Establishment and application of rapid quantification method at the level of Lactobacillus rhamnosus strain HN001
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 102 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.
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