SN/T 5145.5-2019 in English
VALIDRapid Identification of animal ingredient in export food and feed Part 5: Detection of Canis Ingredient PCR-Lateral flow dipstick method
- Issued on:2019-10-25
- Implemented on:2020-05-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$117.00
Introduction
Interpretation of standard technology
| Technical parameters | This method | Conventional PCR | ELISA |
|---|---|---|---|
| Detection principle | PCR-lateral flow chromatography | Electrophoresis analysis | Antibody binding |
| Detection limit | 0.1% | 1% | 5% |
| Operation time | 2.5 hours | 4 hours | 6 hours |
Core Technology Innovations
Using CoxI gene-specific primers (F:5'-GGCTCATTTTCACTATGTGC-3'/R:5'-CGATCCTATAGAGGAGACG-3'), combined with double-labeled probe technology:
1. 5'-end labeled with FITC fluorescein
2. 3'-end labeled with biotin, visual detection through lateral flow chromatography test strips
Key Operation Flow
- DNA Extraction: Samples were treated with the CTAB method and purified with chloroform/isoamyl alcohol
- PCR Amplification: Pre-denaturation at 94℃ for 5min, 30 cycles (94℃ 30s→55℃ 30s→72℃ 30s)
- Chromatographic detection: After the amplified product is mixed with the buffer, the test strip is added and the result is judged in 5 minutes
Implementation points
Quality control requirements
1. Negative control (blank extraction), positive control (known canine DNA) and internal standard control should be carried out simultaneously
2. The A260/A280 ratio should be between 1.7-1.9
3. The laboratory environment should meet the requirements of GB/T 27403-2008
Common problem handling
When a false positive occurs, the following should be carried out:
- Sequencing verification (comparison with NCBI database ≥97%)
- Re-extraction of DNA for testing
- Check for primer dimer contamination
Application value of the standard
This standard is applicable to:
1. Safety supervision of import and export feed
2. Traceability of pet food raw materials
3. Religious food certification (such as halal food testing)
4. Identification of adulteration of animal-derived ingredients
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