One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.
One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.
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DOI：
10.1016/j.aca.2023.341059
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发表时间：
2023-03
影响因子：
6.2
通讯作者：
Ling Deng;Xiaoyi He;Ke Liu;Yuan-li Li;Han Xia;Han-Yu Qian;Xiaona Lu;X. Mao;Yang Xiang
中科院分区：
文献类型：
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作者：
Ling Deng;Xiaoyi He;Ke Liu;Yuan-li Li;Han Xia;Han-Yu Qian;Xiaona Lu;X. Mao;Yang Xiang
类鼻疽伯克霍尔德氏菌是类鼻疽的病原体，类鼻疽是一种可能危及生命的传染病，并在流行地区构成公共卫生风险。由于B.类鼻疽可以实现早期诊断、正确治疗和更好的结果。在此，我们提出了一种用于 B 的即时和视觉检测（称为 OPC-IVD）的 One-Pot CRISPR 集成测定。假鼻疽。重组酶聚合酶扩增与CRISPR-Cas12a识别激活反式裂解相结合，实现了真正的一体式单管反应系统，同时启动扩增和裂解，实现了简便的从样品到答案的检测。这种方法可以通过简化的 DNA 提取来进行，并通过手持反应管在大约 30 分钟内完成。我们的 OPC-IVD 的检测限确定为 2.19 拷贝/uL 质粒 DNA、12.5 CFU/mL B。加标血液样本中的假鼻疽菌和细菌分别为 61.5 CFU/mL。此外，内部扩增对照的引入，有效减少了假阴性的发生，将其纳入反应体系中，与靶标同时扩增，肉眼读取。在临床分离株和样本中进行评估时，该测定显示出 100% 的准确性。我们的 OPC-IVD ofB 的简化工作流程。拟可扩展性是一种可现场部署、无需仪器且超快速的方法，可供类鼻疽分子诊断领域的非专家人员使用，特别是在资源贫乏的环境中。
Burkholderia pseudomalleiis the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification ofB. pseudomalleienables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present aOne-PotCRISPR-integrated assay forInstant andVisualDetection (termed OPC-IVD) ofB. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activatedtrans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL ofB. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD ofB. pseudomalleienables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.