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

开发基于核酸的检测方法来监测和定量活酵母细胞

• 批准号: 518071-2017
• 负责人: Guan, Leluo
• 金额: $ 1.82万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Engage Grants Program
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=640745
• 关键词: Development; nucleic; acid; based; assay

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.

养牛业是加拿大主要农业部门之一，为人类提供动物蛋白（牛奶和肉类）。最近，直接饲喂微生物被认为是通过增强微生物发酵来长期提高动物生产力和健康的有力方法之一。 Biomate 由 AB Mauri North America（拉萨尔，QC）制造，是一种市售的产品。活酵母产品（酿酒酵母品种）可以改善纤维消化并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，保持饲料中活细胞的高水平至关重要。目前，该产品在饲料中的活性评价采用的是培养依赖性方法。为了评估酵母饲料的活细胞，酵母的培养通常在 AB Mauri 的实验室进行，需要 3-4 天，费时费力。此外，由于菌落计数的手动性质，该方法具有较大的误差范围。此外，酵母的培养能力有时会受到饲料中矿物质或其他成分含量高的影响，从而导致样品中活酵母的低估。虽然PCR方法可用于监测目标生物体的数量，但它无法区分死细胞和活细胞的比例，这可能导致样本中活细胞的高估。在这项研究中，我们建议使用微生物基因组学和分子生物学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 个现有酵母基因组来识别 DNA 标记，然后开发单一或多重 PCR 测定。然后将使用 AB Mauri 的混合饲料样品以及基于培养的方法的比较来验证该测定。这种快速有效的基于 DNA/RNA 的方法将为 AB Mauri 实验室的饲料中活酵母细胞的定量和监测提供可靠的检测方法。