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Quantification of Ectopic Fusobacterium Colonisation in Colorectal Cancer Using a Newly Developed nusG-Directed PCR

Janne Becker1, Anna Mertens1, Meikel Duncan Rieger1

  • 1Division of Oral Microbiology and Immunology, Department of Operative Dentistry, Periodontology and Preventive Dentistry, Rheinisch-Westfälische Technische Hochschule (RWTH) University Hospital, 52074 Aachen, Germany.

International Journal of Molecular Sciences
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Summary

A new polymerase chain reaction (PCR) assay targeting the nusG gene can detect Fusobacterium nucleatum (L1) in colorectal cancer (CRC) patients. This non-invasive biomarker shows potential for early CRC diagnostics.

Keywords:
Fusobacterium nucleatum complexN-utilisation substance Gcolorectal cancerendpoint PCRnusGquantitative PCR

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Area of Science:

  • Microbiology and Molecular Diagnostics
  • Gastroenterology and Oncology

Background:

  • The Fusobacterium nucleatum complex, specifically oral lineage 1 (L1) strains, is significantly linked to colorectal cancer (CRC) development.
  • Accurate and sensitive detection of ectopic Fusobacterium L1 colonization is crucial for understanding its role in CRC pathogenesis.

Purpose of the Study:

  • To develop and validate a novel polymerase chain reaction (PCR) assay targeting the universally conserved NusG (N-utilisation substance G) gene.
  • To specifically and sensitively detect ectopic Fusobacterium L1 colonization in clinical samples from CRC patients.

Main Methods:

  • Designed and employed four L1-specific primer pairs targeting the nusG gene in 40 stool samples from CRC patients and healthy controls (HC).
  • Developed species-specific primer pairs for various L1 species and validated assay specificity via Sanger sequencing.
  • Analyzed Ct values and absolute cell counts for statistical significance and effect size, calculating area under the curve (AUC) for diagnostic potential.

Main Results:

  • Two nusG-directed primer pairs demonstrated robust performance, showing statistically significant differences in Ct values and cell counts between CRC patients and HC (padj < 0.05, |r| ≥ 0.5).
  • These primer pairs exhibited promising diagnostic potential with AUC values of 0.909 and 0.883.
  • Fusobacterium L1 abundance in saliva did not significantly differ between groups, limiting conclusions from the salivary sub-cohort.

Conclusions:

  • nusG-based PCR primers represent reliable, non-invasive biomarkers for complementary early CRC diagnostics.
  • Further validation in larger, diverse cohorts is essential for broader clinical application.
  • The assay shows potential for detecting Fusobacterium L1 in other associated diseases.