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16S r RNA ‐targeted oligonucleotide probes for direct detection of P ropionibacterium freudenreichii in presence of L actococcus lactis with multicolour fluorescence in situ hybridization
Author(s) -
MikšKrajnik M.,
Babuchowski A.
Publication year - 2014
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12278
Subject(s) - propionibacterium freudenreichii , lactococcus lactis , biology , 16s ribosomal rna , fluorescence in situ hybridization , propionibacterium , bacteria , lactococcus , oligonucleotide , hybridization probe , cheesemaking , microbiology and biotechnology , oligomer restriction , ribosomal rna , biochemistry , lactic acid , dna , genetics , gene , chromosome
Abstract Multicolour fluorescence in situ hybridization (FISH) has been applied to detect L actococcus lactis and P ropionibacterium freudenreichii cells in mixed populations in medium and skimmed milk, using epifluorescent microscopy. The 16S r RNA ‐targeted 18‐mer oligonucleotide probes, specific for P . freudenreichii were designed and evaluated. Based on multiple alignments of designed sequences, eight 16S r RNA probes were selected for in vitro studies. The permeabilization protocol was optimized for simultaneous hybridization of propionibacteria and lactococci cells. The probes GL O 62 ( 62‐80 ), PEU 64 ( 64‐82 ) and PFX 311 ( 311‐329 ) were found specific for P . freudenreichii in analysis in vitro . L actococcus lactis cells were labelled with L act V 5 ( 822‐840 ), ( S ‐ S ‐ L .lact‐0821‐a‐ A ‐18) probe. The following combinations of oligonucleotide probes: LactV5/GLO 62 , LactV5/PEU 64 and LactV5/PFX 311 , enabled differentiation of L c. lactis and P . freudenreichii cells in culture media and in skimmed milk. Significance and Impact of the Study Results showed that three newly designed 16S rRNA‐targeted oligonucleotide probes specific for P ropionibacterium freudenreichii in combination with a probe specific for L actococcus lactis can be used to differentiate lactic and propionic acid bacteria in mixed communities using multicolour fluorescence in situ hybridization (FISH), without applying permeabilization step. This is a first study on simultaneous detection of both bacterial species with FISH, which was found as rapid, useful and culture‐independent tool for direct visualization of bacteria on single cell level. It might be applied in monitoring of mixed starter cultures in dairy industry.