SN/T 5181-2020 in English
VALIDQuarantine protocol for gold fish haematopoietic necrosis
- Issued on:2020-12-30
- Implemented on:2021-07-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$204.00
Introduction
Analysis of the technical framework of the standard
| Technical module | Traditional method | Innovation of this standard |
|---|---|---|
| Detection method | Virus isolation and culture | Dual PCR system (conventional + fluorescence) |
| Sensitivity | 103 copies/μL | 101 copies/μL |
| Interpretation criteria | Single clinical symptom | Symptom + molecular detection matrix |
Detailed explanation of core detection technology
The standard adopts the CTAB method to extract viral DNA. The key steps include:
- After tissue homogenization, centrifuge at 4℃ 8000r/min to obtain the supernatant
- CTAB solution 25℃ for 2.5 hours to lyse cells
- Double organic solvent extraction (phenol: chloroform: isoamyl alcohol = 25:24:1)
- After ethanol precipitation, store at -20℃
Actual application case
In 2021, Shenzhen Port will implement this standard test on imported ornamental fish. The positive sample with a Ct value of 34.2 was found in the real-time fluorescence PCR test. Sequence comparison confirmed that the homology with the reference strain (Genbank AY939863) was 99.2%, successfully blocking the virus transmission.
Recommendations for the implementation of the standard
- Sampling specifications: Fish with a body length of ＞6cm need to collect multiple organs including kidney, spleen, gills and brain
- Quality control points: Each batch of tests must have positive/negative/blank controls
- Equipment calibration: PCR instrument needs to be regularly verified for temperature gradient
- Result review: Samples with 35＜Ct value＜40 need to be tested repeatedly
Technology evolution background
The original GFHNV virus was renamed CyHV-2 due to taxonomic progress. The characteristics of its genome of 290304bp prompted the detection target to be expanded from the helicase gene (366bp) to the DNA polymerase gene (92bp), and the sensitivity was increased by 10 times. The standard established a dual-gene detection system for the first time to effectively respond to the risk of virus mutation.
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