Pcr-Based Techniques For Detection Of Erwinia Amylovora

This thesis has mainly carried on the detection methods of Erwinia amylovora , the pathogen of pear fire blight.Antiserum were prepared using 0056 strain whole cells as antigen respectively. The specificity and sensitivity of the antrsemm were enough for the immuno-capture-PCR experiment.Based on the standard PCR for detection of Erwinia amylovora published before, we developped the immuno-capture-PCR method, by which could enhanced 10 times of sensitivity of detection pure culture and 1000 times of detection of Erwinia amylovora from samples. This was the first report of using this method for promoting of PCR sensitivity. Using the specific primers REA-FEA designed in this work by ourselves, we proved the specificity and sensitivity of the immuno-capture-PCR method again. The same result has been achieved.Universal PCR primers (L1:5'-AGTCGTAACAAGGTAGCCGT-3' and L2 :5 '-GTGCCAAGGCATCCACC-3') that targeted the intergenic transcribed spacers(ITS) region between 16S and 23 S rDNA gene were utilized to amplify the ITS region from 10 fire blight pathogens.The fragments about 600bp was recovered and purified respectively and cloned.These fragments were sequenced and compared the homology array on the internet. Primer REA-FEA were designed based on the unique sequence of the strains. This pair of specific primer was utilized to amplify the ITS region from 10 fire blight pathogens and 15 other tested bacteria strains and proved the specificity of this pair of primer was high..We developed the one tube Nested-PCR techniques for detection of Erwinia amylovora . Universal PCR primers that targeted the 16SrDNA region and the intergenic transcribed spacers(ITS) region were utilized to amplify the 16SrDNA region and the ITS region from 10 fire blight pathogens and other tested bacteria strains. The fragments were recovered and purified respectively and cloned and sequenced and compared the homology array on the internet. We designed two pairs of primers : a pair of external primers and a pair of internal primers. These two pairs of primers had different annealing temperatures.The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair.Using pure cultures of Erwinia amylovora ,the sensitivity and specificity of the Nested-PCR in one tube was greater than those of standard PCR procedures that used a single primer pair.This one tube Nested-PCR technique can amplified a 270bp fragment only from Erwinia amylovora strains and detection sensitivity can reach 100cfu/ml.