SN/T 5198-2020 in English
VALIDQuarantine protocol for Crithidia bombi
- Issued on:2020-12-30
- Implemented on:2021-07-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$204.00
| Standard No: | SN/T 5198-2020 |
| Document status: | VALID |
| Title in English: | Quarantine protocol for Crithidia bombi |
| Title in Chinese: | 熊蜂短膜虫检疫技术规范 |
| Language: | English |
| File Format: | Electronic (PDF) |
| Delivery: | Via email within 1~3 business days |
| Issued on: | 2020-12-30 |
| Implemented on: | 2021-07-01 |
| Professional Classification: | SN-Import&Export Inspection |
| Related Topics: | bumblebee
Membrane amount detection downstream primer upstream primer upstream primer membrane gene Quarantine technology Kumamoto Quarantine Pupa Inspection and quarantine of quarantine pests and diseases Safety Technical Specification Arbutin |
Introduction
Background of Standard Technology Evolution
This standard replaces SN/T 1162-2002, with major updates: Introduction of real-time fluorescence PCR technology, optimization of DNA extraction process, Addition of ITS gene-specific primers (amplification fragment 447bp/80bp). The revised detection sensitivity is increased by 103 times, and the detection cycle is shortened to 4 hours.
Comparison of core detection methods
| Technical parameters | Conventional PCR | Real-time fluorescence PCR |
|---|---|---|
| Detection limit | 102 copies/μL | 10-1 copies/μL |
| Key equipment | Gradient PCR instrument+electrophoresis system | Fluorescence PCR instrument |
| Interpretation standard | 447bp band+sequencing verification | Ct value ≤ 35 (FAM channel) |
| Biosafety requirements | All operations must be performed in a biosafety cabinet | |
Analysis of key operating points
Typical case of DNA extraction
When the Fuzhou Customs Laboratory used the CTAB/NaCl method to process samples, it was found that liquid nitrogen grinding increased the DNA yield by 42% compared with the PBS homogenization method. It is recommended to use -196℃ quick freezing treatment as a priority. However, please note:
1. Wear antifreeze gloves when operating with liquid nitrogen
2. Add TE buffer immediately after grinding to prevent degradation
Implementation suggestions
- Sampling strategy: Use step-by-step sampling according to the size of the bee colony (full inspection of 10 colonies → 10% of 201 colonies)
- Quality control points: Each batch must contain a positive control (containing the target DNA fragment) and a blank control
- Equipment calibration: The fluorescence PCR instrument needs to be calibrated monthly to ensure the sensitivity of the FAM channel
- Result review: The critical value sample (Ct35-40) should be tested repeatedly and the amplification curve morphology should be analyzed
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