深加工益生菌产品中常用7种乳杆菌的快速鉴定

Rapid identification of 7 kinds of Lactobacillus commonly used in deep processing probiotic products

中文关键词:  提取方法  特异性基因扩增  深加工益生菌产品

英文关键词:Extraction method  Specific gene amplification  Deep processing of probiotic products

中文摘要:

      目的 探讨适用于复杂基质中益生菌产品的DNA提取方法，并通过鉴定7种常见的乳杆菌，比较产品标签标示与实际添加乳杆菌种类的一致性。方法 本研究采用了3种不同的DNA提取方法，分别为十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)法、磁珠法深加工产品基因组DNA抽提试剂盒(磁珠法)和Ezup柱式深加工产品基因组DNA抽提试剂盒(柱式法)；通过测定DNA浓度、纯度以及利用16SrDNA V3/V4基因扩增技术评估DNA质量。此外合成嗜酸乳杆菌、德氏乳杆菌保加利亚亚种、罗伊氏粘液乳杆菌、植物乳植杆菌、干酪乳酪杆菌、鼠李糖乳酪杆菌和副干酪乳酪杆菌特异性引物探针，利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative PCR, qPCR)技术验证方法的特异性、灵敏度和重复性，进一步对市售益生菌产品进行乳杆菌种类的鉴定。结果 综合3个DNA质量评价方法，使用CTAB法提取的DNA质量较高，纯度在1.7~2.1之间，浓度大于25 ng/mL，且16SrDNA V3/V4基因扩增的循环数阈值(cycle threshold, Ct)值均小于35，符合后续qPCR扩增要求；相比之下，柱式法除了饼干类产品DNA纯度为1.96±0.13，浓度为79.07±2.15，扩增Ct值为22.67±3.09能够达到要求外，提取的其他产品DNA和磁珠法提取的DNA无法同时满足3个DNA评价方法要求且重复性较差。本研究所使用的引物和探针对目标乳杆菌有明显的特异性扩增，灵敏度范围为0.0100~1.0000 ng/μL和103~104 CFU/mL，且重复性良好[相对标准偏差(relative standard deviation, RSD)≤2.36%]。检测的1820批市售产品中，6批产品未检出标示的乳杆菌种类，存在标示与实际检出菌种不符的情况。结论 CTAB法因其较高的适应性和低成本，在提取复杂基质中益生菌产品的DNA时表现更优，结合qPCR方法能有效实现产品中乳杆菌种水平鉴定；然而针对干酪乳酪杆菌的检出限需要进一步研究开发更为灵敏的引物和探针。

英文摘要:

      Objective To explore the DNA extraction method suitable for probiotic products in complex substrates, and to compare the consistency between the product label and the actual Lactobacillus species by identifying the common Lactobacillus. Methods In this paper, 3 kinds of different methods of DNA extraction were used, namely cetyltrimethylammonium bromide (CTAB) method, magnetic bead method for genomic DNA extraction kit of deep processing products (magnetic bead method) and Ezup column method for genomic DNA extraction kit of deep processing products (column method). The quality of DNA was evaluated by measuring the concentration and purity of DNA and using 16S rDNA V3/V4 gene amplification technology. In addition, specific primer probes were synthesized for Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus paracasei. The specificity, sensitivity and repeatability of the method were verified by real-time fluorescence quantitative PCR (qPCR), and the species of Lactobacillus were further identified in the commercial probiotic products. Results The purity of DNA extracted by CTAB was 1.7-2.1, the concentration was more than 25 ng/mL, and the cycle threshold (Ct) value of 16S rDNA V3/V4 gene amplification was less than 35, the PCR products meet the requirement of qPCR; in contrast, the column method could meet the requirements except that the purity of biscuit DNA was 1.96±0.13, the concentration was 79.07±2.15, and the amplified Ct value was 22.67±3.09, DNA extracted from other products and DNA extracted by magnetic beads could not meet the requirements of 3 DNA evaluation methods and had poor repeatability. The primers and probes used in this paper had obvious specific amplification for target Lactobacillus, with sensitivity ranges of 0.0100-1.0000 ng/μL and 103-104 CFU/mL, and good repeatability [relative standard deviation (RSD)≤2.36%]. Among the 18 20 batches of commercial products tested, 6 batches of products did not detect the labeled Lactobacillus species, which was inconsistent with the actual detected strains. Conclusion Because of its high adaptability and low cost, CTAB method performs better in extracting DNA from probiotic products in complex substrates, and combined with qPCR method, it can effectively identify the species level of Lactobacillus in products. However, more sensitive primers and probes need to be developed for the detection limit of Lactobacillus casei.