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An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay
Published on: January 26, 2019
Integrated Trinity Test With RPA-CRISPR/Cas12a-Fluorescence for Real-Time Detection of Respiratory Syncytial Virus A
Ling Gong1,2, Xiaowen Wang2, Zhu Li2
1The First Clinical Medical College, Jinan University, Guangzhou, China.
Insights
A new integrated trinity test (IT-RAISE) offers a rapid, sensitive, and inexpensive method for detecting Respiratory Syncytial Virus (RSV) A or B. This assay aids in early diagnosis and management of RSV infections, particularly in vulnerable populations.
Area of Science:
- Molecular diagnostics
- Virology
- Biotechnology
Background:
- Respiratory Syncytial Virus (RSV) causes severe infections, especially in infants, the elderly, and immunocompromised individuals.
- Current diagnostic and treatment options for RSV are limited, with no effective vaccine available.
- RSV A strains typically present with more severe symptoms and complications than RSV B strains.
Purpose of the Study:
- To develop a simple, rapid, and cost-effective diagnostic assay for distinguishing between RSV A and RSV B infections.
- To achieve high amplification efficiency, sensitivity, and specificity in RSV detection.
Main Methods:
- Development of an integrated trinity test (IT-RAISE) assay system utilizing RPA-CRISPR/Cas12a technology.
- The assay detects target sequences via Cas12a-activated cleavage of a reporter ssDNA, generating a fluorescent signal.
- The system was validated using clinical specimens to assess its diagnostic performance.
Main Results:
- The IT-RAISE assay can detect as low as 1.38 × 10^1 copies/μl of target sequences.
- RSV A and RSV B infections were distinguished within 37 minutes.
- Clinical validation showed IT-RAISE sensitivity/specificity of 73.08%/90% for RSV A and 42.86%/93.33% for RSV B.
- The cost per specimen is approximately $2.6, excluding RNA extraction and reverse transcription.
Conclusions:
- The IT-RAISE system is a promising tool for the rapid, sensitive, and specific detection of RSV A or RSV B infections.
- Its simplicity, speed, and low cost offer significant clinical application potential for early disease management.
- The IT-RAISE platform may be adaptable for detecting other viral or bacterial pathogens.
Abstract:
Respiratory syncytial virus (RSV) is a common virus that causes respiratory infection, especially severe respiratory infection in infants and young children, the elderly people over 65 years old, and people with weak immunity. Currently, RSV infection has no effective vaccine and antiviral treatment. The number of deaths due to RSV infection increases every year. Moreover, RSV A infection occurs in a large number and has severe clinical symptoms and complications than RSV B infection. Therefore, the development of a simple, rapid, and inexpensive detection method with high amplification efficiency, high sensitivity, and specificity is very important for the diagnosis of RSV A or RSV B infection, which can help in the early clinical medication and prevent the progress of the disease. Therefore, we developed an integrated trinity test with an RPA-CRISPR/Cas12a-fluorescence (termed IT-RAISE) assay system to detect RSV A or RSV B. The characteristic of the IT-RAISE system is that after target recognition, the reporter single-stranded DNA (ssDNA) is cleaved by Cas12a that is activated by different crRNAs to detect the generated fluorescent signal. This method is simple and helps in adding all reagents rapidly. It is a high-sensitive method that can detect 1.38 × 101 copies/μl of the target sequences, and it can distinguish RSV A or RSV B infection within 37 min. In addition, clinical specimens were detected for IT-RAISE system. It was found that the sensitivity and specificity of RSV A were 73.08 and 90%, respectively, and those of RSV B were 42.86 and 93.33%, respectively. The cost of ONE specimen for IT-RAISE system was approximately $ 2.6 (excluding rapid RNA extraction and reverse transcription costs). IT-RAISE system has good clinical application prospects for detecting RSV A or RSV B infection; it is a simple, rapid, and inexpensive method with high amplification efficiency, high sensitivity, and high specificity. The IT-RAISE system might also detect other viral or bacterial infections.
