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Literature Sharing: A Novel Duplex RT-RAA Method for Rapid and Accurate Detection of Respiratory Syncytial Virus (RSV)
Release date:
2026-01-29
Respiratory syncytial virus (RSV) is the leading pathogen causing lower respiratory tract infections (such as pneumonia and bronchitis) in infants and young children worldwide, with no specific vaccines or antiviral drugs currently available.
Source: Archives of Virology (2020)
DOI: https://doi.org/10.1007/s00705-019-04230-z
I. Research Background and Clinical Need
Respiratory syncytial virus (RSV) is the leading pathogen causing lower respiratory tract infections (such as pneumonia and bronchitis) in infants and young children worldwide, with no specific vaccines or antiviral drugs currently available.
Therefore, rapid and accurate early diagnosis is critical for clinical management and infection control.
• Limitations of current detection methods:
◦ Viral culture: The gold standard with high specificity, but time-consuming (several days) and has low sensitivity.
◦ Serological testing: Low sensitivity, not suitable for early diagnosis.
◦ RT-qPCR: High sensitivity, but requires expensive equipment, specialized laboratories, skilled personnel, and is relatively time-consuming.
This study developed a duplex reverse transcription recombinase-aided amplification (duplex RT-RAA) assay performed in a single closed tube, incorporating an Internal Control (IC), aiming to achieve rapid and accurate detection of RSV.
II. Technical Principle
• Technology: Recombinase-Aided Amplification technology
◦ Principle: An isothermal nucleic acid amplification technique that utilizes recombinase (UvsX), single-stranded DNA-binding protein (SSB), and DNA polymerase to complete amplification within 20–30 minutes at 37–42°C.
◦ Advantages: Low equipment and environmental requirements, simple operation, highly suitable for point-of-care testing (POCT).
III. Experimental Design
1. Innovation: Introduction of a competitive Internal Control (IC)
◦ A competitive internal control was added to the same reaction tube, which uses the same primers as the RSV target for amplification but differs in the probe-binding region.
2. Primer and Probe Design:
◦ Targets a highly conserved region of the RSV L gene to ensure detection of different RSV genotypes.
◦ Design of RSV-specific primers and a FAM-labeled probe.
◦ Design of an IC-specific HEX-labeled probe. The IC template shares primer-binding regions with RSV but has a replaced probe-binding region, enabling competitive amplification.
3. Assay Optimization:
◦ Duplex RT-RAA: Based on the singleplex assay, the internal control was introduced, and its concentration was optimized (finally set at 100 copies/reaction) to ensure stable IC amplification without significantly affecting RSV detection sensitivity.
4. Performance Evaluation:
◦ Sensitivity: Assessed using serially diluted RSV recombinant plasmids (10^5 to 10^0 copies/reaction).
◦ Specificity: Tested with 176 clinical samples positive for other respiratory viruses (e.g., adenovirus, influenza virus).
◦ Clinical Validation: Compared the developed duplex RT-RAA method head-to-head with the gold standard RT-qPCR using 278 clinical nasopharyngeal secretion samples.
IV. Main Findings
1. High Sensitivity:
◦ Limit of detection (LOD) for singleplex RT-RAA: 4.4 copies/reaction.
◦ Limit of detection LOD for duplex RT-RAA: 5.0 copies/reaction, indicating no significant loss of sensitivity upon IC incorporation.
2. High Specificity:
◦ No cross-reactivity observed with 176 non-RSV samples; specificity was 100%.
3. Internal Control Effectiveness:
◦ The IC amplified successfully in all negative samples and negative controls, confirming no reaction inhibition.
◦ When RSV template concentration was extremely high (10^5 copies/reaction), IC amplification was competitively suppressed, which itself indicates a strong positive sample, and the result is interpreted as positive.
4. Clinical Concordance:
◦ In 278 clinical samples, duplex RT-RAA showed complete agreement with RT-qPCR results (102 positive, 176 negative).
◦ Sensitivity, specificity, and agreement with RT-qPCR were all 100% (Kappa value = 1).
V. Conclusion
• This study successfully developed a duplex RT-RAA assay for RSV detection that integrates rapidity (<30 minutes), high sensitivity (5.0 copies/reaction), high specificity, and built-in quality control.
• Key Contribution: The introduction of a competitive internal control effectively eliminates the risk of false negatives due to reaction inhibition, significantly enhancing the reliability of test results. This is a critical step toward practical clinical application.
• Application Prospects: The method is portable, simple to operate, and cost-effective, making it highly suitable as a POCT tool in primary hospitals, clinics, communities, and even remote areas. It provides robust technical support for early RSV diagnosis, outbreak surveillance, and timely intervention.
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