SC/T 7238-2020 in English
VALIDDetection methods for covert mortality nodavirus (CMNV)
- Issued on:2020-08-26
- Implemented on:2021-01-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
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Introduction
Standard Technical Background
The CMNV (CMNV) belongs to the alphavirus genus of the Nodamuraviridae family. The viral VCMD (VCMD) caused by CMNV can cause a 60-80% mortality rate in farmed shrimp. This standard specifies three molecular detection technologies for susceptible hosts such as Litopenaeus vannamei and Penaeus chinensis.
Comparison of core detection methods
| Method | Sensitivity | Time consumption | Equipment requirements | Applicable scenarios |
|---|---|---|---|---|
| Nested RT-PCR | 102 copies/μL | 4-5 hours | PCR instrument + electrophoresis system | Laboratory confirmation |
| RT-LAMP | 101 copies/μL | 1 hour | Constant temperature amplification instrument | Rapid on-site screening |
| Fluorescence quantitative PCR | 100 copies/μL | 2 hours | Real-time fluorescence PCR instrument | Precise quantitative detection |
Key operating points
Sample processing specifications
For shrimp with clinical symptoms, hepatopancreas, gill tissue and muscle tissue should be collected; for asymptomatic individuals, hepatopancreas and gill tissue should be collected. RNA extraction requires the use of RNAiso Plus reagent, and the isopropanol precipitation step should be strictly performed at -20℃.
Primer design features
- Nested PCR: Two pairs of primers (CMNV-nF1/R1 and CMNV-nF2/R2) are used, and the amplified fragments are 619bp and 413bp respectively
- RT-LAMP: Contains FIP/BIP inner and outer primers and loop primers LF/LB, and the betaine concentration must reach 0.8M
- Fluorescence PCR: TaqMan probe is labeled with FAM at the 5' end and TAMRA at the 3' end
Technology evolution analysis
This standard incorporates the RT-LAMP isothermal amplification technology into the CMNV detection system for the first time. Compared with the traditional PCR method:
- Detection time is shortened from 4 hours to 1 hour
- Sensitivity is increased by 10 times
- Get rid of the dependence on precision temperature control equipment
The fluorescent quantitative PCR method uses TaqMan probe technology to accurately measure the viral load and provide data support for epidemiological research.
Implementation recommendations
Key points for quality control
- Each batch of tests must have a positive control (containing CMNV nucleic acid fragments) and a negative control (RNase-free water)
- The RNA extraction OD260/280 ratio should be maintained at 1.8-2.0
- The second step of nested PCR requires a 10-1000-fold dilution of the first-round product
Cross-validation requirements
When a method produces suspicious results, another method should be used for verification. Especially for fluorescent PCR results with a Ct value of 35-40, it is recommended to re-confirm through RT-LAMP.
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