Abstract
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.
Highlights
The specific term, digital polymerase chain reaction first appeared in a report by Kinzler and V ogelstein[1]
If relevant chromosomal DNA is isolated in the least artificially digestive manner during DNA extraction, five copies of 16S rDNA might be located on an intact chromosomal DNA, which can be analysed by digital PCR (dPCR) (Fig. 4a)
When PCR inhibitors existing in an NF supplemented with HK-Lactobacilli to improve host immune defence, DNA isolation is inevitable to circumvent under-estimation results by dPCR
Summary
The specific term, digital polymerase chain reaction (dPCR) first appeared in a report by Kinzler and V ogelstein[1]. DPCR has been applied to viable cells or active viruses in test samples in the fields of clinical, environmental, and food s cience[2,3,4,5,6,7,8,9,10,11]. Macrophage activation accelerates producing cytokines responsible for clinical effects to pathogenic infections[18,19] It was demonstrated the intact and/or close to intact form of Lactobacilli that sustains both the relevant genetic components and PGN could be recommendable to increase host immune r esponse[13]. Some nutritional foods (abbreviated as NFs; a singular form of NF) supplemented with heat-killed Lactobacilli that comprises of both the relevant genetic components and PGN becomes launched in the worldwide market. We believe that the assay should be optimised to stay unaffected even by different DNA recovery rates in the same sample
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