清脆的
嗜血杆菌
重组酶聚合酶扩增
肉眼
化学
病菌
微生物学
病毒学
检出限
聚合酶链反应
细菌
分子生物学
生物
色谱法
基因
遗传学
生物化学
作者
Jie Huang,Manhong Jia,Yiting Liu,Z. Lv,Junming Chen,Wenguang Xiong,Zhenling Zeng
标识
DOI:10.1016/j.aca.2023.342101
摘要
Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions.In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min.We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.
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