快速检测发酵乳中葡萄糖杆菌的赖解旋酶恒温基因扩增方法的建立

Establishment of a Helicase-Dependent Isothermal DNA Amplification Method for Rapid Detection of Gluconobacter in Fermented Milk

建立赖解旋酶恒温基因扩增(helicase-dependent isothermal DNA amplification,HDA)快速检测发酵乳中葡萄糖杆菌的方法。选择葡萄糖杆菌ITS基因序列(基因号为KF896260.1)为目的基因,设计特异性引物,并优化反应体系中UvrD解旋酶和T4 gp32的添加量,建立最优反应体系。采用建立的HDA体系对多种菌株进行扩增和电泳检测,验证方法特异性,通过HDA法直接检测发酵乳中的葡萄糖杆菌,并确定其检出限。结果表明:采用HDA法检测发酵乳中葡萄糖杆菌,反应体系中UvrD解旋酶和T4 gp32的适宜添加量分别为0.10μg和5.0μg,扩增产物与设计序列长度(309 bp)一致,特异性良好,检出限为4.3×10^1 CFU/g。该方法用于检测发酵乳中葡萄糖杆菌的灵敏度高、耗时短。

A rapid method using helicase-dependent isothermal DNA amplification(HDA)for the detection of Gluconobacter in fermented milk was established.The ITS gene sequence(gene number KF896260.1)of Gluconobacter was selected as the target gene for design of specific primers,and the concentrations of UvrD helicase and T4 gp32 in the reaction system were optimized to establish an optimal reaction system.The HDA method was used to directly detect Gluconobacter in fermented milk.The established HDA system was used to amplify and electrophoretically detect various strains in fermented milk to verify the specificity of the method and determine its detection limit.The results showed that the detection of Gluconobacter in fermented milk by the HDA method was consistent with the designed sequence length(309 bp)and the detection limit was 4.3×10^1 CFU/g.The appropriate amounts of UvrD helicase and T4 gp32 added in the reaction system were 0.1 and 5.0μg,respectively.The method is highly sensitive and time saving.