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BACKGROUND: Fusobacterium nucleatum (Fn) has been associated with the risk of colorectal cancer, poor colorectal cancer survival, and tumor attributes. Accurate and sensitive detection of Fn in tumor tissue is critical for evaluating their role in colorectal cancer.METHODS: We developed a droplet digital PCR (ddPCR) assay for detecting Fn using the transcription termination/antitermination gene (nusG) normalized for host tissue (solute carrier organic anion transporter family member 2A1). We assayed Fn(nusG) in matched tumor and normal tissues for 613 participants in the Seattle site of the Colon Cancer Family Registry. We used logistic regression to determine the odds of Fn enrichment in tumor tissue according to the tumor site and stage, adjusting for age, sex, and body mass index.RESULTS: The limit of quantitation for Fn(nusG) was 4.1 copies/10 ng host tissue. Detection of Fn was quenched and poor at low levels in formalin-fixed, paraffin-embedded tissues using qPCR. There was a low agreement between qPCR and ddPCR (Cohen's kappa = 0.46). Fn(nusG) was detected in tumor (21%) and normal (10%) tissues and was enriched in 19% of tumors. Individuals with tumors enriched in Fn were more likely to be female (59% vs. 48%, respectively; P = 0.04) with proximal colon tumors (57% vs. 43%; P = 0.026). In multivariable-adjusted analyses, proximal colon tumors were significantly associated with Fn enrichment (OR vs. rectal tumors: 1.86; 95% confidence interval, 1.11-3.24).CONCLUSIONS: We established a sensitive and specific method to detect Fn enrichment in human tissues.IMPACT: ddPCR enhanced detection of Fn(nusG) for studies targeting tumor-associated bacteria.