解旋酶恒温基因扩增方法快速检测酸乳中醋化醋杆菌

Determination of Acetobacteracetis in yogurt by helicase-dependent isothermal DNA amplification

目的建立解旋酶恒温基因扩增方法(helicase-dependent isothermal DNA amplification,HDA)快速检测酸乳中醋化醋杆菌的分析方法。方法以醋化醋杆菌的16S-23S rRNA ITS序列为目的基因,设计一对特异性引物,优化反应条件UvrD helicase、T4 gp32的浓度,通过解旋酶恒温基因扩增方法直接检测酸乳中醋化醋杆菌,扩增产物通过电泳进行检测。结果 HDA法检测酸乳中醋化醋杆菌的特异性强,实验涉及的其他菌株均未发生扩增,只有醋化醋杆菌得到与设计序列长度一致的197 bp基因片段,方法检出限为3.6×10~1 CFU/g,实验条件优化后T4 gp32、UvrD helicase的终含量分别为4.0、0.15μg。结论该方法灵敏度较高,时间较短,为酸乳中醋化醋杆菌的检测提供了新的思路,同时也为其他食品污染菌的快速检测提供了借鉴意义。

Objective To establish a method for rapid determination of the Acetobacteracetis in yogurt by helicase-dependent isothermal DNA amplification. Methods Taking the 16s-23s rRNA ITS sequence of Acetobacteracetis as the target gene, designing a pair of specific primers, the concentrations of UvrD helicase and T4 gp32 in the reaction conditions were optimized. The Acetobacteracetis in yogurt was detected by helicase-dependent isothermal DNA amplification, and the amplification product was detected by electrophoresis. Results Detection of Acetobacteracetisin in yogurt by helicase-dependent isothermal DNA amplification was in strong specificity, the other bacterias could not been amplificated. Only Acetobacteracetis obtained 197 bp gene fragments with the same length as the design sequence, and the limit of detection was 3.6- 10l CFU/g. The final concentrations of the T4 gp32 and UvrD helicase after the optimization of experimental conditions were 4.0 μg and 0.15 μg, respectively. Conclusion This method has high sensitivity and short time, which provides a new idea for the detection of Acetobacteracetis in yugort, and also provides a reference for the rapid detection of other food.