Analytica chimica acta · 2026
Field-deployable RspCas13d platform for rapid and extraction-free detection of giant panda- and canine-derived Canine Distemper virus.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Tao Qian, Xing Yuxiao, Yang Haidi, Deng Linhua, He Ming, Cheng Yanxi, Wang Qian, Gao Zhan, Wan Haohong, Li Desheng, Ling Shanshan
Аннотация
BACKGROUND: Canine distemper virus (CDV) is a highly contagious RNA virus that causes severe disease in domestic dogs and diverse wildlife species, including endangered giant pandas. Current CDV diagnosis mainly relies on laboratory-based RT-qPCR, which requires nucleic acid extraction, trained personnel, and temperature-controlled instruments, limiting its use in field surveillance and resource-limited settings. Although CRISPR-based diagnostics offer promising alternatives, chemically defined extraction-free workflows compatible with one-pot Cas13d detection remain limited. This study addresses the need for a rapid, visual, extraction-free, and low-infrastructure method for CDV detection.
RESULTS: We developed CLEAR-VISION, an integrated CRISPR diagnostic platform combining CLEAR (Chemical Lysis for Extraction-free Access to RNA) with VISION (Visual Isothermal Single-tube Integrated One-pot Nucleic acid detection). VISION integrates RPA amplification, T7 transcription, and RspCas13d-based detection into a single-tube reaction supported by a chemically defined buffer. CLEAR enabled rapid RNA release at room temperature without extraction kits or heating and was compatible with downstream one-pot detection. The PAM- and PFS-independent property of RspCas13d allowed flexible target selection, while lyophilized reagents improved storage and transportation convenience. CLEAR-VISION enabled CDV detection within 30 min and provided dual visual readouts, including fluorescence and lateral flow assays. Clinical evaluation in the current sample set showed consistent results with RT-qPCR for samples from giant pandas, stray dogs, and pet dogs. The assay also maintained stable performance at physiological temperature (37 °C), reducing reliance on temperature-controlled equipment.
SIGNIFICANCE AND NOVELTY: CLEAR-VISION provides a chemically defined, extraction-free, and low-infrastructure CRISPR diagnostic workflow for rapid CDV detection. Its novelty lies in integrating room-temperature chemical lysis with single-tube RPA-T7-RspCas13d detection, lyophilized reagents, and dual visual readouts. This platform supports the potential application of CRISPR-based diagnostics for on-site CDV surveillance in domestic animals and wildlife.
RESULTS: We developed CLEAR-VISION, an integrated CRISPR diagnostic platform combining CLEAR (Chemical Lysis for Extraction-free Access to RNA) with VISION (Visual Isothermal Single-tube Integrated One-pot Nucleic acid detection). VISION integrates RPA amplification, T7 transcription, and RspCas13d-based detection into a single-tube reaction supported by a chemically defined buffer. CLEAR enabled rapid RNA release at room temperature without extraction kits or heating and was compatible with downstream one-pot detection. The PAM- and PFS-independent property of RspCas13d allowed flexible target selection, while lyophilized reagents improved storage and transportation convenience. CLEAR-VISION enabled CDV detection within 30 min and provided dual visual readouts, including fluorescence and lateral flow assays. Clinical evaluation in the current sample set showed consistent results with RT-qPCR for samples from giant pandas, stray dogs, and pet dogs. The assay also maintained stable performance at physiological temperature (37 °C), reducing reliance on temperature-controlled equipment.
SIGNIFICANCE AND NOVELTY: CLEAR-VISION provides a chemically defined, extraction-free, and low-infrastructure CRISPR diagnostic workflow for rapid CDV detection. Its novelty lies in integrating room-temperature chemical lysis with single-tube RPA-T7-RspCas13d detection, lyophilized reagents, and dual visual readouts. This platform supports the potential application of CRISPR-based diagnostics for on-site CDV surveillance in domestic animals and wildlife.