Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material

摘要

Three PCR methods, referred in this study as ?conventional“, ?nested“ and ?chromosomal“ PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.9′105 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.5′103 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material.