鼠李糖乳杆菌HN001菌株水平快速定量方法的建立及应用

Establishment and application of rapid quantification method at the level of Lactobacillus rhamnosus strain HN001

目的应用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qPCR),建立鼠李糖乳杆菌HN001菌株水平的快速定量检测方法。方法通过比对鼠李糖乳杆菌HN001的近缘菌株的基因组,筛选出鼠李糖乳杆菌HN001菌株水平的特异基因,以菌株水平的特异基因为靶标设计引物及探针,优化反应体系和条件,建立鼠李糖乳杆菌HN001株水平的qPCR检测方法。对方法的特异性、灵敏度、检出限进行验证,并采用已建立的qPCR方法进行模拟添加样品和实际样品的检测。结果所建立的方法特异性强,与近缘菌株无交叉反应,鼠李糖乳杆菌HN001纯培养液检出限为10^(2)CFU/mL,灵敏度可达到650 copies/μL,可在2 h内完成样品检测。对模拟添加样品以及实际样品检测中qPCR和平板计数法结果的对数值进行显著性分析检验,两种方法的测定值结果均无显著性差异(P>0.05)。结论本研究建立的qPCR检测方法可有效应用于益生菌产品中鼠李糖乳杆菌HN001菌株水平定量检测,具有快速、灵敏、准确的特点。

Objective To establish a rapid quantitative assay for Lactobacillus rhamnosus HN001 at the strain level based on real-time fluorescence quantitative polymerase chain reaction(qPCR).Methods By comparing the genomes of the related strains of Lactobacillus rhamnosus HN001,the specific genes at the level of Lactobacillus rhamnosus HN001 were screened,primers and probes were designed with the specific genes at the strain level as the target,the reaction system and conditions were optimized,and a qPCR method was established for the detection of Lactobacillus rhamnosus HN001 strain.The specificity,sensitivity,and limit of detection of the method were verified,and the established qPCR method was used to detect the simulated added samples and the actual samples.Results The established method was highly specific and had no cross-reactivity with closely related strains.The limit of detection of Lactobacillus rhamnosus HN001 was 10^(2)CFU/mL in pure culture solution,and the sensitivity could reach 650 copies/μL,and the sample detection could be completed within 2 h.The logarithm of the results of qPCR and plate counting method in the simulated spiked samples and actual samples were analyzed for significance,and there was no significant difference between the results of the two methods(P>0.05).Conclusion The qPCR method established in this study can be effectively used for the quantitative detection of Lactobacillus rhamnosus HN001 at the strain level in probiotic products,which is characterized by rapidity,sensitivity and accuracy.