摘要
通过依赖解旋酶恒温基因扩增(helicase-dependent isothermal DNA amplification,HDA)技术建立一种快速检测发酵乳中植物乳杆菌的方法。根据植物乳杆菌scrB基因序列(基因号为AJ579541.1)设计特异性引物,通过实验筛选出检测体系中UvrD解旋酶和T4 gp32蛋白的较优含量,达到最优反应条件。所建立的HDA方法通过人工添加植物乳杆菌确定检出限,通过多种菌株扩增验证方法特异性,通过扩增产物电泳和序列比对分析验证方法一致性和稳定性。结果表明:采用HDA法检测发酵乳中植物乳杆菌,反应体系中UvrD解旋酶和T4 gp32蛋白的适宜添加量分别为0.15μg和5.0μg,特异性良好,与涉及的其他菌株均未发生扩增,检出限为2.8×10^(1) CFU/g,扩增序列与设计序列长度(273 bp)一致且序列同源性为100%;本研究建立的方法可以快速检测发酵乳中植物乳杆菌,具有灵敏度高、操作简单、应用前景广等特点。
A rapid detection method based on helicase-dependent isothermal DNA amplification (HDA) was established for Lactobacillus plantarum in fermented milk.Specific primers were designed according to the scrB gene sequence of Lactobacillus plantarum (Genbank Accession NO.AJ579541.1),and the optimal concentrations of UvrD helicase and T4 gp32 in the reaction system were determined through experiments.The limit of detection (LOD),specificity,consistency and stability of the proposed method were evaluated by use of L.plantarum-spiked samples,amplification of various strains,and electrophoresis of amplified products and sequence alignment analysis,respectively.The results showed that the optimized of UvrD helicase and T4 gp32 in the reaction system were found to be 0.15 and 5.0μg,respectively.The HDA method had high specificity with no amplification of other strains tested.The detection limit was 2.8×10^(1) CFU/g.The amplified product was consistent with the designed sequence length (273 bp) and the sequence homology was 100%.In conclusion:this method is rapid,simple,sensitive and suitable for the detection of Lactobacillus plantarum in fermented milk.
作者
张雅伦
李永波
张涛
陈晨
张捷
周巍
张岩
ZHANG Yaun;LI Yongbo;ZHANG Tao;CHEN Chen;ZHANG Jie;ZHOU Wei;ZHANG Yan(Hebei Engineering Research Center for Special Food Safety and Health,Key Laboratory of Special Food Supervision Technology for StateMarket Regulation,Hebei Food Safety Key Laboratory,Hebei Food Inspection and Research Institute,Shijiazhuang 050071,China)
出处
《乳业科学与技术》
2023年第1期30-34,共5页
JOURNAL OF DAIRY SCIENCE AND TECHNOLOGY
基金
河北省市场监督管理局科研计划项目(2021ZD16)。
关键词
依赖解旋酶恒温扩增技术
发酵乳
植物乳杆菌
快速检测
helicase-dependent isothermal DNA amplification
fermented milk
Lactobacillus plantarum
rapid detection
