Optimization of PCR in application of hot start <Emphasis Type="Italic">Taq</Emphasis> DNA polymerase for detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> with primers FER1-F and FER1-R1 |
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Authors: | D Obradovic S Kevresan |
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Institution: | 1.Department of Biology,University of Montenegro, Faculty of Science,Podgorica,Montenegro;2.University of Novi Sad, Faculty of Agriculture,Novi Sad,Serbia |
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Abstract: | There are two approaches in detection of bacterium Erwinia amylovora by PCR. One is based on detection of plasmid pEA29 and the other is based on detection of a chromosomal DNA sequence, specific
for E. amylovora, in a sample. Since pathogenic strains without pEA29 have been isolated from the environment, methods based on this plasmid
have been compromised and PCR methods based on chromosomal DNA species specific sequences became only reliable methods. PCR
method with chromosomal primers FER1-F and FER1-R is currently the most reliable method due to its high sensitivity and specificity.
The goal of this research is to make a significant improvement of the method by optimization of PCR in application of hot
start DNA Taq polymerase, instead of wax, to obtain a hot start reaction. This enzyme, which is currently widely applied, can provide simpler
achievement of hot start, saving labor and time and decreasing possibility of cross contamination of samples. Experiments
showed that simple replacement of a regular recombinant Taq DNA polymerase by a hot start Taq DNA polymerase leads to complete failure of the reaction. Many optimization experiments had to be carried out to obtain an
operational and reliable PCR which simultaneously has high sensitivity and specificity. Content of the reaction mixture, as
well as temperature and time parameters of PCR, were significantly changed to achieve proper optimization. |
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