SN/T 5385-2021 in English
VALIDDetection and identifiction of Blueberry scorch virus
- Issued on:2021-06-18
- Implemented on:2022-01-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$321.00
Introduction
Analysis of the Standard Technical Framework for Blueberry Scorch Virus
| Detection Method | Sensitivity | Time Consumption | Applicable Scenarios |
|---|---|---|---|
| DAS-ELISA | Medium (ng level) | 4-6 hours | Large-batch screening |
| Immunochromatographic Test Strips | Low (μg level) | 3-10 minutes | On-site Rapid Detection |
| Nested RT-PCR | Ultra-high (pg level) | 5-6 hours | Laboratory confirmation |
| Real-time fluorescence RT-PCR | Ultra-high (pg level) | 2 hours | Precise quantitative detection |
Detailed explanation of the biological characteristics of the virus
Blueberry scorch virus (BlScV) belongs to the Beta-Filoviridae family. Its genome is a single-stranded RNA of 8.5kb. It is mainly transmitted by aphids in a non-persistent manner. Typical symptoms are sudden wilting of mosaic leaves during flowering (see Figure A.1), which can lead to poor fruit development. Hidden symptom varieties account for about 15%.
Key points of detection technology operation
Sample preparation specifications
Add extraction buffer (containing 1.3% sodium sulfite + 2% PVP) at a ratio of 1:10 (W/V), and centrifuge at 10000g for 10 minutes at 4℃ to obtain the supernatant. Be careful to avoid RNase contamination, and it is recommended to use a grinding instrument for low-temperature operation.
Key parameters of nested RT-PCR
- First round of amplification: BlScV-FP1/R primer pair, 667bp product, 53℃ annealing
- Second round of amplification: BlScV-FP2/R primer pair, 448bp product, 58℃ annealing
- Electrophoresis conditions: 1.5% agarose gel, 0.5×TBE buffer, 35V/cm
Recommendations for the implementation of the standard
Cross-border quarantine scenario: It is recommended to use the combination of immunochromatographic test strips + DAS-ELISA for initial screening, and then use real-time fluorescence RT-PCR to verify positive samples. In 2021, Fuzhou Customs applied this solution, and the detection rate increased to 99.2%.
Research institutions: Nested RT-PCR combined with sequence determination (NCBI BLAST comparison) is recommended to obtain virus strain differentiation data at the same time.
Technology evolution analysis
Compared with the IC-RT detection technology in 2016 (Xie Lixue et al.), this standard adds a new real-time fluorescence RT-PCR method, which shortens the detection cycle by 60%. The probe is designed for the conserved region of the CP/RdRp gene (see Table E.1), and the cross-reaction rate is <0.1%.
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