Re-Evaluation of Conventional PCR Primers and Sampling Methods for The Detection of Erwinia amylovora in Contaminated Pear Pollen

Abstract

Detection of Erwinia amylovora (Ea) in contaminated pollen has raised phytosanitary concerns, however, the most efficient primer and sampling method to diagnose it by conventional polymerase chain reaction (cPCR) from contaminated pear pollen remain insufficiently evaluated. Therefore, in this study, eleven primer pairs reported previously were re-evaluated for their sensitivity, reproducibility, and efficiency using genomic DNA (gDNA) and cell suspensions from three different E. amylovora strains, including two domestic and one foreign strain. The selected primer pair was also tested with pollen isolates and artificially inoculated pollen. EAPSGL3961/EAPSGL4610c showed the most favorable overall analytical performance under the conditions tested, producing a distinct 649-bp amplicon at 0.02 ng/reaction of gDNA and 105 colony-forming unit (cfu)/ml (approximately 2×102 cfu/reaction) in cell assays. EAPSGL3961/EAPSGL4610c primer was shown to neither amplify nonspecifically nor prevent target detection with any isolates collected from pollen. Regarding sampling method, indirect method in which 1 μl of supernatant from two blocks (0.0003 g per block) suspended and boiled in 10 mM MgCl₂ was used as template in a total of 25 μl of cPCR solution was shown to be the most efficient to detect Ea from contaminated pear pollen. Therefore, the EAPSGL3961/EAPSGL4610c primer and procedures for the indirect sampling method are likely applicable to the detection of Ea in contaminated pear pollen in the quarantine field.