Diagnostic microbiology and infectious disease · 2025
Rapid detection of respiratory syncytial virus using RT-LAMP-CRISPR/Cas12b on a gravity-driven microfluidic chip.
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Liu Yuanzhu, Mei Haokun, Gao Chang, Yang Yingqi
Аннотация
AIMS: Respiratory syncytial virus (RSV) is the most significant pathogen causing acute lower respiratory tract infections in children. Early detection of RSV can help control disease progression and reduce complications. However, RT-qPCR-based detection methods cannot provide accurate results within one hour and are not suitable for use in resource-limited settings. Therefore, there is a pressing need to develop a rapid and precise bedside RSV detection assay to meet clinical demands.
METHODS AND RESULTS: Firstly, we developed a RT-LAMP-assisted CRISPR/Cas12b method for detecting RSV M gene, capable of identifying target RNA with a limit of detection as low as 100 copies/μL within 40 min. Secondly, we created and tested a sampling lysis reagent, demonstrating its effectiveness in enabling direct detection without the need for nucleic acid extraction, thus improving bedside detection efficiency. Lastly, to facilitate use in resource-limited areas, we designed and developed a gravity-driven microfluidic chip that simplifies the stepwise process of RT-LAMP amplification and CRISPR/Cas12b detection. This chip allows for visual recognition of results without the need for an external power source when used in point-of-care (POC) settings. This assay showed 99% agreement with RT-qPCR, highlighting its potential for practical application. Additionally, no cross-reactivity was observed with other respiratory pathogens infection, demonstrating good clinical specificity.
CONCLUSIONS: In summary, the platform we developed is faster and more user-friendly than RT-qPCR, while achieving comparable sensitivity.
IMPACT STATEMENT: Our finding fills the gap in the inability to detect RSV in POC setting, safeguarding the health of children, and offers new insights for the innovation of CRISPR diagnostics.
METHODS AND RESULTS: Firstly, we developed a RT-LAMP-assisted CRISPR/Cas12b method for detecting RSV M gene, capable of identifying target RNA with a limit of detection as low as 100 copies/μL within 40 min. Secondly, we created and tested a sampling lysis reagent, demonstrating its effectiveness in enabling direct detection without the need for nucleic acid extraction, thus improving bedside detection efficiency. Lastly, to facilitate use in resource-limited areas, we designed and developed a gravity-driven microfluidic chip that simplifies the stepwise process of RT-LAMP amplification and CRISPR/Cas12b detection. This chip allows for visual recognition of results without the need for an external power source when used in point-of-care (POC) settings. This assay showed 99% agreement with RT-qPCR, highlighting its potential for practical application. Additionally, no cross-reactivity was observed with other respiratory pathogens infection, demonstrating good clinical specificity.
CONCLUSIONS: In summary, the platform we developed is faster and more user-friendly than RT-qPCR, while achieving comparable sensitivity.
IMPACT STATEMENT: Our finding fills the gap in the inability to detect RSV in POC setting, safeguarding the health of children, and offers new insights for the innovation of CRISPR diagnostics.