Use of checkerboard DNA–DNA hybridization to study complex microbial ecosystems

Abstract

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe-target formats, such as checkerboard DNA-DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin-labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10(4) cells. In all, 93.5% of potential cross-reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross-reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA-DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions

“…Failure to detect a signal was recorded as zero, although counts in the 1-1000 range could have been present. The sensitivity and specificity of the probes were determined as reported by Socransky et al (2004). The sensitivity of the assay was adjusted to permit detection of 10 4 cells of a given species by adjusting the concentration of each DNA probe.…”

Dysbiosis and Alterations in Predicted Functions of the Subgingival Microbiome in Chronic Periodontitis

“…Early studies using culture-based techniques were labor-intensive (21), and many organisms remained uncultivable despite improvements in culturing techniques (22). Subsequent molecular approaches based on DNA-DNA hybridization and microarray techniques substantially improved our understanding of these microbial communities (23)(24)(25)(26)(27). However, since knowledge of nucleic acid signatures or hybridization probes are required a priori, these methods precluded detailed analyses of entire microbial communities.…”

Presence of Helicobacter pylori and Campylobacter ureolyticus in the oral cavity of a Northern Thailand population that experiences stomach pain

“…The use of the CKB method with its low sensitivity and a detection level of >10 4 cells [25,27] for evaluation of the subgingival microbiota in this study needs to be commented on. The CKB method was chosen because it is a convenient method in field studies, when sensitivity is a minor problem in samples with high bacterial number due to poor oral hygiene.…”

“…After a series of stringency washes at 70°C, hybrids were detected using phosphatase-conjugated anti-digoxigenin antibodies and the signals were visualized with a chemiluminescent substrate (CDP Star, Roche Diagnostics). The number of bacteria in the samples was compared to standard samples containing 10 6 and 10 5 cells of each species [27]. The readings were performed with the visual score method (0–5) and using the percent method (based on BLU-signals intensities expressed as a fraction of the signal intensity of the high standard) according to Dahlén et al [25] as control.…”