清脆的
副溶血性弧菌
放大器
计算生物学
Cas9
化学
重组酶聚合酶扩增
聚合酶链反应
生物
细菌
遗传学
基因
生物化学
作者
Yubo Peng,Pengpeng Xue,Renjing Wang,Huijie Shang,Bangben Yao,Zhi Zheng,Chao Yan,Wei Chen,Jianguo Xu
出处
期刊:Talanta
[Elsevier]
日期:2024-01-01
卷期号:266: 125061-125061
被引量:2
标识
DOI:10.1016/j.talanta.2023.125061
摘要
Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans-cleavage of a reporter molecule. By combining these processes, the target genomic DNA extracted from V. parahaemolyticus is able to activate many CRISPR/Cas12a units (CRISPR/Cas12an) simultaneously, resulting in a greatly amplified target signal to indicate the presence and concentration of V. parahaemolyticus. This unique model offers more advantages compared to traditional amplification models that use one RPA amplicon to activate one CRISPR/Cas12a unit. Under optimized conditions, our method enables the detection of target V. parahaemolyticus within a linear range of 1 × 102-1 × 107 CFU/mL, with an impressive limit of detection as low as 12.4 CFU/mL. It is conceivable that the adaptive tandem CRISPR/Cas12a molecular amplifier could be adapted as routine diagnostic kits in future for in-field detection of pathogens.
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