Detection of Erwinia amylovora by nested PCR and PCR-dot-blot and reverse-blot hybridizations.
The sensitivity and specificity of 4 methods based on the polymerase chain reaction (PCR) for detection of E. 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 2 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 directly from E. amylovora cells but not from other bacteria associated with apple. From pure culture <1 cell of E. amylovora was detected with nested PCR, an increase in sensitivity of 1000-fold compared with single-round PCR. The lower limit of detection of PCR-dot-blot and reverse-blot hybridization methods was c. 20 cells. Weak positive signals were sometimes produced by bacteria other than E. amylovora in hybridization experiments but only if nonspecific PCR products were visible on ethidium bromide-stained agarose gels. 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. The potential applications of each method are discussed.