Rapid and Sensitive Detection of Brucella spp. by Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Dipstick

Abstract:

In this study，we established a rapid Brucella spp. detection method by combining loop-mediated isothermal amplification（LAMP） and lateral flow dipstick（LFD） examination. The method used four specific primers targeting to Brucella spp. OMP25 gene for LAMP and one fluorescein isothiocyanate（FITC）-labeled probe to specifically hybrid to the biotin-labeled LAMP product for LFD examination. The optimized amplification temperature and time were 63 ℃ and 50 min，respectively and the total procedure time was only 80 min by taking sample process into account. The established LAMP-LFD method could specifically detect Brucella spp.. Its detection sensitivity for pure bacterial culture reached 5.4×100 CFU/mL，100-fold lower than that of conventional PCR established using the outer primer set. Overall，the established LAMP-LFD method could rapidly and specifically detect Brucella spp. and is simple，economical and intuitive. In addition，it requires no other auxiliary equipment，thus it is suitable for basic laboratory tests，emergency use and in on-site monitoring and has high promotion value.