López-Enríquez Lorena, Rodríguez-Lázaro David, Hernández Marta
Instituto Tecnológico Agrario de Castilla y León, Carretera de Burgos, km. 119, 47071 Valladolid, Spain.
Appl Environ Microbiol. 2007 Jun;73(11):3747-51. doi: 10.1128/AEM.02642-06. Epub 2007 Apr 20.
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.
我们开发了一种实时荧光定量PCR检测方法，用于定量检测酪丁酸梭菌，该菌已被确定为奶酪后期产气的主要致病因子。该检测方法具有100%的特异性，在40%的反应中分析灵敏度为1个基因组当量。在5个对数动态范围内，定量呈线性（R²>0.9995），低至10个基因组当量，PCR效率>0.946。通过在离心和核酸提取前对样品进行优化的去污剂处理和酶预处理，该检测方法可检测低至300个酪丁酸梭菌孢子，相对准确度为82.98至107.68，并且在25毫升人工污染的生牛奶或超高温处理的全脂牛奶中能检测到低至25个孢子。
