SN/T 5227.4-2019 in English
VALIDRapid detection of duck derived ingredient in food for exportReal-time recombinase- -aid amplification ( RAA) method
- Issued on:2019-12-27
- Implemented on:2020-07-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$127.00
Introduction
Technical Standard Background and Evolution
As one of the 11 animal-derived detection standards issued by the General Administration of Customs in 2019, this standard marks the transformation of my country's export food detection technology from traditional PCR to isothermal amplification technology. Compared with traditional methods, the RAA method shortens the detection time from 2 hours to 20 minutes and increases the sensitivity to 0.01% (w/w).
Core Detection Principle
| Technical Elements | Traditional PCR | RAA Method |
|---|---|---|
| Reaction Temperature | 55-95℃ Variable Temperature | 37-42℃ Constant Temperature |
| Key Enzyme | Taq DNA Polymerase | Recombinase (SC-recA/BS-recA) + Bsu Polymerase |
| Detection Time | 120min | 15-20min |
| Equipment requirements | Thermal cycler | Constant temperature fluorometer |
Key operation procedures
DNA extraction optimization plan
When using the CTAB method for extraction, it is necessary to keep the sample fully suspended during the 70℃ incubation stage, and strictly control the 4℃ environment during the isopropanol precipitation stage. Actual cases show that it is recommended to increase the number of chloroform extractions for samples containing oil.
Amplification system configuration
The standard recommended system contains: 300ng/μL recombinase + 75ng/μL Bsu polymerase. In actual applications, it is found that the magnesium ion concentration (280mM) has a significant effect on the amplification efficiency.
Key points of quality control
| Quality control indicators | Fluorescence PCR instrument | Constant temperature detector |
|---|---|---|
| Positive judgment value | Ct≤35.0 | T≤15min |
| Critical value range | 35.0| 15min | |
| Repeat detection requirements | Critical value results need to be retested and confirmed | |
Implementation suggestions
- Primer verification: Sequencing should be performed to confirm the 253bp amplification product for the first use
- Equipment calibration: The temperature deviation of the constant temperature module must be ≤0.5℃
- Cross-contamination prevention and control: Establish a zoning operation process according to GB/T 27403-2008
- Method validation: It is recommended to conduct heat treatment tolerance testing on processed foods
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