GB/T 40251-2021 in English
VALIDCode of diagnosis for MSX disease of oysters - In situ hybridization method
- Issued on:2021-05-21
- Implemented on:2021-12-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$233.00
《GB/T 40251-2021牡蛎单孢子虫病诊断规程 原位杂交法》由TC156（全国水产标准化技术委员会）归口，TC156SC11（全国水产标准化技术委员会水产养殖病害防治分会）执行，主管部门为农业农村部。
Introduction
Technical background and significance of the standard
GB/T40251-2021 is my country's first national standard for in situ hybridization diagnosis of oyster monosporidiosis. The disease is caused by Haplosporidium nelsoni, which can lead to more than 90% mortality in bivalve shellfish such as oysters. The establishment of the standard fills the gap in my country's molecular detection of this pathogen, and is of milestone significance for the prevention and control of aquaculture diseases.
Core Technology Principles
The in situ hybridization method uses the DIG-labeled probe MSX1347 (sequence 5'-ATG-TGT-TGG-TGA-CGC-TAA-CCG-3') to achieve tissue in situ visualization of pathogen nucleic acids through the principle of base complementary pairing. Compared with traditional microscopic examination methods, its detection sensitivity is increased by 100 times, and trace pathogens in the early infection stage can be detected.
Comparison of key technical parameters
| Technical indicators | In situ hybridization method | Traditional tissue printing method (SC/T7205.1) |
|---|---|---|
| Detection limit | 10^2 copies/g tissue | 10^4 copies/g tissue |
| Specificity | 100%(species level) | Genus level(80%) |
| Sample processing time | 48 hours | 2 hours |
| Result stability | Can be stored for a long time | Requires immediate observation |
Key experimental operation specifications
1. Sample processing
The stomach, intestine, adductor muscle and mantle tissues of adult shellfish need to be collected and fixed with DAFA fixative (ethanol: formaldehyde: glacial acetic acid = 33:11:5), and then processed through gradient dehydration (50%-100% ethanol), transparency (xylene), and wax immersion (52-54℃).
2. Probe hybridization
Key parameters:
- Protease K digestion: 37℃ 15 minutes
- Probe concentration: 2ng/μL
- Hybridization temperature: 42℃ 16-18 hours
Implementation recommendations
Application scenarios: It is recommended to carry out monitoring in the high-incidence season when the seawater temperature is ＞10℃ and the salinity is ＞15‰. For farmed populations with abnormal deaths, the visceral mass of spat should be collected for testing first.
Quality control points: Each batch of experiments must have a positive control (known infected samples) and a negative control, and the color development time must be controlled within 1-3 hours to avoid non-specific coloring.
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