发酵乳中沙门氏菌依赖解旋酶恒温基因扩增快速检测方法的建立

建立依赖解旋酶恒温基因扩增（helicase-dependent isothermal DNA amplification，HDA）快速检测发酵乳中沙门氏菌方法。以沙门氏菌invA基因序列为目的基因，设计特异性引物，优化反应体系中UvrD解旋酶及T4 gp32添加量，建立最优反应体系。通过HDA方法直接检测发酵乳中沙门氏菌，扩增其产物后进行电泳检测，验证方法特异性。结果表明：采用HDA快速检测法检测发酵乳中沙门氏菌特异性良好，优化后反应体系体积50 μL时，UvrD解旋酶添加量为0.10 μg，T4 gp32添加量为5.0 μg，得到与设计序列长度（304 bp）一致的扩增产物，检出限为2.6×102 CFU/g；该方法用于快速检测发酵乳中沙门氏菌能够满足检测需求，具有较高的灵敏度、易操作，可作为一种基础且快速的方法检测发酵乳中沙门氏菌。

Helicase-Dependent Isothermal DNA Amplification for Rapid Detection of Salmonella in Fermented Milk

A rapid detection method for Salmonella in fermented milk was developed by helicase-dependent isothermal DNA amplification (HDA). In this method, the invA gene sequence of Salmonella was used as the target gene to design specific primers, and the concentrations of UvrD helicase and T4 gp32 in the reaction system were optimized to establish the optimal reaction system. The HDA method was used to directly detect Salmonella in fermented milk, and the amplification products were detected by electrophoresis to verify the specificity of this method. The results showed that the proposed method exhibited high specificity for Salmonella in fermented milk. The optimized reaction system contained 0.10 μg of UvrD helicase and 5.0 μg of T4 gp32 per 50 μL of sample. The amplification product was consistent with the designed sequence length (304 bp). The detection limit was 2.6 × 102 CFU/g. The method can meet the requirements for the rapid detection of Salmonella in fermented milk, and thanks to its high sensitivity and easy operation, it can be used as a basic method for rapid detection of Salmonella in fermented milk.