Development of a nucleic acid based assay to monitor and quantify viable yeast cells

养牛业是加拿大主要的农业部门之一，为人类提供动物蛋白（牛奶和肉类）。近年来，直接饲喂微生物被认为是通过强化微生物发酵来提高动物生产力和健康的长期有效途径之一。（LaSalle，QC）是市售的活酵母产品之一（酿酒酵母品种），能改善纤维消化，防止瘤胃代谢功能障碍，从而使牛具有更高的生产力和更好的健康状况。在农场，Biomate通常与饲料混合，并全天提供给动物，以实现产品的最大效果。因此，在饲料中保持高水平的活细胞是至关重要的。目前，本品在饲料中的活性评价采用培养依赖性方法。为了评估酵母蛋白饲料的活细胞，酵母的培养通常在AB Mauri的实验室进行，这需要3-4天的时间，这是耗时和劳动力成本。此外，由于人工计数菌落的性质，该方法具有较大的误差范围。此外，酵母菌的可培养性有时会受到饲料中高含量矿物质或其他成分的影响，从而导致样品中活酵母菌的低估。虽然PCR方法可以用来监测目标微生物的数量，但它不能区分非活细胞和活细胞的比例，这可能会导致高估样品中的活细胞。在这项研究中，我们建议使用微生物基因组学和基于分子生物学的方法来识别和靶向活酵母细胞的“标记基因”。我们将通过比对约120个现有的酵母基因组来鉴定DNA标记，然后开发单一或多重PCR检测。然后将使用AB Mauri的混合饲料样品验证该测定法，并与基于培养的方法进行比较。这种快速有效的DNA/RNA检测方法将为AB Mauri实验室提供一种可靠的定量和监测饲料中活酵母细胞的方法。

Cattle industry is one of the major Canadian agriculture sectors, which provides the animal proteins (milk andmeat) to humans. Recently, the direct fed microbes have been considered as one of the powerful approaches tohave long term effect to improve the animal productivity and health through enhancing microbial fermentation.Biomate, manufactured by AB Mauri North America (LaSalle, QC) is one of the commercial available liveyeast products (Saccharomyces cerevisiae variety) that can improve fiber digestion and prevent the ruminalmetabolic dysfunction, resulting cattle with greater productivity and better health. On farm, the Biomate isusually mixed with the feed and provided to the animals through the day to achieve the maximum effect of theproduct. Therefore, it is essential to maintain the high level of the viable cells in feed. Currently, the evaluationof the activity of this product in feed uses the culture dependent methods. To evaluate the viable cells of yeastin feed, the cultivation of the yeast is usually performed at AB Mauri's lab, which takes 3-4 days that is timeconsuming and labor costly. Also, this method has a large error margin due to the manual nature of countingcolonies. In addition, the cultivability of yeast sometime is affected by the high content of minerals or othercomponents in feed, which results in an underestimation of viable yeast in the sample. Although the PCRmethod can be used to monitor the population of targeted organisms, it can't differentiate the proportion ofnon-viable and viable cells, which could lead to over-estimation of the viable cells in the sample. In this study,we propose to identify and target "marker genes" for viable yeast cells using microbial genomics and molecularbiological based approaches. We will identify the DNA markers by aligning ~120 existing yeast genomes andthen develop a single or multiplex PCR assay. The assay will then be validated using the mixed feed samplesfrom AB Mauri, together with the comparison of the cultured based methods. The rapid and effectiveDNA/RNA based method will provide a reliable assay to quantify and to monitor the alive yeast cells in feedfor AB Mauri lab.