Development of species-specific primers and probes in silico for the detection of periodontal pathogens

Introduction. In recent decades, the prevention of infections associated with cancer development has shifted toward targeted control systems based on quantitative hygienic prenosological risk assessment. A risk-oriented approach to oral microbiota monitoring is of particular importance, since chronic dysbiosis and inflammation caused by periodontal pathogens contribute to the carcinogenesis of the oral mucosa.

Objective. To perform in silico selection of species-specific primers and probes for multiplex real-time PCR that, after subsequent clinical validation, could be used for quantitative hygienic prenosological risk assessment of oral mucosal cancer and other inflammatory diseases in healthy persons.

Materials and Methods. Complete genome sequences of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Prevotella intermedia were retrieved from the NCBI RefSeq database. Conserved genomic regions were identified using Mauve v2.4.0, and comparative analysis with related taxa was performed using BLASTn. Species-specific primers and probes were designed in Geneious Prime^® 2020.2.5 considering optimal parameters for real-time PCR. Primer specificity was assessed in silico using NCBI Primer-BLAST.

Results. Primer and probe sets were developed for the detection of four key periodontal pathogens, including two independent sets for P. gingivalis (rgpA and 16S rRNA), as well as primers for P. intermedia, T. forsythia, and T. denticola. The use of fluorescent labels (FAM, JOE, ROX, Cy5) enables four-plex PCR for simultaneous quantitative assessment of bacterial load. Inclusion of the rgpA locus provides an in-depth prenosological evaluation of the pathogenic potential of the microbiota.

Limitations. The study is limited to in silico analysis of primer specificity. This work represents a stage of selection and theoretical justification of primers and probes intended for the creation of a test system. Further clinical validation on patient samples is required to confirm sensitivity and specificity under real-world conditions, as well as establish a correlation between the detected levels of periodontal pathogens and the risk of developing malignant neoplasms of the oral mucosa.

Conclusion. The selected multiplex PCR panel, after mandatory clinical validation, may become a basis for early diagnosis, monitoring, and hygienic prenosological risk assessment of oral mucosal cancer. Implementation of a risk-oriented approach and quantitative PCR diagnostics will enable personalized strategies for the prevention and control of oral diseases.

Compliance with ethical standards. This study did not require approval from a biomedical ethics committee.

Contributions:
Kazimov A.E. – concept and design of the study, collection and processing of material, writing text;
Vysochanskaya S.O. – collection and processing of material, writing text;
Petrova D.A. – collection and processing of material, writing text, text editing;
Malinovskaya A.D. – collection and processing of material, writing text;
Zhernov Yu.V. – concept and design of the study, collection and processing of material, writing text.
All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.

Conflict of interest. The authors declare no conflict of interest.

Funding. The study had no sponsorship.

Received: March 30, 2026 / Revised: May 25, 2026 / Accepted: June 18, 2026 / Published: July 31, 2026

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