SN/T 5227.7-2019 in English
VALIDRapid detection of horse 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
$127.00
Introduction
Technical background of the standard
This standard belongs to Part 7 of the SN/T 5227 series. It aims to meet the demand for rapid detection of horse (Equus caballus)-derived ingredients in exported food. It adopts recombinase-mediated chain replacement nucleic acid amplification technology (RAA), which has the advantages of constant temperature amplification (39°C) and rapidity (completed within 20 minutes) compared with traditional PCR technology. The minimum detection limit is 0.1% (W/W).
Analysis of core methods
| Technical elements | Traditional PCR | RAA method | Advantage comparison |
|---|---|---|---|
| Reaction temperature | Thermal cycle (95℃-60℃) | Constant temperature 39℃ | No temperature change equipment required |
| Reaction time | ≥90min | ≤20min | Efficiency increased by 4.5 times |
| Enzyme system | Taq DNA polymerase | Recombinase + SSB protein + Bsu polymerase | Higher chain replacement efficiency |
| Detection limit | 0.1%-1% | 0.1% | Equivalent sensitivity |
Typical application scenario
A meat export company found that the T value of the constant temperature fluorescence detector was 14.5min. After review and confirmation, it was determined to be positive. Tracing back to the source found that horse meat was mixed in the raw materials, thus avoiding major trade disputes.
Key Operation Points
- DNA extraction: The CTAB method requires strict control of the phenol:chloroform extraction ratio (25:24:1)
- Primer design: Targeting mitochondrial ATP synthase 6 gene (211bp), the probe contains FAM-dT/THF modification
- System configuration: The concentration of recombinase (SC-recA/BS-recA) needs to be maintained at 300ng/μL±5%
Quality control requirements
| Control type | Fluorescence PCR instrument standard | Constant temperature detector standard |
|---|---|---|
| Positive control | Ct≤30.0 | T≤15min |
| Negative control | No Ct value | No T value |
| Blank control | No amplification curve | No signal growth |
Implementation suggestions
- Laboratories need to establish a molecular detection quality control system in accordance with GB/T 27403
- Regularly verify the activity of Bacillus subtilis recombinase (BS-recA)
- For complex matrix samples, it is recommended to add a PEG precipitation purification step
Technology evolution analysis
Since RAA technology was incorporated into the inspection and quarantine standard system in 2016, the detection species have been expanded to 11 types of animal-derived ingredients. In the future, it may develop in the following directions:
- Development of multiple RAA detection systems
- Application of freeze-dried reagent technology
- Integration with microfluidic chips
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