Table 3 dPCR measurement results due to double colours of FAM (16S rDNA specific) and HEX (hsp60) DNA probes for chromosomal DNA that were extracted from heat-killed (HK) L. paracasei-supplemented nutritional food products.

From: Exclusive use of digital PCR allows an absolute assay of heat-killed Lactobacilli in foods targeting multiple copies of 16S rDNA

Test samplea

DNA extraction

16S rDNA (copies/µl) in the direct master mix

hsp60 gene (copies/µl) in the direct master mix

Ratio (16S rDNA/ hsp60 gene)

Actually chromosomal DNA conc. with Test sample (ng/µl) due to OD260b

Chromosomal DNA in the direct master mix (molecules/µl) due to OD260b

HK-L. paracasei-NFY

1st

3,914.5

546.3

7.2

1.50 × 10–1

3.00 × 103

2nd

3,629.7

525.5

6.9

1.55 × 10–1

3.10 × 103

3rd

3,112.8

460.1

6.8

1.35 × 10–1

2.70 × 103

HK-L. paracasei-NFS

1st

1948.9

347.9

5.6

1.30 × 10–1

2.60 × 103

2nd

1915.6

352.4

5.4

1.10 × 10–1

2.20 × 103

3rd

2,808.8

422.9

6.6

1.45 × 10–1

2.90 × 103

HK-L. paracsei-NFM

1st

3,157.1

455.0

6.9

1.15 × 10–1

2.30 × 103

2nd

2,834.2

392.2

7.2

1.50 × 10–1

3.00 × 103

3rd

2,706.5

422.9

6.4

1.15 × 10–1

2.30 × 103

HK-L. paracasei-NFO

1st

1,483.7

296.7

5.0

6.50 × 10–2

1.30 × 103

2nd

2,207.7

356.8

6.2

1.00 × 10–1

2.00 × 103

3rd

2,174.9

311.1

7.0

9.00 × 10–2

1.80 × 103

Test samplea

DNA extraction

DNA recovery rate due to OD260c

Absolute dPCR assay (cells/ml)d

Absolute dPCR assay for products

Mean (cells/ml)

SD (cells/ml)

RSD (%)

HK-L. paracasei-NFY

1st

0.60

7.60 × 107

7.27 × 107

2.83 × 106

3.9

2nd

0.62

7.11 × 107

3rd

0.54

7.11 × 107

HK-L. paracasei-NFS

1st

0.52

5.61 × 107

6.17 × 107

5.75 × 106

9.3

2nd

0.44

6.76 × 107

3rd

0.58

6.15 × 107

HK-L. paracsei-NFM

1st

0.46

8.34 × 107

7.18 × 107

1.49 × 107

20.8

2nd

0.60

5.50 × 107

3rd

0.46

7.71 × 107

HK-L. paracasei-NFO

1st

0.26

9.57 × 107

8.09 × 107

1.28 × 106

15.8

2nd

0.40

7.47 × 107

3rd

0.36

7.24 × 107

  1. aProducts, nutritional foods with yogurt (NFY), strawberry (NFS), milk tea (NFM)or orange (NFO) flavour originally supplemented with HK-L. paracasei cells were applied to DNA extraction. In view of originally supplemented concentration of HK-L. paracasei, a tenfold dilution of purified DNA concentration (ng/µl) used for the absolute dPCR was 0.25 ng/µl if 100% recovery obtained, which corresponds to 5.0 × 103 chromosomal DNA molecules/µl of the direct master mix.
  2. bThe values correspond to 1–10th of the relevant data of Table 1 owing to the tenfold dilution of purified DNA solutions. “Chromosomal DNA molecules in the direct master mix (molecules/µl) due to OD260” is calculated, quoting one molecule = 5 fg of chromosomal DNA for prokaryotic cell.
  3. cDNA recovery rate due to OD260 was calculated using “Chromosomal DNA molecules in the direct master mix (molecules/µl) due to OD260/just upper-mentioned 5.0 × 103 chromosomal DNA molecules/µl of the direct master mix”.
  4. dAbsolute assay data was obtained from the successive numerical formula: “16S rDNA (copies/µl) in the direct master mix/Ratio (16S rDNA/hsp60 gene) × a dilution (tenfold) of purified DNA solution × addition of diluted DNA to the direct master mix at a tenfold dilution × purified DNA solution volume (0.2 ml)/volume for diluted test samples that were supplied for DNA extraction (1.5 ml) × original dilution factor (3) for nutritional foods × 1,000 (owing to a conversion of cells/µl to cells/ml) × 1/DNA recovery rate × 100 /Mean value of 47.7 of reaction rate (%) in Supplementary Table S2”.
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