动物双歧杆菌乳亚种i797菌株特异性量化分析方法研究

Real-time PCR assays for the strain-specific detection of Bifidobacterium animalis subsp.lactis i797

动物双歧杆菌乳亚种i797(Bifidobacterium animalis subsp.lactis i797)是一株具有改善小儿腹泻和消化不良功能的潜在益生菌。通过与其它22株动物双歧杆菌乳亚种菌株进行全基因组测序-单核苷酸多态性(Whole Genome Sequencing-Single Nucleotide Polymorphism,WGS-SNP)分析,确定了i797菌株位于K1717_01615基因上的一个特异性位点。根据该位点设计引物及探针,基于荧光定量PCR建立了i797菌株的量化分析方法。结果表明,所选位点可以有效区分i797与其它18株双歧杆菌,且在传代40次过程中表现出较强的稳定性。该检测方法对于i797菌株DNA浓度和菌液浓度的灵敏度分别为1.5 pg和10^(4)CFU/mL,重复性和重现性检测结果的相对标准偏差分别小于2%和6%,并且对于高浓度同亚种菌株ATCC 27673的抗干扰能力较强。利用该方法可以实现复合益生菌产品中i797菌株的准确量化分析,为益生菌产品的质量控制和合规性评估提供了有效的解决方案。

Bifidobacterium animalis subsp.lactis i797,a probiotic strain with potential therapeutic applications in paediatric diarrhoea and dyspepsia,was subjected to a comprehensive Whole Genome Sequencing-Single Nucleotide Polymorphism(WGS-SNP)analysis among a set of 23 strains.This analysis identified a unique genomic locus on the K1717_01615 gene that is exclusive to strain i797.Utilizing this locus,we developed primers and probes for a fluorescence quantitative PCR-based quantitative analysis method.The method was capable of distinguishing strain i797 from 18 other Bifidobacterium strains,and demonstrated stability over 40 passages.Sensitivities for DNA concentrations and cell densities of strain i797 were established at 1.5 pg and 10^(4) CFU/mL,respectively.In terms of repeatability and reproducibility,the relative standard deviations of the method were found to be below 2%and 6%,respectively.In addition,the method demonstrated strong anti-interference properties for high cell densities of the same subspecies of strain ATCC 27673.This method enables precise quantitative analysis of strain i797 in complex probiotic products.The study presents an innovative strain-specific quantitative analysis method that can be used as a potent tool for ensuring the quality control and compliance assessment of probiotic products.