Detection of Mycobacterium bovis in goat milk samples by Loop-Mediated Isothermal Amplification

ROCHA V.,; STICOTTI E,; BIGI F.; EIRIN ME; MACIAS A,; MACIÓ, M.; TRANGONI M,; ZUMÁRRAGA MJ,; MAGNANO G,; SCHNEIDER M,; SIOYA B,

Congreso; 3rd International Congress of Asian-African Society of Mycobacteriology; 2021

Asian-African Society of Mycobacteriology

Introduction: Mycobacterium bovis is the causative agent of bovine tuberculosis. It is also a pathogen for other animals such as goats and humans. Molecular methods are applied to detect members of the M. tuberculosis complex in different clinical samples as milk. The Loop-Mediated Isothermal Amplification (LAMP) proceeds at a constant temperature using a strand displacement reaction. LAMP does not require a thermal cycler. It is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity and specificity. The primers are specifically designed to recognize 6 distinct regions on the target gene. The amplified product can be detected by naked eye as a white precipitate or a color change of the reaction solution. Objective: To detect species of the Mycobacterium tuberculosis complex in goat milk samples by IS6110-LAMPMaterial and methods: Twenty goat milk samples from animals reacting to the tuberculin test were studied by IS6110-LAMP, Touch-Down (TD) IS6110 and Rv2807 PCR. These goats had been slaughtered, their organs and milk cultured. The isolates obtained were typed by spoligotyping. DNA extraction was performed using 500uL of milk sample. It was incubated with sodium dodecyl sulfate / proteinase K and treated twice with phenol:chloroform:isoamylic alcohol and once with chloroform:isoamylic alcohol. Then NaCl was added and DNA was precipitated with isopropanol and washed with ethanol. The remaining ethanol was evaporated and the pellet resuspended in water. IS6110-LAMP reaction was performed in a 25uL volume containing Bst DNA polymerase, betaine, magnesium sulfate, dNTPs, and primers (F3, B3, FIP, BIP, LF, LR). SYBR Gold solution was added inside of the cup of the tubes that were incubated at 63°C during one hour. After that the tubes were shacked and a yellowish color indicated a positive reaction. The specificity of IS6110-LAMP was evaluated using DNA from different species of Non Tuberculous Mycobacterias (NTM) (Mycobacterium smegmatis, M. phlei, M. fortuitum, M. gordonae, M. intracellulare, M. porcinum, M. avium subsp. avium, M. avium subsp. paratuberculosis) and the close related Nocardia sp. TD IS6110 and TDRv2807 PCR were also performed with an initial step of 96°C for 3 min, followed by 8 cycles of 96°C for 1 min, annealing temperatures starting at 72°C for 1 min (decreasing by 1 °C/cycle), and 72°C for 1 min for extension. This step was followed by 30 cycles of 96°C for 1 min, 65°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 8 min. The amplification products were 245 and 443 bp, respectively.Results: While 17/20 goats were confirmed by bacteriology, no isolates were obtained from milk culture. The 17 isolates were further confirmed as M. bovis by spoligotyping. Neither NTM nor Nocordia sp. templates amplified by IS6110-LAMP. The 40 % (8/20) of the samples were IS6110-LAMP positive. There was only one discordant result between IS6110-LAMP, TD-Rv2807 and TD-IS6110. Conclusion: IS6110-LAMP is a useful tool to detect species of the M. tuberculosis complex in goat milk samples that could be implemented in laboratories with low technological complexity.