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

The Fusobacterium nucleatum complex, which comprises oral lineage 1 (L1) strains, is strongly associated with colorectal cancer (CRC). NusG (N-utilisation substance G) is a transcription elongation factor that is universally conserved. This study aimed to develop and validate a novel nusG-directed polymerase chain reaction (PCR) assay to specifically and sensitively detect ectopic Fusobacterium L1 colonisation in clinical CRC patient samples. Four L1-specific primer pairs targeting the nusG gene were designed using MEGA11 software (Molecular Evolutionary Genetics Analysis, version 11.0.13) and successfully employed in 40 stool samples from CRC patients and healthy controls (HC). Additionally, five species-specific primer pairs were designed for the L1 species F. animalis clades 1 and 2, F. nucleatum, F. polymorphum, and F. vincentii, and were successfully applied to stool and saliva samples. Their specificity was verified via Sanger sequencing. Two L1-specific primer pairs (NusG5-F/NusG6-R and NusG2a-F/NusG5-R) demonstrated robust performance in our cohort, showing statistical significance (padj < 0.05) and a large effect size (|r| ≥ 0.5) in the difference in Ct values and absolute cell counts between CRC patients and the HC group. These primer pairs also exhibited promising preliminary diagnostic potential, with respective area under the curve (AUC) values of 0.909 and 0.883. However, Fusobacterium L1 abundance in saliva samples did not differ significantly between groups, indicating that definitive conclusions cannot be drawn due to the limited power of the salivary sub-cohort. The data indicates that nusG-based PCR primers could be used as reliable, non-invasive biomarkers as a complementary tool for early CRC diagnostics. While potentially applicable in the context of other Fusobacterium-implicated diseases, further validation in larger, ethnically diverse cohorts remains essential.