RPA-CRISRP/Cas12a技术快速检测实验动物感染肺炎克雷伯杆菌方法的建立及应用

Establishment and Application of RPA-CRISRP/Cas12aTechnology for Rapid Detection of Klebsiella pneumoniae Infection in Laboratory Animals

摘要: 目的：将RPA扩增技术与CRISPR/Cas12a检测技术相融合，建立实时、灵敏、特异的检测肺炎克雷伯杆菌的快速方法。方法：根据肺炎克雷伯杆菌phoE基因保守序列设计并合成特异性的RPA扩增引物和CRISPR RNA (crRNA)，通过RPA等温扩增技术放大待测靶标基因，利用Cas12a蛋白的反式切割(trans-cleavage)活性检测靶标基因。对RPA-CRISPR/Cas12a检测体系的主要参数进行优化，确定最佳反应体系。结果：成功筛选出RPA扩增引物和crRNA，优化了体系中三个重要参数的最佳反应浓度crRNA (40 uM)、Cas12a (250 nM)和ssDNA (10 uM)。肺炎克雷伯杆菌基因组DNA检测灵敏度可达10⁻⁵ ng/uL，与实验动物常见的致病菌无交叉反应。该方法应用于实验动物临床检测时，具有较高的灵敏度和特异性。结论：本研究建立的RPA-CRISRP/Cas12a检测方法，操作简便、实时快速、成本低且特异性好，能够实现对实验动物感染肺炎克雷伯杆菌的快速检测。

Abstract: Objective: To integrate RPA amplification technology with CRISPR/Cas12a detection technology and establish a real-time, sensitive and specific rapid method for the detection of Klebsiella pneumoniae. Methods: Specific RPA amplification primers and CRISPR RNA (crRNA) were designed and synthesized based on the conserved sequence of the phoE gene of Klebsiella pneumoniae. The target gene to be tested was amplified by RPA isothermal amplification technology, and the trans cleavage activity of Cas12a protein was used to detect the target gene. The main parameters of the RPA-CRISPR/Cas12a detection system were optimized to determine the optimal reaction system. Result: RPA amplification primers and crRNA were successfully screened out, and the optimal reaction concentrations of three important parameters in the system, namely crRNA (40 uM), Cas12a (250 nM), and ssDNA (10 uM), were optimized. The genomic DNA detection sensitivity of Klebsiella pneumoniae can reach 10⁻⁵ ng/uL, and there is no cross-reaction with the common pathogenic bacteria in experimental animals. When this method is applied to the clinical detection of experimental animals, it has high sensitivity and specificity. Conclusion: The RPA-CRISRP/Cas12a detection method established in this study is simple to operate, real-time and rapid, low in cost and good in specificity, and can achieve rapid detection of Klebsiella pneumoniae infection in experimental animals.