实时荧光定量PCR检测金山醋酸乳杆菌的方法与应用

Establishment and application of real-time fluorescence quantitative PCR for detection of Acetilactobacillus jinshanensis

摘要:

[目的] 建立基于实时荧光定量PCR （RT-qPCR）的金山醋酸乳杆菌特异性检测方法，并将其应用于食醋和白酒发酵过程样品的快速检测。[方法] 筛选金山醋酸乳杆菌基因组中特异性强的基因序列作为模板设计特异性引物，通过标准菌株、醋醅样品进行PCR验证引物的特异性和准确性，建立RT-qPCR方法分析金山醋酸乳杆菌在不同地区酒醅、醋醅和食醋样品中的含量。[结果] 以金山醋酸乳杆菌的苯丙氨酰-tRNA合成酶α亚基（编码pheS基因）为参考序列，设计了一对GC含量55%、T_m值59℃、可扩增199 bp片段的引物。建立的RT-qPCR方法特异性强、灵敏度高且重复性好，检测浓度范围为2.24-10.24 lg （copies/μL），成功应用于4个地区的酒醅、醋醅和食醋样品检测。对镇江香醋醋酸发酵过程的研究表明，醋醅中的金山醋酸乳杆菌数量先迅速增加，随后稳定在10⁸ copies/g干醅。[结论] 本研究建立的RT-qPCR方法可对食醋和白酒发酵过程中金山醋酸乳杆菌进行特异性鉴定和快速定量。

关键词:镇江香醋;金山醋酸乳杆菌;特异性引物;RT-qPCR

Abstract:

[Objective] To establish a real-time quantitative method PCR (RT-qPCR) for the detection of Acetilactobacillus jinshanensis in the fermentation of Chinese traditional fermented vinegar and baijiu.[Methods] The specific gene in the Acetilactobacillus jinshanensis genome was screened for the design of PCR-sequence specific primer. The specificity and accuracy of specific primers were verified by PCR with isolated strains and vinegar Pei. The RT-qPCR method was established to analyze the content of Ac. jinshanensis in vinegar, jiupei and cupei from different regions.[Results] We designed a pair of primers with product size of 199 bp, GC content of 55% and T_m value of 59℃ with phenylalanine-tRNA ligase subunit α (encoding pheS gene) of Ac. jinshanensis. The RT-qPCR method has potent specificity, high sensitivity, good repeatability and versatility, and its detection range is 2.24-10.24 lg(copies/μL). The method can be applied for the detection of Ac. jinshanensis in the fermentation of cupei and jiupei from different regions.[Conclusion]] The RT-qPCR method can be used for the specific identification and accurate quantification of Ac. jinshanensis in the fermentation of vinegar and baijiu.

Key words:Zhenjiang aromatic vinegar;Acetilactobacillus jinshanensis;specific primers;RT-qPCR