Comparison of specificity and sensitivity of immunochemical and molecular techniques for reliable detection ofErwinia amylovora

Abstract

Erwinia amylovora [(Burrill)Winslowet al.] (Ea), the causal agent of fire blight, was detected in plant samples and pure bacterial cultures by means of PCR, IFAS and ELISA. Polyclonal antibodies ofNeogen Europe Ltd. were used for IFAS and PTA-ELISA and laboratory-generated primers EaF72 and EaR560 for PCR. Using theBIOLOG system and an immature pear fruit assay, identities of allEa strains were confirmed as the fire blight bacterium. In assays of pureEa cultures, PTA-ELISA, and both IFAS and PCR were sensitive to concentrations 10⁶-10⁵ and 10⁵-10⁴ CFU/mL, respectively. When saprophytic bacteria associated withEa 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 ofEa was more reliably detected by IFAS (at a dilution of 1:1000) than by PTA-ELISA (to dilution 1:100). The capacity to detectEa 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 (Qiagen). In this case the PCR was sensitive to a concentration of 10⁵ CFU/mL. PCR was much more specific than either immunochemical technique, because no false positives were observed when primers EaF72 and EaR560 were used.