Comparison of Specificity and Sensitivity of Immunochemical and Molecular Techniques for Reliable Detection of Erwinia amylovora

摘要

iErwinia amylovora [(BurrILL) Winslow iet al.] (iEa), the causal agent of fire blight, was detected in plant samples and pure bacterial cultures by means of PCR, IF AS and ELISA. Polyclonal antibodies of iNeogen Europe Ltd. were used for IF AS and PTA-ELISA and laboratory-generated primers EaF72 and EaR560 for PCR. Using the iBIOLOG system and an immature pear fruit assay, identities of all iEa strains were confirmed as the fire blight bacterium. In assays of pure iEa cultures, PTA-ELISA, and both IFAS and PCR were sensitive to concentrations 10u6-10u5 and 10u5-10u4 CFU/mL, respectively. When saprophytic bacteria associated with iEa in plant samples were tested as potentially cross-reacting bacteria, PTA-ELISA and IFAS gave 20 and 14 % cross-reactions, respectively. In plant samples, the presence of iEa was more reliably detected by IFAS (at a dilution of 1:1000) than by PTA-ELISA (to dilution 1:100). The capacity to detect iEa might be increased using an optimized PCR, but for PCR prepared from infected plant samples it was necessary to use the bacterial DNA isolated with a DNeasy Plant Mini Kit (iQiagen). In this case the PCR was sensitive to a concentration of 10u5 CFU/mL. PCR was much more specific than either immunochemical technique, because no false positives were observed when primers EaF72 and EaR560 were used.