SN/T 5227.11-2019 in English
VALIDRapid detection of rat derived ingredient in food for export-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
$156.00
Introduction
Core interpretation of technical standards
This standard adopts a dual-platform verification system of constant temperature fluorescence detector and real-time fluorescence PCR instrument, and uses specific primers to amplify a 198bp fragment of rat mitochondrial cytochrome b gene (Cytb), with a minimum detection limit of 0.1% (W/W).
Comparison of Method Principles
| Technical Parameters | Real-time Fluorescence RAA | Traditional PCR |
|---|---|---|
| Reaction Temperature | 39℃ Constant Temperature | 95℃-55℃ Variable Temperature |
| Reaction Time | within 20 minutes | more than 2 hours |
| Core Enzyme System | SC-recA/BS-recA Recombinase + Bsu Polymerase | Taq DNA Polymerase |
Key Operation Points
Pay special attention during the DNA extraction phase: CTAB buffer needs to maintain pH 8.0, and the transfer of water phase during phenol-chloroform extraction should avoid interfacial protein contamination. A laboratory case showed that the amplification efficiency is best when the A260/A280 ratio is 1.82.
When configuring the amplification system, the timing of adding buffer B (magnesium acetate) directly affects the enzyme activity, and it is recommended to add it in the last step.
Quality Control Standard Analysis
- Positive Control: Ct value ≤ 30.0 (PCR instrument) or T value ≤ 15min (thermostat)
- Gray Zone Determination: 35.0
- Blank Control must have no fluorescence growth, otherwise it indicates contamination
Technology Evolution Background
RAA technology originated from the research and development results of Jiangsu Inspection and Quarantine Bureau in 2016. Compared with traditional PCR: Equipment requirements are reduced by 80% (no thermal cycle module is required), which is particularly suitable for on-site rapid testing at ports. This standard, as Part 11 of the SN/T 5227 series, forms technical mutual recognition with EU Regulation EU/2015/2285.
Implementation recommendations
- Laboratories should regularly verify the recombinant enzyme activity, and it is recommended to perform a positive standard gradient test before each batch of testing
- Add ddH2O washing steps for complex matrix samples (such as condiments)
- Establish a CT value-copy number standard curve to improve quantitative accuracy
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