Functional Food Ingredients Group, Food Ingredients and Technology Institute, R & D Division, Morinaga Milk Industry Co. Ltd., 5-1-83, Higashihara, Zama, Kanagawa, 252-8583, Japan.
Sci Rep. 2020 Jul 29;10(1):12691. doi: 10.1038/s41598-020-69206-5.
The real-time PCR (qPCR) and digital PCR (dPCR) to amplify a single-copy of house-keeping genes (i.e., hsp60, pheS or tuf) are used for the assay of limited microbial species. In general, with a single-copy gene, there are obviously varied DNA sequences for even the same microbial species, which could cause difficulties with design of primers and probes for PCR when targeting various single copy genes. In general, for identification by dPCR (as a representative case: Lactobacillus paracasei), accumulated DNA sequence information of 16S rDNA, which is much more frequently used, should be targeted. In contrast, next-generation sequencing revealed that there are five copies of 16S rDNA in a live L. paracasei MCC1849. Therefore, we aimed to reveal, if heat-killed L. paracasei supplemented in nutritional foods that aid the host immune system have the relevant five copies per chromosomal DNA, and if the relevant copies remain unchanged on the same chromosomal DNA or remain to be different chromosomal DNA fragments. So, we revealed the actual distribution of the potential original five copies of 16S rDNA using our innovative dPCR, in which both 16S rDNA and hsp60 genes were simultaneously elongated. The molecular ratios of 16S rDNA/hsp60 dispersed in the dPCR chip were then estimated. The 16S rDNA/hsp60 molecular ratios of the heat-killed L. paracasei in foods, resultantly ranged from 5.0 to 7.2, being the same or higher than that of the five copies determined by next-generation sequencing. The 16S rDNA copy number/ratio indicated the chromosomal DNA molecular number and the associated cell number. As significance, different nutritional foods could potentially cause the loss of chromosomal DNA of supplemented beneficial microbes to a much greater degree. Our absolute dPCR does not require standard correlative samples for the estimation of final products. The estimation principle of the ratio of 16S rDNA/a house-keeping single-copy gene by our absolute dPCR could lead to a useful and accurate assay for various nutritional foods.
实时聚合酶链反应（qPCR）和数字聚合酶链反应（dPCR）扩增单个管家基因（即 hsp60、pheS 或 tuf）用于有限微生物物种的检测。通常，对于单个拷贝基因，即使是相同的微生物物种，其 DNA 序列也有明显的差异，这可能会导致针对各种单拷贝基因设计引物和探针时出现困难。一般来说，对于 dPCR 的鉴定（以乳杆菌属 paracasei 为例），应针对使用更频繁的 16S rDNA 积累的 DNA 序列信息进行靶向。相比之下，下一代测序表明，活的 L. paracasei MCC1849 中有 5 个拷贝的 16S rDNA。因此，我们旨在揭示在有助于宿主免疫系统的营养食品中添加热灭活的 L. paracasei 是否具有相关的每条染色体 DNA 五个拷贝，如果相关拷贝在同一染色体 DNA 上保持不变或仍然是不同的染色体 DNA 片段。因此，我们使用我们的创新 dPCR 揭示了潜在的原始 16S rDNA 五个拷贝的实际分布情况，其中同时延长了 16S rDNA 和 hsp60 基因。然后估计 dPCR 芯片中分散的 16S rDNA/hsp60 的分子比。结果，食品中热灭活的 L. paracasei 的 16S rDNA/hsp60 分子比范围为 5.0 至 7.2，与通过下一代测序确定的五个拷贝相同或更高。16S rDNA/hsp60 的拷贝数/比指示染色体 DNA 分子数和相关细胞数。意义在于，不同的营养食品可能会导致补充有益微生物的染色体 DNA 大量丢失。我们的绝对 dPCR 不需要标准相关样本来估计最终产物。我们的绝对 dPCR 对 16S rDNA/管家单拷贝基因比值的估计原理可以为各种营养食品提供有用且准确的检测方法。
