Comparison of 16S rDNA-based PCR and checkerboard DNA–DNA hybridisation for detection of selected endodontic pathogens

Abstract

Molecular methods have been used recently to investigate the bacteria encountered in human endodontic infections. The aim of the present study was to compare the ability of a 16S rDNA-based PCR assay and checkerboard DNA-DNA hybridisation in detecting Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Peptostreptococcus micros, Porphyromonas endodontalis, Por. gingivalis and Treponema denticola directly from clinical samples. Specimens were obtained from 50 cases of endodontic infections and the presence of the target species was investigated by whole genomic DNA probes and checkerboard DNA-DNA hybridisation or taxon-specific oligonucleotides with PCR assay. Prevalence of the target species was based on data obtained by each method. The sensitivity and specificity of each molecular method was compared with the data generated by the other method as the reference -a value of 1.0 representing total agreement with the chosen standard. The methods were also compared with regard to the prevalence values for each target species. Regardless of the detection method used, T. denticola, Por. gingivalis, Por. endodontalis and B. forsythus were the most prevalent species. If the checkerboard data for these four species were used as the reference, PCR detection sensitivities ranged from 0.53 to 1.0, and specificities from 0.5 to 0.88, depending on the target bacterial species. When PCR data for the same species were used as the reference, the detection sensitivities for the checkerboard method ranged from 0.17 to 0.73, and specificities from 0.75 to 1.0. Accuracy values ranged from 0.6 to 0.74. On the whole, matching results between the two molecular methods ranged from 60% to 97.5%, depending on the target species. The major discrepancies between the methods comprised a number of PCR-positive but checkerboard-negative results. Significantly higher prevalence figures for Por. endodontalis and T. denticola were observed after PCR assessment. There was no further significant difference between the methods with regard to detection of the other target species.

Endodontic Therapy Associated with Calcium Hydroxide As an Intracanal Dressing: Microbiologic Evaluation by the Checkerboard DNA-DNA Hybridization Technique

“…Of the Porphyromonas species, P. gingivalis was detected in 75% of the cases; however, P. endodontalis was present in 22% of the samples at baseline. Our data are in agreement with those of Siqueira et al (22), who also found high levels of P. gingivalis by using two molecular methods, polymerase chain reaction and checkerboard. The relatively high prevalence of T. forsythensis (50%), a species related to alveolar bone loss in patients with destructive periodontal disease (15), is in contrast to previous studies where the isolation and identification of this microorganism from canals with pulp necrosis was carried out by conventional cultural techniques (3).…”

Molecular Analysis of Microbial Diversity in Advanced Caries

“…However, these lactate-utilizing bacteria were found in other lesions, particularly those containing Prevotella (Table 2). Interestingly, Pseudoramibacter alactolyticus, which can also ferment both carbohydrates and lactate, is found in 56% of endodontic infections (29) and was present at high levels in samples 9 and 10 where no lactobacilli were detected.…”

The microbiology of the acute dental abscess

“…Firstly, the use of molecular cloning and sequencing techniques to identify uncultivable micro-organisms using 16s rRNA or rDNA has led to the identification of several novel species (Dymock et al, 1996). The second approach utilizes PCR or DNA-DNA hybridization chequerboard techniques (Siqueira et al, 2001d(Siqueira et al, , 2002a) and more recently 16S rRNA gene sequencing and species-specific primers searching for the presence of specific microbes (Dymock et al, 1996;Riggio et al, 2006;Sakamoto et al, 2006;Siqueira et al, 2001bSiqueira et al, , c, 2002bSiqueira et al, , 2003. This approach has demonstrated the higher prevalence of more fastidious organisms such as Treponema species in the acute dental abscess.…”