Classification and identification of propionibacteria based on ribosomal RNA genes and PCR.
A rapid method was developed to differentiate the genus Propionibacterium from other genera by using a modified multiplex-PCR (MPCR) approach. Three 16S rRNA-targeted oligonucleotide primers were designed to amplify simultaneously two DNA-fragments in the MPCR assay. The universal primer pair bak11w and bak4 (corresponding to the E. coli 16S rRNA positions 8-25 and 1522-1540, respectively) was used in combination with the primer pair bak4 and gd1 (5'-TGCTTTCGATACGGGTTGAC-3'). The later sequence corresponding to a 16S rRNA motif that is unique for the genus Propionibacterium. Propionibacteria were identified by the amplification of a Propionibacterium-genus specific 900-bp fragment whereas MPCR with DNA from other bacteria generated only a DNA fragment of 1500 bp in amplifications with the two universal primers. The whole procedure including cell lysis, MPCR amplification and analysis can be performed within 1 day, detection limits are at approximately 10(3) cfu propionibacteria (or 35 pg DNA). In addition, the taxonomic situation of the genus Propionibacterium was reexamined using a cycle sequencing strategy. Based on the 16S rDNA, a phylogenetic tree of all the Propionibacterium type strains was reconstructed.
Institut für Lebensmittelwissenschaften, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
G
G
Dasen
J
J
Smutny
M
M
Teuber
L
L
Meile
D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D004275:DNA, Ribosomal / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011424:Propionibacterium / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D012313:RNA / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; C034477:RNA primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012313:RNA