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Sensitive and rapid detection of Pectobacterium atrosepticum by targeting the gyr B gene using a real‐time loop‐mediated isothermal amplification assay
Author(s) -
Hu L.X.,
Yang Z.H.,
Zhang D.,
Zhao D.M.,
Zhu J.H.
Publication year - 2016
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12618
Subject(s) - biology , microbiology and biotechnology , blackleg , loop mediated isothermal amplification , genetics , botany , dna , brassica
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 gyr B 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 gyr B gene of P. atrosepticum worked most efficiently at 61·0°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°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 gyr B gene of P. atrosepticum, which can be easily performed and real‐time monitored, was established. Significance and Impact of the Study Potato blackleg caused by Pectobacterium atrosepticum ( P. atrosepticum ) which is mainly transmitted through the seed potato leads to the decline in potato production. To reduce yield loss, rapid detection of P. atrosepticum in seed potato remains essential. Based on the gyr B gene of P. atrosepticum , species‐specific primers were designed. A real‐time, loop‐mediated isothermal amplification (RealAmp) assay was established for the detection of P. atrosepticum . The RealAmp assay is a specific, rapid and sensitive method for P. atrosepticum detection. Therefore, it provides an effective diagnosis of potato blackleg in both the growing and stored potato.