SN/T 5204-2020 in English
VALIDPlant pathogenic bacteria screen MALDI-TOF MS method
- Issued on:2020-12-30
- Implemented on:2021-07-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$146.00
Introduction
Technical background of the standard
This standard was formulated by the General Administration of Customs to meet the needs of rapid detection of pathogenic bacteria in import and export plant quarantine. It uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology to establish a standardized screening process. Compared with traditional culture and identification methods, the detection cycle is shortened from 5-7 days to within 24 hours.
Core Detection Principles
| Technical Elements | Parameter Requirements | Biological Basis |
|---|---|---|
| Detection Targets | 200-6000 Proteins | Species-specific Proteins Accounting for 50% of the Dry Weight of Bacteria |
| Mass Range | 1000-20000 Da | Small Molecular Peptides Such as Ribosomal Proteins |
| Ion Mode | Linear Positive Ion | HCCA Matrix Optimization of Protonation Efficiency |
Key Operation Specifications
Bacteria pretreatment process
- Water washing and impurity removal: 1-2mg bacteria + 1mL deionized water centrifugation
- Solvent fixation: 300μL water + 900μL ethanol gradient treatment
- Protein extraction: 70% formic acid + acetonitrile ultrasonic lysis
Mass spectrometry parameter setting
- Laser energy dynamic adjustment range: 0-100%
- Calibration standard: Peptide/Protein Calibration Standard mixed solution
- Spotting requirements: 4 technical replicates + 1 calibration control
Result judgment criteria
| log(score) value | Judgment level | Subsequent processing |
|---|---|---|
| ≥2.2 | Species level identification | Report can be issued directly |
| 2.0-2.2 | Genus level identification | Supplementary biochemical test is recommended |
| <2.0 | Undeterminable | Traditional methods need to be reviewed |
Implementation points
Quality control
Before each test, the standard calibration solution must be used for instrument calibration. It is recommended to insert 1 known strain for every 20 samples as process control.
Database construction
The standard requires the use of the instrument's own database or a self-built equivalent database. It is recommended that quarantine agencies establish a local fingerprint library of common quarantine pathogens.
Sample preservation
Positive samples need to be stored at ultra-low temperatures (-80°C) for more than 6 months, and the original mass spectrometry data should be backed up to an independent storage device.
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