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      Quantitative detection of Clostridium tyrobutyricum in milk by real-time PCR.

      Applied and Environmental Microbiology
      Animals, Cattle, Clostridium tyrobutyricum, genetics, isolation & purification, Colony Count, Microbial, methods, DNA, Bacterial, analysis, Milk, microbiology, Polymerase Chain Reaction, Sensitivity and Specificity, Spores, Bacterial

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          We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R(2) > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

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