News & Events

Published in《Frontiers in Cellular and Infection Microbiology》

DOI: 10.3389/fcimb.2025.1621012

Bluetongue Virus (BTV), a highly pathogenic agent causing severe infectious diseases in ruminants such as cattle and sheep, has been widely distributed across all continents except Antarctica. More than 42,000 cases were reported in Europe alone between 2023 and 2024, inflicting severe losses on the livestock industry. Although no large-scale outbreaks have occurred in China, antibodies against multiple serotypes have been detected, posing persistent pressure on epidemic prevention and control. Traditional detection methods either rely on expensive equipment, are time-consuming (4-6 hours), or fail to cover all 36 BTV serotypes, which severely limits the demand for rapid field screening. However, the emergence of an innovative detection technology is revolutionizing this landscape.

1. Sample Preparation: Rapid lysis using nucleic acid release reagents (15 minutes) for faster and more accurate results, or traditional TRIzol extraction (30 minutes);

2. Isothermal Amplification: RT-ERA amplification completed at a constant temperature of 37°C for 20 minutes, eliminating the need for repeated temperature cycling with complex equipment;

3. Specific Detection: Cas13a reaction initiated at 40°C for 20 minutes, with results readable via three methods—fluorescence quantitative analysis, visual fluorescence under blue light, and naked-eye interpretation using lateral flow dipsticks (colloidal gold).

1. Exceptional Sensitivity and Specificity

• Sensitivity: Achieves a detection limit of 20 RNA copies per reaction, comparable to laboratory-grade RT-qPCR, enabling accurate capture of samples with low viral loads.

• Specificity: No cross-reactivity with 9 closely related or common pathogens (including EHDV and AKAV), eliminating misdiagnosis risks.

• Comprehensive Serotype Coverage: By designing crRNAs targeting BTV conserved genes, the assay successfully detects 30 known BTV serotypes (comprising 10 strains + 20 synthetic sequences), addressing the challenge of monitoring serotype diversity.

2. Outstanding Clinical Validation Performance

• Fluorescence Value/Signal Readout: Clinical sensitivity of 100% and specificity of 100%, fully consistent with the national standard RT-qPCR assay.

• Lateral Flow Dipstick Readout: Sensitivity of 96% and specificity of 100%, meeting the requirements for rapid on-site screening.

• Field Adaptability: Using NARR-treated crude samples (no nucleic acid purification required), the assay maintained a sensitivity of 97% and specificity of 100%. Detection performance remained unaffected even in the presence of sample inhibitors.

  

  

3. No Specialized Equipment Required: Directly Deployable in Field Settings

In contrast to virus isolation that relies on BSL-3 laboratories and large-scale instrumentation, this method offers the following advantages:

• No sophisticated instruments needed: Fluorescent signals can be observed via a simple blue light imaging device, and lateral flow dipsticks enable direct naked-eye interpretation.

• Simplified sample processing: Crude extract can be obtained through 5-minute heat denaturation with nucleic acid release reagents, operable by farmers or grassroots veterinarians.

• Controllable costs: The integrated reaction workflow reduces consumable requirements, making it suitable for large-scale field monitoring.