SN/T 5227.9-2019 in English
VALIDRapid detection of fox derived ingredient in food for exportRecombinase- -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 background of the standard
This standard, as the 9th part of the SN/T 5227 series, uses the recombinase-mediated strand replacement nucleic acid amplification technology (RAA) to establish a rapid and specific detection system for the possible contamination of fox-derived ingredients in exported food. Compared with the traditional PCR method, this technology has three advantages: constant temperature amplification (37-42℃), fast reaction (completed within 20 minutes) and low equipment requirements.
Core Technology Innovation
| Technical Indicators | Traditional PCR | RAA Method | Improved Effect |
|---|---|---|---|
| Reaction Temperature | Requires variable temperature cycle (95-60℃) | Constant temperature 39℃ | Reduce equipment complexity |
| Reaction Time | ≥90min | 15-20min | Efficiency increased by 4 times |
| Limit of Detection (LOD) | 0.1% | 0.01% | Sensitivity increased 10 times |
Key Operation Points
DNA Extraction Optimization Plan
The standard recommended CTAB method has been specially optimized:
1) 70℃ incubation for 15min: fully lyse the cell membrane containing fox derived components
2) Phenol:chloroform:isoamyl alcohol (25:24:1): improve protein removal efficiency
3) 0.7 times isopropanol precipitation: ensure the recovery rate of trace DNA
RAA reaction system configuration
Key component concentration control:
- Recombinase protein (SC-recA/BS-recA): 300ng/μL
- Bsu DNA polymerase: 75ng/μL
- Fluorescent probe: 0.6μL (10μM)
Quality control system
Standard three-level quality control: 1. Laboratory classification: It is recommended that Class I port laboratories be equipped with constant temperature fluorescence detectors, while Class II ports can use ordinary PCR instruments for modification
1) Instrument calibration: A260/A280 ratio is required to be strictly controlled between 1.7-1.9
2) Control setting: Positive control Ct value ≤30.0 (T value ≤15min)
3) Gray zone retest: Samples with 35.0
Implementation suggestions
2. Staff training: Focus on mastering the precision control of CTAB extraction and RAA reaction system configuration
3. Method validation: LOD validation experiments are required for newly introduced commercial kits
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