Determination of Lactobacillaceae by PCR methods

摘要

This work was focused on determination of possibilities of method PCR (polymerase chain reaction) to detect Lactobacillus. PCR was compared to several other methods of molecular biology, especially to FISH (fluorescence in situ hybridization). 13 Lactobacillus genera from collection were chosen. Their identities were confirmed by API CH50 tests. While biochemical features of all chosen Lactobacillus genera are known, the complete genomic DNA sequence is known only for some of them. Several commercially available kits based on PCR were used for their identification. Due to knowledge of a sequence of well-conserved region of different Lactobacillus gDNAs we could design primers for this region. These primers were used for non-specific PCR reaction with lower annealing temperature. The reaction was used for detection of the sequences from the genera with not known sequence of gDNA. Such amplified DNA fragments were cloned into pCR?4 vector by TOPO? TA kit and afterwards sequenced. Exact knowledge of the sequence allowed us to design specific primers. The same region for amplification was used also for Real-time PCR. Real-time PCR is an advanced and highly specific method so we expected to obtain lot of important data. These data we expected to use for consequent distinction of individual lactobacillus genera. For measurement we have used LightCyclerTMRoche?. Another method used for lactobacillus genera determination was FISH based on detection of specific DNA probe marked with fluorescent dye. We have used fluorescent probes from commercially available kit Vermicon.