Lomonaco Sara, Furumoto Emily J, Loquasto Joseph R, Morra Patrizia, Grassi Ausilia, Roberts Robert F
Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, 10095 Grugliasco TO, Italy; Department of Food Science, The Pennsylvania State University, University Park 16802.
Department of Food Science, The Pennsylvania State University, University Park 16802.
J Dairy Sci. 2015 Feb;98(2):804-12. doi: 10.3168/jds.2014-8509. Epub 2014 Dec 26.
Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing protocol, resulting in a SNP profile matching the profile for the strain BB-12.
当向食品中添加益生菌微生物时，在属、种和菌株水平上进行鉴定是很有必要的。动物双歧杆菌乳亚种（BAL）的菌株在全球范围内常用于添加了益生菌菌株的乳制品中。然而，由于该亚种不同基因组之间的基因组同一性程度很高（99.975%），因此菌株鉴别很困难。通过靶向信息性单核苷酸多态性（SNP），可以有效地对单态性物种进行分型。先前一项分析BAL参考菌株和商业菌株的研究结果确定了可用于将常见菌株分为8组的SNP。本文描述了一种基于引物延伸反应（PER）的微测序分析方法的开发，该方法靶向多个SNP，可实现BAL的菌株分化。基于先前的数据，选择了6个信息性SNP进行进一步测试，并优化了多重初步PCR以扩增包含所选SNP的DNA区域。开发了与所选SNP紧邻退火的延伸引物（EP），并在单重和多重PER中进行测试以评估其性能。选择了属于动物双歧杆菌乳亚种9个不同基因组簇的25个菌株，并使用开发的微测序分析方法进行分析，同时靶向6个所选SNP。随后进行了重复的片段分析，结果表明该分析产生了8种特定图谱，可区分最常用的商业菌株。这种新颖的多重PER方法提供了一种简单、快速、灵活的基于SNP的分型方法，用于正确表征和鉴定发酵乳制品中BAL的商业益生菌菌株。为了评估该方法的实用性，从添加和未添加动物双歧杆菌乳亚种BB-12的酸奶中提取DNA。然后将提取的DNA进行微测序分析，得到的SNP图谱与BB-12菌株的图谱相匹配。
