Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability - up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

中文翻译：

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液滴数字PCR是一种可行的益生菌菌株高质量计数的改进替代方法。

长期以来，传统的微生物计数方法一直被用作益生菌微生物的标准评估程序。这些方法是劳动密集型的，需要很长时间才能获得结果，并且固有地具有很高的可变性-高达35％。随着临床益生菌和微生物组科学的不断发展和发展，研究人员彻底定义和提供目标益生菌剂量变得越来越重要。此外，为了建立高质量的商业产品，必须向消费者施用相同的剂量水平。已经建立了使用流式细胞术的ISO方法，该方法确实加快了结果的产生时间并减少了变异性，但是该方法尚未在整个益生菌行业中得到广泛采用。这可能是由于在已建立的质量体系中实施和维护新的测试平台所需的专业知识。在这项研究中，我们将使用板计数和流式细胞术的计数与使用液滴数字PCR（ddPCR）进行了比较，这不仅比板计数提供了更快的结果时间，而且比板计数和流式细胞术具有更少的变异性，还具有其他优势，例如作为特定菌株的计数。ddPCR的使用提供了设计引物以靶向缺失和单碱基对差异的能力，这将允许在微生物组分析中进行菌株谱分析。我们证明了ddPCR益生菌的计数结果与平板计数和流式细胞仪结果均呈正相关，因此应被视为评估益生菌的可行的下一代计数方法。