AimsCurrently, there is no direct method for detecting Clostridium thermocellum in the insoluble medium. In this study, a quantitative real-time PCR assay was developed for the direct growth detection of C.thermocellum at the single-cell level in lignocellulosic biomasses. Methods and ResultsThe assay targeted the cipA gene and was able to distinguish C.thermocellum from other species with good reproducibility which quantitative detection limit was 10 cell equivalents (CE) per reaction. OD600-based counting and qPCR quantification of C.thermocellum cultured in soluble medium were compared and an excellent consistency was revealed, indicating the appropriateness of the developed qPCR method. Analysis based on yellow affinity substrate and fermentation products may incorrectly estimate its population. ConclusionsThe developed assay can serve as a specific, sensitive and reproducible method for the detection of C.thermocellum in lignocellulosic biomass at the single-cell level. Significance and Impact of the StudyWith the ability to rapidly detect C.thermocellum, this method will contribute substantially to the understanding of the lignocellulosic biomass degradation mechanism. Moreover, it can also be applied to detect C.thermocellum growth in certain co-culture system for the understanding of the metabolic interactions.