Department of Clinical Microbiology and Immunology, College of Pharmacy and Laboratory Medicine Science, Third Military Medical University (Army Medical University), Chongqing, 400038, China.
College of Basic Medical Science, Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Anal Chim Acta. 2023 Apr 29;1252:341059. doi: 10.1016/j.aca.2023.341059. Epub 2023 Mar 6.
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.
伯克霍尔德氏菌是类鼻疽病的病原体，这是一种潜在的致命性传染病，在流行地区对公共健康构成威胁。由于高死亡率、固有抗生素耐药性和非典型表现，快速、准确、敏感地鉴定伯克霍尔德氏菌能够实现更早的诊断、适当的治疗和更好的类鼻疽病结果。在这里，我们提出了一种用于即时和可视化检测伯克霍尔德氏菌的 One-Pot CRISPR 整合检测（OPC-IVD）。通过整合重组酶聚合酶扩增和 CRISPR-Cas12a 识别激活的转切割，实现了真正的一体式单管反应系统，同时启动扩增和切割，实现了简单的样本到答案检测。这种方法可以通过简化 DNA 提取来进行，并且可以通过手持反应管在大约 30 分钟内完成。我们的 OPC-IVD 的检测限分别确定为质粒 DNA 的 2.19 拷贝/μL、伯克霍尔德氏菌的 12.5 CFU/mL 和血液样本中掺入细菌的 61.5 CFU/mL。此外，引入内部扩增对照有效地减少了假阴性的发生，这是在反应系统中引入的，并与目标同时扩增，肉眼可读。该检测在临床分离物和样本中的准确性达到 100%。我们的伯克霍尔德氏菌 OPC-IVD 的简化工作流程可实现现场部署、无仪器和超快的方法，特别在资源匮乏的环境中，非专业人员也可以在分子诊断领域中使用。
