FIRST STEPS TO DEVELOP A DETECTION METHOD OF BRUCELLA SP., APPLICATING THE MOLECULAR TECHNOLOGY OF ISOTHERMAL AMPLIFICATION

HERMIDA, P; L. LAROCCA; STOLOWICS FG; WERBAJH S; ZORREGUIETA, A; VOJNOV A; CARRILLO C

Jornada; . XX Jornadas Anuales de la Sociedad Argentina de Biología y XVII Jornadas de la Sociedad Uruguaya de Biociencias; 2018

Brucellosis is a chronic infectious disease that has a big impact on veterinary and human health. This zoonosis is caused by bacteria of the genus Brucella sp., with a strong affinity for reproductive organs, making sexually mature and pregnant mammals more susceptible to infection. Currently, the disease can be diagnosed either: i- directly by blood, marrow or other tissue cultures; or ii- indirectly through a serological reaction, being more frequently chosen because it is simpler and safer than the first one. Our purpose is to develop new detection methods for Brucellosis with highly specific and sensitive molecular techniques such as PCR. Yet, PCR and other similar techniques have one major drawback: they need to be performed at a lab with specific infrastructure and trained personnel. Thus, our main goal is to develop and set up a Loop mediated isothermal amplification (LAMP) to detect Brucella sp., which should share the benefits of PCR but needing just a simple heater. Three primer sets were designed with a specific software (LAMP Design) and tested in a LAMP basic reaction using DNA from B. abortus? (human pathogen) as a template. The three sets were functional, with different sensitivity and specificity parameters. Later, we adjusted the reaction components, designing a mix that avoids having to manipulate various reagents; while all three sets maintained their functionality. Afterwards, the optimal conditions (time and temperature) of the reaction were determined for each primer set, finding a range of temperature from 62 to 66°C and 50 to 60 minutes of time. Finally, we developed a simple result-reading method with a sensitivity comparable to that of the analytical method of agarose gel electrophoresis. These results encourage us to continue working and test artificially-inoculated samples, and subsequently test clinical samples, to bring us closer to the development of a simple and effective test kit to detect Brucellosis.