SN/T 5227.8-2019 in English
VALIDRapid detection of donkey 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
$146.00
Introduction
Technical background and evolution of the standard
This standard belongs to Part 8 of the SN/T 5227 series, and establishes the recombinase-mediated strand replacement nucleic acid amplification method (RAA) for donkey-derived ingredients in exported food. This technology has the following advantages over traditional PCR:
| Technical parameters | RAA method | Conventional PCR |
|---|---|---|
| Reaction temperature | 37-42℃ constant temperature | Temperature cycling required |
| Detection time | within 20 minutes | 1.5-2 hours |
| Equipment requirements | Constant temperature fluorometer | Thermal cycler |
Core detection principle
Use class=instrument>Bacillus subtilis recombinase (BS-recA) forms a complex with the primer, opens the DNA double helix with the assistance of single-stranded DNA binding protein (SSB), and realizes exponential amplification of the target sequence through the mitochondrial cytochrome b gene (Cytb) specific primer (146bp fragment).
Typical application scenario
A company that exports donkey hide products uses this standard to test the purity of raw donkey hides and finds that horse-derived ingredients are mixed in batch samples, so timely traceability can be used to avoid trade disputes.
Key Operation Points
DNA Extraction Specifications
Note when using the CTAB method for extraction:
1. The degree of sample crushing affects the extraction efficiency
2. The centrifugation parameters must be strictly controlled during the phenol:chloroform extraction stage
3. The A260/A280 ratio should be maintained within the range of 1.7-1.9
Amplification System Configuration
| Components | Concentration Requirements |
|---|---|
| dNTPs | 2.5 mmol/L |
| SSB Protein | 225 ng/μL |
| recA recombinase | 300 ng/μL |
Implementation suggestions
1. The laboratory needs to establish a cross-contamination prevention and control system, referring to Appendix D of GB/T 27403-2008
2. It is recommended to set up three-line controls (positive/negative/blank) for each batch of testing
3. Critical value samples (Ct35-40) should be repeatedly verified
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