J P Burton1, J K McCormick, P A Cadieux, G Reid. 1. Canadian Research and Development Center for Probiotics, The Lawson Health Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada. jburton@lri.sjhc.london.on.ca
Abstract
AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots. METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe. After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE. CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples.
AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots. METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe. After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE. CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples.