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

Abstract

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.