In Situ Polymerase Chain Reaction Visualization of Vibrio halioticoli Using Alginate Lyase Gene AlyVG2

A prokaryotic in situ polymerase chain reaction (PI-PCR) technique was applied to visualize Vibrio halioticoli cells using alginate lyase gene alyVG2 as a target gene. Prior to PI-PCR, a primer set, VG2-OS3, for specific amplification of an approximately 1.0-kb fragment from V. halioticoli genomic D...

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Bibliographic Details
Published in:Marine biotechnology (New York, N.Y.) N.Y.), 2000-01, Vol.2 (1), p.74-79
Main Authors: Sugimura, I, I, Sawabe, T, Ezura, Y
Format: Article
Language:English
Subjects:
Alginate lyase
Alginic acid
alyVG2 gene
Amplification
Bacteria
Cells
Deoxyribonucleic acid
Detection
Digoxigenin
DNA
Fluorescence
Fragmentation
Fragments
Marine
Nucleotide sequence
PCR
Phosphatase
Polyimide resins
Polymerase chain reaction
Seaweed meal
Vibrio fischeri
Vibrio halioticoli
Vibrio pelagius
Waterborne diseases
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Summary:A prokaryotic in situ polymerase chain reaction (PI-PCR) technique was applied to visualize Vibrio halioticoli cells using alginate lyase gene alyVG2 as a target gene. Prior to PI-PCR, a primer set, VG2-OS3, for specific amplification of an approximately 1.0-kb fragment from V. halioticoli genomic DNA was developed with amplified fragments from V. pelagius and V. fischeri DNAs as reference strains. One-stage PI-PCR using the primer set, digoxigenin-labeled dUTP, and indirect alkaline-phosphatase-linked fluorescence detection technique (HNPP/Fast Red TR as a substrate) failed to differentiate V. halioticoli IAM14596(T) cells from ATCC25916(T) cells of the closely related species V. pelagius. However, two-stage PI-PCR adding the extension and digoxigenin-labeling step of the amplified fragment into the first amplification stage allowed us to differentiate V. halioticoli cells from V. pelagius cells.
ISSN:1436-2228
1436-2236
DOI:10.1007/s101269900009