Specific identification of Mycobacterium bovis by Loop-Mediated Isothermal Amplification (LAMP) targeting the Region of Difference 12 (RD12) of the M. tuberculosis complex

Abstract

Bovine tuberculosis is a prevalent zoonotic disease that causes high risks for production animals, dairy producers and consumers, together with significant economic losses. Thus, methods for easy, fast and specific detection of Mycobacterium bovis in small and medium-sized livestock under field conditions are very required. In this work, a Loop-Mediated Isothermal Amplification LAMP-PCR targeting the Region of Difference 12 (RD12) of M. bovis genome was designed for the purpose of identification. A set of six primers designed for the isothermal amplification of five different genomic fragments led to the specific identification of M. bovis from other mycobacterial species. A basic colorimetric reaction was clearly observed at first sight under natural light, indicating positive identification of M. bovis in a maximum of 30 min of isothermal amplification at 65 °C. The limit of detection was near 50 fg of M. bovis genomic DNA, corresponding approximately to 10 copies of the genome. • The proposed LAMP-PCR amplification of M. bovis genomic DNA might be performed by untrained laboratory personnel. • Specific identification of M. bovis LAMP is possible in 30 min at 65.. C using a simple water bath. • The basic colorimetric reaction for M. bovis identification could be observed with the naked eye under natural light.

Keywords

• Mycobacterium bovis
• RD12 genomic region
• M. tuberculosis complex
• LAMP-PCR
• Phenol red colorimetric reaction
• point-of-care method