定量PCR技术检测杀菌型产品中乳酸菌

Determination of lactic acid bacteria in bactericidal products by quantitative PCR

目的利用定量PCR(quantitative PCR,qPCR)技术检测杀菌型产品中乳酸菌,并与标签标示相比较,查探产品中各类菌种的异同性及数量。方法以市售的发酵乳及含乳饮料作为研究对象,设计可区分产品中常见的6种乳酸菌特异性引物及探针,利用qPCR技术对方法的特异性、灵敏度进行验证,并建立标准扩增曲线;利用模拟样品对方法进行检验,最后对市场上购买的实际样品进行检测。结果除去干酪乳杆菌,其他5种引物都能特异性扩增出其目标菌,并且对其他的乳酸菌均无扩增现象;DNA检测的灵敏度可达到2~4 pg;单一标准菌种扩增曲线线性较好,相关系数r2值在0.954~0.992之间;对模拟样品的检测,与平板法差异性比较,P值均大于0.05,无显著性差异。对市售样品进行检测,产品中标记的干酪乳杆菌、双歧杆菌和嗜热链球菌与检测结果存在不匹配现象。结论本研究建立的qPCR方法可快速、准确地对产品中德氏乳杆菌保加利亚亚种、嗜热链球菌、乳酸乳球菌、嗜酸乳杆菌、双歧杆菌的鉴定和定量的分析。

Objective To detect lactic acid bacteria in bactericidal products by quantitative PCR(qPCR),and compare the results with the label to find out the heterogeneity and quantity of various types of bacteria in the products.Methods Taking commercially available fermented milk and milk-containing beverages as research objects,totally 6 kinds of primers and probes were designed to distinguish common lactic acid bacteria in the products.The specificity and sensitivity of the method were verified by qPCR,and the standard amplification curve was established.The simulation sample method was used to test,and the actual samples purchased in the market were tested.Results Except Lactobacillus casei,the other 5 primers could amplify their target bacteria specifically,and there was no amplification of other lactic acid bacteria.The sensitivity of DNA detection could reach 2-4 pg.The linearity of the single standard strain amplification curve was good,with r2 between 0.954-0.992.Compared with the plate method,P>0.05,and there was no significant difference.In the detection of commercial samples,Lactobacillus casei,Bifidobacteria and Streptococcus thermophilus labeled in the product did not match the test results.Conclusion This qPCR method can quickly and accurately identify and quantitatively analyze the products,including Lactobacillus delbrueckii subsp.Bulgaricus,Streptococcus thermophilus,Lactococcus lactis,Lactobacillus acidophilus and Bifidobacteria.