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Dairy product residual volume in water discharged from the washing process of dairy production lines

Series: Ernestly measurement

Dairy product residual volume in water discharged from the washing process of dairy production lines

These days, a single line is used for producing various products. Lines are cleaned after manufacturing a product and then used to manufacture a different product.
Some people may wonder if the line is actually clean. This is especially worrisome when dealing with allergens.
We checked the actual condition using a mobile PCR device.

[First step: Creation of a calibration curve]

Sample

Milk

Pretreatment

Only dilution to achieve the following concentrations
106,104,103,102,101 pL/reaction as well as negative control

Reaction composition
μL Remarks (final concentration)
Enzyme: KAPA 3G Plant (2.5 U/μL) 0.6 1.5U/reaction
Buffer (×2) 8 x1
F-Primer: Bovine (100 μM) 0.06 400nM
R-Primer: Bovine (100 μM) 0.13 800nM
Probe: Bovine (100 μM) 0.03 200nM
MgCl2(25mM) 0.8 1.25mM
Sample 1
PCR grade water 5.4

Reaction sample total 16μL

Program
Process Temperature Time
Initial denaturation 95℃ 15sec
Annealing/extension 65℃ 20sec 50cycle
Denaturation 95℃ 4sec
Unit: pl/rxn Ct value
1×106 28.6
1×104 35.5
1×103 39.4
1×102 42.0
1×101 45.3
Negative control 0.0
slope -3.34
intercept 48.84
R2 1.00
Amplification efficiency 99%

[Second step: Measurement of residual milk volume]

Sample

Water discharged from the cleaning process for dairy production lines

Pretreatment

None

Template Ct Residual milk volume
(pl/6ul)
ppm
A 39.52 334.8 55.3
B 39.48 345.7 57.1
C 41.34 91.1 15.1
Negative control Not detected
*1 Primer/probe sequence
Tm
Cow-Primer F    5′-CCCGATTCTTCGCTTTCCAT-3′ (20mer) 66.8
Cow-Primer R    5’-CTACGTCTGAGGAAATTCCTGTTG-3′ (24mer) 65.4
Cow-probe         5’FAM-CATCATAGCAATTGCCATAGTCCAC-3’BHQ1 (25mer) 72.9

Made by Nihon Gene Research Laboratories Inc.
Tm values are calculated using the nearest neighbor method. However, the calculation method of Nihon Gene Research Laboratories was used for the probes.
Reference paper:Tanabe et al., A Real-Time Quantitative PCR Detection Method for Pork, Chicken, Beef, Mutton, Horseflesh in Foods.
Biosci. Biotechnol. Biochem., 2007, 71(12),3131-3135