Sensitive and rapid detection of Pectobacterium atrosepticum by targeting the gyrB gene using a real-time loop-mediated isothermal amplification assay

This study reports the development of a real-time, loop-mediated isothermal amplification (RealAmp) assay for the detection of Pectobacterium atrosepticum (P. atrosepticum). A phylogenetic tree was constructed based on the gyrB gene of P. atrosepticum and related species. Pectobacterium atrosepticum from different sources can be clustered in the same branch with 100% support rate. The RealAmp primers targeting the gyrB gene of P. atrosepticum worked most efficiently at 61.0 degrees C. Compared with 55 related bacterial strains, the eight P. atrosepticum strains displayed positive reaction in the RealAmp assay. The melting temperature (Tm) of P. atrosepticum amplified products was about 85.0 degrees C. The detection limit of the RealAmp assay for the detection of P. atrosepticum in pure culture was approx. 3 CFU reaction(-1). The detection limit of the RealAmp assay for the detection of P. atrosepticum in artificially contaminated samples was 22 CFU reaction(-1). The detection rate of the RealAmp assay for the detection of potato tubers was 28.5-32.0% higher than that of the conventional PCR. In summary, a specific, sensitive and rapid RealAmp assay based on the gyrB gene of P. atrosepticum, which can be easily performed and real-time monitored, was established.