Development of a new multiplex quantitative real-time polymerase chain reaction method for Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum detection in cheese

Late blowing is a type of microbiological spoilage that causes unwanted changes in the taste, odour and texture of cheese due to the metabolic activities of Clostridium contaminating raw milk. This study aimed to develop a multiplex quantitative real-time polymerase chain reaction (PCR) analysis method for the rapid and simultaneous detection of Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum using specific primers and probes. The optimised method was determined to work with high sensitivity and specificity to quantify late-blowing Clostridium species in cheese samples. The lowest detectable gene copy number was 4.9 × 101, 7.8 × 101 and 8.5 × 101 for C. tyrobutyricum, C. butyricum and C. sporogenes, respectively. These results demonstrated that this method is a powerful tool for detecting and quantifying late-blowing agents in cheese. This is also the first multiplex qPCR study involving C. butyricum for detecting Clostridia, a late-blowing agent in cheese.