LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis.
In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
Marcos D
MD
Trangoni
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
Andrea K
AK
Gioffré
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Pathobiology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
María E
ME
Cerón Cucchi
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
Karina C
KC
Caimi
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
Paula
P
Ruybal
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
Martín J
MJ
Zumárraga
Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.
Silvio L
SL
Cravero
D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods; D002002:Brucella / Q000235:genetics / Q000302:isolation & purification*; D002006:Brucellosis / Q000175:diagnosis; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010283:Paratuberculosis / Q000175:diagnosis; D013997:Time Factors
D017931:DNA Primers
brucellosis; loop-mediated isothermal amplification; molecular typing; paratuberculosis