Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods |
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Authors: | Bang-Ce Ye K Ikebukuro and I Karube |
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Institution: | (1) Research Institute of Biochemistry, Key State Laboratory of Bioreactor Engineering, East China University of Science and Technology, MeiLong Road 130, 200237 Shanghai, China e-mail: bcye@hotmail.com,;(2) Research Center for Advanced Science and Technology, University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153, Japan, JP |
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Abstract: | Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP)
by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes
are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is
used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in
the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the
PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR
(in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization
strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious
agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains.
Received: 28 December 1998 / Revised: 22 April 1999 / Accepted: 24 April 1999 |
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