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Detection of AYU(sweetfish)

Series: Brief measurement

Detection of AYU(sweetfish)

AYU lives in clean rivers and it tastes great when grilled with salt. However, how can you actually identify AYU?
It may be difficult to accurately determine and confirm ayu by its appearance. An easy way of determination is to check for the AYU gene using a mobile PCR device.
We examined the gene using a mobile PCR device to check if the sample was genuine AYU.

Sample

De-icing fluid from frozen AYU

Pretreatment

De-icing fluid was diluted 1,000 fold and the solution was stirred using a vortex mixer.
(1) 1,000 fold diluted solution
(2) 100,000 fold diluted solution
(3) 10,000,000 fold diluted solution
The solutions above were used as samples.

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  (100μM)※1 0.14 900nM
R-Primer  (100μM)※1 0.14 900nM
Probe  (100μM)※1 0.04 250nM
MgCl2(25mM) 0.8 1.25mM
Sample 1
PCR grade water 5.3

Reaction sample total 16μL

Program
Process Temperature Time
Initial denaturation 95℃ 15sec
Annealing/extension 60℃ 10sec 50cycle
Denaturation 95℃ 3sec
Results

The AYU gene was detected even in the 10,000,000 fold diluted de-icing fluid.
This indicates that the gene can be detected even when a slight amount is mixed in food.
Furthermore, there is a possibility that ayu may be detected even when it is in a river.

*1 Instruction was kindly given by Dr. Doi from the University of Hyogo.
Tm
Paa-CytB-Primer F    5′-CCTAGTCTCCCTGGCTTTATTCTCT-3′ (25mer) 65.5
Paa-CytB-Primer R    5′-GGTTACTAGTGGGTTTGCTGGG-3′ (21mer) 65.8
Paa-CytB-probe    5’FAM-ACTTCACGGCAGCCAACCCCC-BHQ1-3′ (21mer) 76.1

Made by Nihon Gene Research Laboratories Inc.
Tm values are calculated using the nearest neighbor method.
Reference paper:Doi et al., Environmental DNA analysis for estimating the abundance and biomass of stream fish.
Freshwater Biology, Volume62, Issue1