Quantitative real-time PCR using TaqMan and SYBR Green forActinobacillus actinomycetemcomitans,Porphyromonas gingivalis,Prevotella intermedia,tetQgene and total bacteria

Abstract

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.

The effects of feed blocks containing tomato and cucumber by-products on <i>in vitro</i> ruminal fermentation, microbiota, and methane production

“…The DNA samples were used as templates to quantify the copy numbers of 16SrRNA (for bacteria) and of methyl coenzyme M reductase A (mcrA) gene for methanogens. For each microbial group, three different aliquots of DNA were analysed using qPCR with the following primer pairs: forward: 5′-GTGSTGCAYGGYTGTCGTCA-3′ and reverse: 5′-ACGTCRTCCMCACCTTCCTC-3′ for total bacteria (Maeda et al, 2003), and forward: 5′-TTCGGTGGATCDCARAGRGC-3′ and reverse: 5′-GBARGTCGWAWCCGTAGAATCC-3′for methanogens (Denman et al, 2007). A negative control (sterile distilled water) was also loaded on each plate run to screen for possible contamination and dimer formation and to set the background fluorescence for plate normalization.…”

“…The external standards used for the qPCR amplifications had been validated previously for rumen bacteria (Maeda et al, 2003) and methanogenic archaea (Denman et al, 2007).…”

Total bacterial counts on oral mucosa after using a commercial saliva substitute in patients undergoing hematopoietic cell transplantation

“…The gene encoding the small subunit of bacterial 16S rDNA has been used frequently as a target of PCR examination because of its structural characteristics [2,5]. The nucleotide sequences of some portions of the 16S rDNA are highly conserved through evolution [3]. The conserved sequences can provide PCR primers for amplification of 16S rDNA from all bacterial species [3].…”

“…The nucleotide sequences of some portions of the 16S rDNA are highly conserved through evolution [3]. The conserved sequences can provide PCR primers for amplification of 16S rDNA from all bacterial species [3]. Quantitative real-time PCR has been demonstrated to be a powerful tool for quantitative microbiological examination [3].…”

“…The conserved sequences can provide PCR primers for amplification of 16S rDNA from all bacterial species [3]. Quantitative real-time PCR has been demonstrated to be a powerful tool for quantitative microbiological examination [3]. Therefore, we evaluated the changes in total bacterial counts on the buccal mucosa after using Oralbalance® by real-time PCR quantification of 16S rDNA.…”