SN/T 5145.7-2019 in English
VALIDRapid ldentifieation of animal ingredient in export food and feed Part 7 : Detection of Ovis aries 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
Technical background of the standard
As an important part of the entry-exit inspection and quarantine industry standard system, this standard has established a rapid screening technology of PCR-test strip method to meet the regulatory needs for the accuracy of animal-derived ingredient identification in international trade. After its release and implementation in 2019, it effectively solved the problems of traditional detection methods, such as long time consumption (sequencing confirmation is required) and high cost.
Principle of the core method
| Technical links | Key parameters | Mechanism of action |
|---|---|---|
| DNA extraction | CTAB lysis + phenol chloroform purification | Removal of protein/polysaccharide interference |
| PCR amplification | CYT-B gene specific primers | FITC/Biotin dual labeling |
| Test strip detection | Gold-labeled antibody-streptavidin system | Dual-line colorimetric determination |
Typical application case: A mutton product exporting company used this method to detect 0.5% sheep bone meal mixed in feed, thus avoiding trade disputes caused by halal certification issues.
Key operating specifications
- Primer design: Targeting sheep mitochondrial CYT-B gene (GenBank: AF010406.1), forward primer 5'-TCCATATATTGGCACAAACC-3' contains FITC label
- PCR system: 30 cycle amplification (94℃ 30s→55℃ 30s→72℃ 30s), positive and negative controls need to be set
- Quality control requirements: A260/A280 ratio 1.7-1.9, amplification products need to be verified by sequencing (homology ≥97%)
Comparison of technical advantages
| Detection method | Time consumption | Sensitivity | Equipment requirements |
|---|---|---|---|
| PCR-test strip method | 3 hours | 0.1% | Conventional PCR instrument |
| Real-time fluorescence PCR | 2 hours | 0.01% | Requires fluorescence quantification instrument |
| DNA sequencing method | 24 hours | 0.1% | Sequencing platform |
Implementation suggestions
- Sample pretreatment: For high-salt/high-sugar samples, additional washing steps are required. For feed samples, the sampling volume is adjusted according to the particle size (200 mg for 20 mesh)
- Pollution prevention measures: Strictly implement the laboratory zoning requirements of GB/T 27403-2008, and waste products need to be incinerated
- Verification requirements: New batches of test strips need to use standard substances to verify the color validity of the quality control line/detection line
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