Improved PCR for Detection of Xanthomonas euvesicatoria pv. perforans in tomato seeds

Abstract
The detection efficiency of the PCR methods for Xanthomonas euvesicatoria pv. perforans (Xep) was determined. Xep specific primer set; HpaF–f/HpaF–r was compared with the previously reported Bs–XpF/Bs–XpR primers in multiplex PCR reaction and singleplex PCR for detection of Xep were tested. The improvement of PCR amplification efficacy was investigated by adding PCR additives or either 5% DMSO or 1 M betaine. Adding both PCR additives gave the same lowest detection limit of Xep detection in both multiplex and singleplex PCR at 100 fg. However, multiplex PCR gave no equal amplification rate for both sets of primers and also showed cross–amplification to X. vesicatoria. Then singleplex PCR with both PCR additives was further tested in a tomato seed lot. The artificially inoculated tomato seeds with various concentrations of Xep from 1.05 x 10⁵ to 10¹ CFU/ml were tested with singleplex PCR. The lowest detection limit was 10¹ CFU/ml of the 5 replications of each inoculated seed. One artificially inoculated tomato seed from each bacterial cell suspension was mixed with 2,000 healthy seeds making 0.05% (1/2,000) contaminated tomato seeds sample and further tested with singleplex PCR, results showed the detection limit of 10¹ CFU/ml in 0.05% Xep contaminated seed lot.