organisms directly from tissue samples is understudied. In this study, tissue samples were collected from buffaloes that were positive to a single intradermal test. Three different direct DNA extraction methods were undertaken to find a simple procedure for rapid detection of M. bovis from tissue: 1) a tissue genomic extraction kit; 2) a combination of enzymatic (lysozyme) extraction and the tissue genomic extraction kit; and 3) boiling the tissue for 30 min before using a combination of lysozyme extraction and the tissue genomic extraction kit. The DNA samples were then used to identify M. bovis using PCR. The greatest yield of DNA concentration was obtained from the combination of enzymatic extraction and the tissue genomic extraction kit. In addition, this method also provided the highest percentage of positive results for M. bovis.