Quantitative detection of Clostridium tyrobutyricum in milk by real-time PCR.
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.
López-Enríquez
Lorena
L
Instituto Tecnológico Agrario de Castilla y León, Carretera de Burgos, km. 119, 47071 Valladolid, Spain.
Rodríguez-Lázaro
David
D
Hernández
Marta
M
D000818:Animals; D002417:Cattle; D048014:Clostridium tyrobutyricum / Q000235:genetics / Q000302:isolation & purification*; D015169:Colony Count, Microbial / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013171:Spores, Bacterial / Q000302:isolation & purification
D004269:DNA, Bacterial