DETECTION OF ERWINIA AMYLOVORA BY NESTED PCR AND PCR-DOT-BLOT AND REVERSE BLOT HYBRIDIZATIONS

The sensitivity and specificity of four methods based on the polymerase chain reaction (PCR) for detection of Erwinia amylovora were compared.
A previously developed single-round PCR assay was used to amplify a specific 1-kb DNA fragment of plasmid pEA29, known to be unique to and conserved in E. amylovora. The ends of the 1-kb single-round PCR product were sequenced, and two new oligonucleotide primers were designed from sequences internal to the original primers, and used in nested PCR. These primers directed amplification of a 844-bp fragment from the 1-kb first-round PCR product or directly from E. amylovora cells, but not from other bacteria associated with apple.
Less than one cell of E. amylovora from pure culture was detected with nested PCR, an increase in sensitivity of 1,000 fold compared to single-round PCR. The lower limit of detection of PCR-dot-blot and reverse-blot hybridization methods was approximately 20 cells.
When asymptomatic apple tissue was screened, E. amylovora was detected in 61, 84, and 100% of leaf samples; 0, 66, and 80% of axillary bud samples; and 4, 27, and 75% of mature fruit calyx samples by first-round PCR, PCR-dot-blot hybridization, and nested PCR, respectively.

VII International Workshop on Fire Blight

P.S. McManus, A.L. Jones

https://doi.org/10.17660/ActaHortic.1996.411.20