Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers |
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Authors: | D Obradovic J Balaz S Kevresan |
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Institution: | (1) Faculty of Science, Department of Biology, University of Montenegro, P.O. Box 211, Podgorica, 81000, Montenegro;(2) Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovica 8, Novi Sad, 21000, Serbia |
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Abstract: | A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and
enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular
PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was
identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed.
The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose
gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by
electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6
CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29
has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid. |
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Keywords: | PCR molecular test bacterium chromosome pEA29 Erwinia amylovora |
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