Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

Orrù, Germano; Marini, Mario; Ciusa, Maria Laura; Isola, Daniela; Cotti, Marina; Baldoni, M; Piras, Vincenzo; Pisano, Elisabetta; Montaldo, C

doi:10.1186/1471-2334-6-98

Cited by 40 publications

(48 citation statements)

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“…Although several PCR-based methods for detection of the oral pathogens have been reported (18,19,(21)(22)(23)(24)(25)(26), we hereby report the first designed duplex PCR and real time PCR assays in Iran for sensitive, specific and rapid detection of the two pathogens. To address an improved PCR assay potentially capable for application in clinic, recommendations of CLSI (Clinical Laboratory Standards Institute) for evaluation and validation of the molecular assays such as analytic sensitivity and analytic specificity were noted.…”

Section: Objectivesmentioning

confidence: 99%

Development and Comparison of Conventional PCR and SYBR Green Real Time PCR for Detection of Aggregatibacter actinomycetemcomitans and Tannerella for Sythensis

Soleimani

Zolfaghari

Morovvati

2013

Jundishapur J Microbiol

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Background: Aggregatibacter actinomycetemcomitans and Tannerella forsythensis are two major pathogens in destructive periodontal disease in humans. The detection of these bacteria is needed for diagnosis and management of the mentioned diseases. Objectives: We aimed to develop and compare improved multiplex conventional and SYBR green real time PCR assays for a specific diagnosis of the organisms based on specific marker genes. Materials and Methods: Both PCR approaches were performed with primers targeting diagnostic genes of the organisms, hbpA gene of A. actinomycetemcomitans and 16S rRNA gene of T. forsythensis. For preparation of a stable positive control, the PCR products were cloned into pTZ57R/T plasmid. The test specificity was evaluated using the same PCR reactions but in the presence of genomes of various negative control bacteria. Results: As expected, agarose gel electrophoresis of PCR products of the hbpA and 16S rRNA genes showed 160 bp and 250bp bands respectively. Temperature melting analyses of the SYBR green real time PCR assays showed the Tm at 78.02 °C and 84.62 °C for hbpA and 16S rRNA genes respectively. The amplification results using negative control genomes as template was negative showing the specificity of both designed assays. The detection limits of the conventional PCR assay for hbpA and 16S rRNA genes were 5200 and 1200 copies of each gene, respectively. The designed SYBR green PCR assays tenfold increased the test sensitivity. Conclusions: The designed assays provide simple, reliable, and rapid procedures that identify two main periodontal pathogens. Theses assays are new diagnostic opportunities in our laboratory and also are working as important supplements of the current time consuming phenotypic assays.

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Section: Objectivesmentioning

confidence: 99%

Development and Comparison of Conventional PCR and SYBR Green Real Time PCR for Detection of Aggregatibacter actinomycetemcomitans and Tannerella for Sythensis

Soleimani

Zolfaghari

Morovvati

2013

Jundishapur J Microbiol

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“…To detect the specific 530-bp sequence that determines the JP2 or non-JP2 promoter region, the primers used for DNA amplification were designed in accordance with the work of Orrù et al (35) and were synthesized by a commercial service (Invitrogen, Carlsbad, CA). Specifically, the pair of primer sequences (forward, 5Ј-CATTCTCGGCGAAAAAACTA-3Ј, and reverse, 5Ј-CCC ATAACCAAGCCACATAC-3Ј) circumscribe a fragment of 530 bp present in the A. actinomycetemcomitans non-JP2 strain 652 but absent in the JP2 strain.…”

Section: Vol 47 2009 Jp2 a Actinomycetemcomitans Treatment 2019mentioning

confidence: 99%

“…As a result, non-JP2 clones (e.g., A. actinomycetemcomitans strains 652 and ␥4) appear to be associated with a lesser severity of periodontal disease (11,30). In contrast, subjects with severe forms of periodontal disease have tended to harbor deletion-positive A. actinomycetemcomitans clones (7,8,17,18,19,35). Haubek et al (19) found a higher rate of progression of periodontal disease among JP2-strain-positive patients, while Wu et al (43) reported that detection of the A. actinomycetemcomitans LTX gene (lktA), but not the gene for the fimbria-associated protein (fap) of A. actinomycetemcomitans, in the subgingival sulcus correlates with the severity of chronic periodontal disease in China.…”

mentioning

confidence: 99%

Diminished Treatment Response of Periodontally Diseased Patients Infected with the JP2 Clone ofAggregatibacter(Actinobacillus)actinomycetemcomitans

