To investigate whether the new RNA in situ hybridization test (RNAscope) could be a reliable alternative to immunohisto­chemistry (IHC) in detection of Porphyromonas gingivalis in esophageal squamous cell carcinoma (ESCC) tissues. The speci­ficity of RNAscope probe was tested in esophageal cell lines in­fected by P. gingivalis. Thirty-five formalin-fixed, paraffin-em­bedded (FFPE) tissues from patients who were firstly diagnostic as ESCC in the First Affiliated Hospital of Henan University of Science and Technology were collected. P. gingivalis was de­tected by IHC and RNAscope using FFPE tissues. The ability and clinical prognostic value of RNAscope technique were ana­lyzed and compared with that of IHC. RNAscope was specific for the detection of P. gingivalis in esophageal cells. Compared with IHC, the percentage of the sensitivity and specificity of RNAscope in detection of P. gingivalis in the cancerous tissues of ESCC patients were 92.9% and 95.2%, respectively. The concordance rate was 94.3% between IHC and RNA­scope (kappa = 0.881; P < 0.001). Importantly, the overall survival (OS) periods estimated by Kaplan­Meier analysis using the results of RNAscope or ref­erence IHC test were similar (IHC, P = 0.001; RNA­scope, P = 0.004). RNAscope is a novel and reliable method in detection of P. gingivalis and investigation of the pathogenesis of ESCC. 

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