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Does chicken bone broth contain only chicken?

Series: Brief measurement

Does chicken bone broth contain only chicken?

Authentic chicken bone broth is made by slowly boilied chicken meat and bones.
It is a classic soup and anyone can very easily create a light yet rich taste. Is this taste really delivered from the components of chicken?
We checked it using a mobile PCR device.

Sample

Suspension of 0.1 g of chicken bone broth (made by company F) and 0.9 mL of purified water mixed at this ratio

Pretreatment

The suspension was stirred using a vortex mixer for 5 minutes and left for 5 minutes (no centrifugation).
1 μL was taken from near the center and was used as a sample.

Reaction composition
μL Remarks (final concentration)
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 64℃ 20sec 45cycle
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 chicken increased, indicating that chicken is used as the ingredient.
Real-time PCR amplification curves observed in the measurement are shown in the figure below.

Measurement was performed using a multiplex of cow, pig, 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