Cortelli

Costa

Kawai³

et al. 2009

J Clin Microbiol

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This longitudinal study evaluated the response to periodontal treatment by subjects infected with either JP2 (n ‫؍‬ 25) or non-JP2 (n ‫؍‬ 25) Aggregatibacter (Actinobacillus) actinomycetemcomitans. Participants were treated during the first 4 months by receiving (i) scaling and root planing, (ii) systemic antibiotic therapy, and (iii) periodontal surgery. Probing depth (PD), clinical attachment level (CAL), and gingival and plaque indices (GI and PI, respectively) were monitored at baseline and at 12 months, along with DNA-PCR-based subgingival detection of JP2 or non-JP2 A. actinomycetemcomitans. At baseline, PD, CAL, and GI scores were statistically higher in the JP2 strain-positive group than the non-JP2-strain-positive group. At 12 months, PD, CAL, and GI scores had decreased significantly for both groups, but the reduction rates of PD and CAL were higher in the non-JP2-strain-positive group. Among JP2-strain-positive patients in the baseline, patients who remained JP2 strain positive at 12 months showed significantly higher GIs than did the patients who had lost the detectable JP2 clone. Patients who remained JP2 strain positive at 12 months appeared to be more resistant to mechanical-chemical therapy than did those who were still non-JP2 strain positive, while the elimination of JP2 A. actinomycetemcomitans remarkably diminished gingival inflammation. Early identification and elimination of the JP2 clone of A. actinomycetemcomitans will enable practitioners to effectively predict the outcome of treatments applied to periodontal patients.Compared to gingivitis, or disease-free periodontium, an increased incidence of Aggregatibacter (Actinobacillus) actinomycetemcomitans has been reported in the lesions of localized aggressive periodontitis (LAP) or severe chronic periodontitis (9,13,16,41,44,45). It is also true that while polymicrobial infection is implicated in the pathogenesis of chronic adult periodontitis, oral colonization by A. actinomycetemcomitans is more directly associated with the pathogenesis of LAP. Such pathogenic outcomes related to A. actinomycetemcomitans infection seem to be partially derived from leukotoxin (LTX) produced by A. actinomycetemcomitans. LTX is a potent virulence factor, which is encoded in an operon consisting of four genes (5) and typically causes cytotoxicity in neutrophils (26) and macrophages (37). Furthermore, in vitro experiments revealed that a specific 530-bp sequence in the LTX promoter region, rather than the LTX operon, impacts the transcription regulation of LTX (5), whereas there are several factors that regulate the expression of LTX from A. actinomycetemcomitans (25). It is also true that there may be highly leukotoxic A. actinomycetemcomitans strains other than just the type with the 530-bp deletion (24). Nonetheless, the deletion of this 530-bp promoter region, which is found in some pathogenic strains of A. actinomycetemcomitans, also called JP2 strains, has critical ramifications. For example, faster expression of the LTX gene with the deletion has been o...

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“…Then, an isolation of whole genomic DNA from oral samples was done with the use of a modified CTAB method (called: cetyltrimethylammonium bromide, hexadecyltrimethylammonium bromide). 40 Each paper point was placed in a 1.5 mL Eppendorf tube filled with 400 μL of sterile water (Sigma Aldrich Reagent Water Molecular Biology), and the mixture was stirred in a vortex apparatus. Afterwards, paper points were removed from the tubes and 70 μL of 10% SDS (sodium dodecyl sulfate), and 50 μL of proteinase K at a concentration of 1 mg/mL (SigmaAldrich) were added.…”

Section: Dna Extraction Protocolmentioning

confidence: 99%

Porphyromonas gingivalisin periodontal pockets and heart valves

et al. 2014

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Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

Cited by 40 publications

References 50 publications

Development and Comparison of Conventional PCR and SYBR Green Real Time PCR for Detection of Aggregatibacter actinomycetemcomitans and Tannerella for Sythensis

Development and Comparison of Conventional PCR and SYBR Green Real Time PCR for Detection of Aggregatibacter actinomycetemcomitans and Tannerella for Sythensis

Diminished Treatment Response of Periodontally Diseased Patients Infected with the JP2 Clone ofAggregatibacter(Actinobacillus)actinomycetemcomitans

Porphyromonas gingivalisin periodontal pockets and heart valves

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