COMPARISON OF DNA STANDARDS FOR REAL-TIME PCR-BASED QUANTIFICATION OF LACTOBACILLUS ACIDOPHILUS IN DAIRY PRODUCTS: DNA standards for quantification of probiotics

Monir-sadat Shakeri
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Abstract

Probiotic bacteria are an essential part of the healthy gut microbiota. Fermented foods as potential sources of health-promoting bacteria can regulate the intestinal microbial population. However, the exact quantification of these bacteria in such multiple-strain matrixes continues to remain elusive. In this study, we evaluated the reliability of genomic DNAs and cloned recombinant plasmids as standard controls for absolute real-time PCR assay. The associated standard curves were constructed and used for the quantification of Lactobacillus acidophilus probiotics. All stages from the design and construction of standards and related curves met the criteria for high-quality products. There were no significant differences between the two enumeration methods. However, plasmid-based standard curves resulted in a lower detection limit than the curves of genomic DNA standards. Our findings showed that the non-linearized recombinant plasmids had long-term stability at high concentrations during storage at -20 °C, which strongly depended on the purification methods. We propose that the recombinant plasmid standards can supersede the traditional genomic DNA standards for accurate quantification of probiotic bacteria.
乳制品中嗜酸乳杆菌实时pcr定量的DNA标准比较:益生菌定量的DNA标准
益生菌是健康肠道菌群的重要组成部分。发酵食品作为促进健康的细菌的潜在来源,可以调节肠道微生物群。然而,这些细菌在这种多菌株基质中的确切定量仍然难以捉摸。在这项研究中,我们评估了基因组dna和克隆的重组质粒作为绝对实时PCR检测的标准对照的可靠性。建立相关标准曲线,用于嗜酸乳杆菌益生菌的定量。从设计和施工的各个阶段的标准和相关曲线都达到了高质量产品的标准。两种计数方法间无显著差异。但质粒标准曲线的检出限低于基因组DNA标准曲线。我们的研究结果表明,非线性重组质粒在-20°C的高浓度下具有长期稳定性,这在很大程度上取决于纯化方法。我们认为重组质粒标准物可以取代传统的基因组DNA标准物来精确定量益生菌。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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