Verification of immunofluorescence colony‐staining of Erwinia carotovora subsp. atroseptica by reisolation and immunodiffusion or PCR

Abstract

In this study, the reliability and efficiency of three procedures for verification of IFC‐positive colonies of Erwinia carotovora subsp. atroseptica were compared: (1) PCR amplification, (2) reisolation on a non‐selective medium (trypticase soy agar) followed by direct immunodiffusion (TSA‐DID), developed for isolation of target and cross‐reacting bacteria, and (3) reisolation on a selective medium (crystal‐violet pectate) and characterization of selected isolates with Ouchterlony double diffusion (DLCVP‐ODD), developed for isolation of pectinolytic Erwinia spp. The reliability of a PCR amplification procedure for characterization of E.c. atroseptica was evaluated. Specific amplification products could be produced from DNA of all 187 European strains of the bacterium, while no amplification products were obtained from DNA of four distinctive serological groups of bacteria cross‐reacting with antibodies against E.c. atroseptica, nor from DNA of randomly selected saprophytic bacteria isolated from potato peel extracts. All 60 immunofluorescent‐positive target colonies from a potato peel extract with added E.c. atroseptica tested were positive by PCR compared with 68 and 72% successful determinations by TSA‐DID and DLCVP‐ODD, respectively. PCR enabled verification of fluorescent colonies from IFC preparations of naturally infected seed lots with an efficiency of 93%, compared with 48 and 71% successful determinations by TSA‐DID and DLCVP‐ODD, respectively. It is concluded that PCR is useful for routine confirmation of the identity of fluorescent colonies in IFC.