基于重组酶聚合酶扩增和CRISPR/Cas12a系统的2种快速检测奇异变形杆菌的方法

Two rapid detection methods for Proteus mirabilis based on recombinase polymerase amplification and the CRISPR/Cas12a system

摘要:

【背景】 奇异变形杆菌(Proteus mirabilis)是一种重要的致病菌，能够引起人类和动物感染。目前针对该菌的快速检测技术研究较为匮乏，这为临床早期诊断和精准防治工作带来挑战。【目的】 基于不同原间隔序列临近基序(protospacer adjacent motif, PAM)序列策略，构建基于重组酶聚合酶扩增(recombinase polymerase amplification, RPA)和CRISPR/Cas12a系统的2种检测平台，实现对奇异变形杆菌的快速检测。【方法】 针对奇异变形杆菌保守基因建立基于RPA和CRISPR/Cas12a系统的一步法和两步法检测平台。一步法平台在39 ℃条件下同时进行RPA与CRISPR/Cas12a反应，实时收集荧光信号；两步法平台则首先在39 ℃条件下进行RPA扩增，扩增完成后通过瞬时离心将CRISPR/Cas12a体系移至管底，继续在39 ℃条件下进行反应以产生荧光信号。优化反应条件后，评估2种方法对奇异变形杆菌临床分离株及其他常见细菌样本的检测灵敏度和特异性。【结果】 成功建立了基于RPA和CRISPR/Cas12a的2种检测平台，均可在30 min内检测到最低2.8 copies/μL的标准质粒。在使用热裂解法对样本进行预处理时，一步法灵敏度为10⁶ CFU/mL，而两步法灵敏度为10³ CFU/mL；在对模拟样本进行基因组DNA提取后，一步法和两步法灵敏度分别为10² CFU/mL和10¹ CFU/mL。两种方法在30例临床分离株样本中均表现出良好的检出能力，与其他常见致病菌无交叉反应，具有良好的特异性。【结论】 本研究基于不同的PAM序列设计策略，构建了针对奇异变形杆菌的RPA-CRISPR/Cas12a一步法和两步法检测平台，具有快速、灵敏、特异的优点，有望在奇异变形杆菌现场检测中应用。

Abstract:

[Background] Proteus mirabilis is a major pathogen capable of causing infections in both humans and animals. Currently, the research on rapid detection techniques for this bacterium is scarce, posing challenges to early clinical diagnosis and targeted prevention and control efforts. [Objective] We developed two detection platforms based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems, utilizing distinct protospacer adjacent motif (PAM) sequence strategies, to achieve rapid detection of P. mirabilis. [Methods] One-step and two-step detection platforms based on RPA and CRISPR/Cas12a systems were developed for P. mirabilis by targeting its conserved genes. The one-step platform simultaneously performs RPA and CRISPR/Cas12a reactions at 39 ℃, collecting fluorescence signals in a real-time manner. The two-step platform first conducts RPA at 39 ℃, and then transfers the CRISPR/Cas12a system to the bottom of the tube through brief centrifugation and continues the reaction at 39 ℃ to generate fluorescence signals. After optimization of the reaction conditions, we used the clinical isolates of P. mirabilis and other common bacterial samples to evaluate the sensitivity and specificity of both methods. [Results] Two detection platforms based on RPA and CRISPR/Cas12a were successfully established, both capable of detecting a minimum of 2.8 copies/μL of standard plasmid within 30 min. During sample pretreatment by thermal lysis, the one-step method achieved the sensitivity of 10⁶ CFU/mL, while the two-step method demonstrated the sensitivity of 10³ CFU/mL. After genomic DNA extraction from simulated samples, the sensitivity of the one-step and two-step methods reached 10² CFU/mL and 10¹ CFU/mL, respectively. Both methods demonstrated excellent detection ability across 30 clinical isolates and showed no cross-reactivity with other common pathogenic bacteria, showcasing good specificity. [Conclusion] We developed one-step and two-step RPA-CRISPR/Cas12a detection methods for P. mirabilis based on two distinct PAM sequence design strategies. These methods demonstrate rapid, sensitive, and specific detection performance, showing promising potential for on-site detection of P. mirabilis.