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What are meat balls made of?

Series: Brief measurement

What are meat balls made of?

Meat balls taste great. Some people may wonder what kind of meat is used to make them?
Some people with allergies may want to know the ingredients, and people in the service industry may want to know if they have indicated ingredients correctly.
You can easily measure the ingredients by using a mobile PCR device.

Sample

Suspension containing 0.1 g of chicken dumpling and 0.9 mL of purified water mixed at this ratio

Pretreatment

The suspension was boiled for approx. 5 minutes, 1 mL of the broth was taken, and it was centrifuged using a tabletop centrifuge for 5 minutes.
(1) 1 μL was taken from near the center and was used as a sample.
(2) The material described in (1) was diluted 100 fold and 1 μL was taken as a sample.

Reaction composition
μL Remarks
Enzyme: KAPA 3G Plant (2.5 U/μL) 0.8 2U/reaction
Buffer (×2) 8 x1
Primer F: beef/pork/chicken (100 μM each) 0.06/0.06/0.1 400/400/600nM
Primer R: beef/pork/chicken (100 μM each) 0.13/0.14/0.1 800/900/600nM
Probe: beef/pork/chicken (100 μM each) 0.03 each 200nM each
MgCl2(25mM) 0.8 1.25mM
Sample 1
PCR grade water 4.7

Reaction sample total 16μL

Program
Process Temperature Time
Initial denaturation 95℃ 15sec
Annealing/extension 60℃ 30sec ※2 50cycle
Denaturation 95℃ 4sec

*2) Reaction condition at the initial development stage. Amplification at 63 degrees Celsius in 15 seconds has been confirmed later. (Please refer to technical data.)

Results

Only the indicator for Poultry increased, indicating the ingredient was chicken.
PCR amplification curves observed in the measurement are shown in the figure below.

Measurement was performed using a multiplex of beef, pork, and chicken.

*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-BHQ1-3’ (25mer) 72.9
Pig-Primer F    5′-CTTGCAAATCCTAACAGGCCTG-3′ (22mer) 66.2
Pig-Primer R   5′-CGTTTGCATGTAGATAGCGAATAAC-3′ (25mer) 65.0
Pig-probe     5’ROX-AACAGCTTTCTCATCAGTTACACACAT-BHQ2-3′ (27mer) 70.2
Chicken-Primer F    5′-TCTGGGCTTAACTCTCATACTCACC-3′ (25mer) 65.6
Chicken-Primer R    5′-GGTTACTAGTGGGTTTGCTGGG-3′ (22mer) 65.6
Chicken-probe    5’Cy5-CATTCCTAACACTAGCCCTATTCTCC-3’BHQ3 (26mer) 70.3

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