Diagnostic microbiology and infectious disease · 2026
Enhanced stability of RPA-CRISPR-Cas12a system for respiratory pathogen detection using Trehalose-Carboxymethyl Chitosan Lyoprotectant.
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Yang Huifeng, Li Xiaoyi, Su Yimin, Guo Jianxin, Wang Tengfei, Wang Zhigang, Fu Hai, Chen Zhi, Xie Zhongjian, Li Defa
Аннотация
PURPOSE: Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection.
MATERIALS AND METHODS: A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis, SARS-CoV-2, Streptococcus pneumoniae, and human adenovirus) using real-time fluorescence and gel electrophoresis.
RESULTS: This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR-Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR-preliminarily supporting the system's potential diagnostic accuracy-we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance.
CONCLUSION: The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.
MATERIALS AND METHODS: A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis, SARS-CoV-2, Streptococcus pneumoniae, and human adenovirus) using real-time fluorescence and gel electrophoresis.
RESULTS: This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR-Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR-preliminarily supporting the system's potential diagnostic accuracy-we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance.
CONCLUSION: The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.