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目的建立葡萄酒中酒香酵母实时荧光PCR检测方法。方法将滴度为3.0×10~6cfu/ml、3.0×10~5cfu/ml、3.0×10~4cfu/ml、3.0×10~3cfu/ml、3.0×10~2cfu/ml、30 cfu/ml的1 ml菌液加至45 ml酒样中,离心洗涤后利用碱裂解法提取DNA,进行实时荧光PCR检测,测定检测方法的灵敏度;提取革兰阳性细菌、革兰阴性细菌以及5株葡萄酒中相关菌株(包含1株酒香酵母等效菌株)的DNA,进行实时荧光PCR检测,测定检测方法的特异性。结果该实时荧光PCR方法的定量限为6.67 cfu/ml,检出限为0.67 cfu/ml;该方法对酒香酵母及其等效菌株均可检出,对革兰阳性细菌、革兰阴性细菌以及其余相关菌株均无交叉反应。结论该实时荧光PCR方法灵敏度高、特异性好,可准确快速地检测出葡萄酒中的酒香酵母菌。
Objective To establish a real-time fluorescent PCR detection method of wine yeast in wine. Methods Titers of 3.0 × 10 ~ 6 cfu / ml, 3.0 × 10 ~ 5 cfu / ml, 3.0 × 10 ~ 4 cfu / ml, 3.0 × 10 ~ 3 cfu / ml, 3.0 × 10 ~ 2 cfu / ml, 30 cfu / ml 1 ml of broth was added to 45 ml of wine samples, centrifuged and washed by alkaline lysis method for DNA extraction, real-time fluorescence PCR detection to determine the sensitivity of detection methods; Gram-positive bacteria, Gram-negative bacteria and five wines Strains (including one S. cerevisiae equivalent strain) were subjected to real-time fluorescence PCR and the specificity of the assay. Results The limit of quantitation of this real-time PCR method was 6.67 cfu / ml, with a detection limit of 0.67 cfu / ml. The method could detect both Gram-positive bacteria and Gram-negative bacteria As well as the other related strains did not cross-reaction. Conclusion The real-time fluorescence PCR method has the advantages of high sensitivity, good specificity and accurate and rapid detection of S. lividans in wine.