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 appl ied 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 ampl ification of an approximately 1.0-kb fragment from V. halioticoli genomic D NA was developed with amplified fragments from V. pelagius and V. fischeri DNAs as reference strains. One-stage PI-PCR using the primer set, digoxigen in-labeled dUTP, and indirect alkaline-phosphatase-linked fluorescence dete ction technique (HNPP/Fast Red TR as a substrate) failed to differentiate V . halioticoli IAM14596(T) cells from ATCC25916(T) cells of the closely rela ted species V. pelagius. However, two-stage PI-PCR adding the extension and digoxigenin-labeling step of the amplified fragment into the first amplifi cation stage allowed us to differentiate V. halioticoli cells from V. pelag ius cells.