A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system
A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system
复制标题
使用 LbCas12a 变体系统现场检测园艺作物检疫性病原菌的灵敏视觉方法
DOI：
10.1016/j.jhazmat.2021.128038
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发表时间：
2022-03-15
影响因子：
13.6
通讯作者：
Zheng, Xianbo
中科院分区：
文献类型：
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作者：
Jiao, Jian;Yang, Mengjie;Zheng, Xianbo
种植前对种子和幼苗进行检疫病原体检测是一项重要的植物检疫措施。目前正在开发用于病原体检测的 CRISPR 介导的分子诊断方法，但仍然存在许多挑战。在这里，我们分析了一种工程化的 Crispr/LbCas12a 变体 (LbCas12a-5M)，它比野生型具有更强大的反式切割活性和更广泛的 PAM 序列 (TNTN) 偏好。我们开发了一种筛选细菌植物病原体特定序列的程序，所设计的物种特异性 crRNA 与其他细菌物种没有交叉反应。结合细菌 DNA 的简单提取，建立并优化了基于 LbCas12a-5M 的视觉检测技术，用于检测检疫性病原体火梨欧文氏菌和西瓜食酸菌，检测限高达 40 CFU/反应，灵敏度与 qPCR 检测一致。该方案比 qPCR 更快、更简单，样品制备仅需 40 分钟或更短时间。我们进一步验证了该方法的潜在应用，表明该方法可用于西瓜种子上西瓜菌的快速准确诊断，与 qPCR 检测结果 100% 一致。所开发的方法简化了病原体的检测，并为检疫干预措施提供了具有成本效益的对策。
Pre-planting testing of seeds and plantlets for the existence of quarantine pathogens is an important phytosanitary measure. The CRISPR-mediated molecular diagnostic methodologies are being developed for pathogens detection, but many challenges remain. Here, we profiled an engineered Crispr/LbCas12a variant (LbCas12a-5M) that has more robust trans-cleavage activity and a wider PAM sequences (TNTN) preference than wild type. We developed a procedure for screening specific sequences of bacterial plant pathogens, and the designed speciesspecific crRNA displayed no cross-reactions with other bacterial species. Combined with a simple extraction of bacterial DNA, an LbCas12a-5M-based visual detection technique was established and optimized for detecting quarantine pathogens Erwinia amylovora and Acidovorax citrulli with detection limits up to 40 CFU/reaction and a sensitivity consistent with qPCR assay. This protocol was faster and simpler than qPCR, requiring 40 min or less from sample preparation. We further validated the potential application of the method by showing that it can be used for rapid and accurate diagnosis of A. citrulli on seeds of watermelon, with 100% agreement with the results of qPCR assay. The developed method simplifies the detection of pathogens and provides cost-effective countermeasures to quarantine interventions.