Burkholderia pseudomallei is 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 of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-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 of B. 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 of B. pseudomallei enables 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.

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，在流行地区构成公共卫生风险。由于高死亡率、内在抗生素耐药性和非典型表现，建立快速、准确和灵敏的B. pseudomallei鉴定可以实现类鼻疽的早期诊断、适当治疗和更好的结果。在此，我们提出了一种O ne- P ot C RISPR 集成测定法，用于B. pseudomallei的即时和视觉检测（称为 OPC-IVD）. 整合重组酶聚合酶扩增和 CRISPR-Cas12a 识别激活的反式切割，实现了真正的一体式单管反应系统，同时启动扩增和切割，实现了轻松的样品到答案测定。这种方法可以通过简化的 DNA 提取来执行，并通过手持反应管在 30 分钟左右完成。我们的 OPC-IVD 的检测限被确定为 2.19 拷贝/uL 的质粒 DNA，12.5 CFU/mL 的类鼻疽杆菌, 和 61.5 CFU/mL 的加标血样中的细菌。此外，内部扩增对照的引入有效降低了假阴性的发生，将其纳入反应体系，与靶标同步扩增，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B. pseudomallei OPC-IVD 的简化工作流程实现了一种可现场部署、无需仪器和超快速的方法，可供类鼻疽分子诊断领域的非专家人员使用，尤其是在资源贫乏的环境中.