Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.

The use of molecular techniques to detect Yersinia pestis has enabled remarkable progress in the provision of necessary information on the occurrence of plague. In Tanzania, despite the long history of plague, DNA confirmation on the presence of Y. pestis in human specimens has not been done. This study was conducted in Mbulu district in Northern Tanzania where plague outbreaks have recently been reported. Nine human bubo specimens were investigated for Y. pestis plasminogen activator gene by using polymerase chain reaction (PCR), and two were found to be positive. The two positive amplicons, together with three previously obtained PCR positive rodent samples, were sequenced using a 3130 genetic analyzer and then compared with those available in GenBank by basic local alignment search tool (BLAST). All sequences obtained from both human and rodent samples showed 99% sequence similarity to Y. pestis plasmid pPCP1, detected from ancient DNA, confirming the presence of Y. pestis in humans that possibly sourced from rodents in Tanzania.

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