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Conventionally in-situ hybridization (ISH) is used for the study of chromosome structure/function and cytological analysis. With the growing availability of microbial genomic maps and requirement of high sensitivity, specificity and short time for microbiological diagnostic assays, ISH has been introduced into clinical microbiology for rapid diagnosis of sepsis; diverse of ISH-based methods have been promoted by means of novel probe, label and automated hybridization system. ISH has become a leading diagnostic technique for sepsis diagnosis. This review summarizes common hybridization assays in relation to sepsis diagnosis.