葡萄酒中酒香酵母实时荧光PCR检测方法的研究

Research on the detection of Brettanomyces bruxellensis in wines with real-time PCR

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 develop a real-time PCR detection method for Brettanomycesbruxellensis in wines. Methods 1 ml of strain liquid with the 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 and 30 cfu / ml were added to 45 ml wine samples respectively,and the mixed wine samples were centrifuged and washed. The DNA of these samples was extracted by alkaline lysis method after centrifugal purifying for the sensitivity detection by real-time PCR. The DNA of gram-positive bacterium,gram-negative bacterium and five related strains in wine（ including one equivalent strain） were extracted and detected by real-time PCR to determine the specificity of the method. Results The quantification limit of this method was 6. 7 cfu / ml and the detection limit was 0. 7 cfu / ml. The method was specific for Brettanomycesbruxellensis and the equivalent strains,with no cross-reaction with gram-positive bacterium,gram-negative bacterium or other five related strains. Conclusion The real-time PCR method has high sensitivity and good specificity,and can detect Brettanomyces bruxellensis in wines quickly and accurately.