Yang Rui, Tang Tian, Liu Peiran, Wang Xiaoxia, Jiang Yihao, Zhang Shirong, Wang Meijuan, Yang Luo, Wang Chuan
/ （ 610041） Department of Laboratory Sciences, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China.
/ （ 610041） Translational Preventive Medical Research Institute, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2025 Mar 20;56(2):549-555. doi: 10.12182/20250360105.
To establish a one-step detection method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a protein for the rapid and sensitive detection of human influenza B virus.
RPA amplification primers were designed according to the conserved gene ( gene) of human influenza B virus (Victoria lineage). The reaction system was established using the standard plasmid as the template. First of all, the reaction system was incubated at 37 ℃ for 15 minutes for RPA amplification. Then, the CRISPR/Cas12a system on the tube cap was thoroughly mixed with the RPA amplification product at the bottom of the tube through fast centrifugation, and real-time fluorescence detection was carried out at 37 ℃. The reaction conditions were optimized to establish a one-step RPA-CRISPR/Cas12a detection method for human influenza B virus. The sensitivity of the testing method was evaluated using standard plasmids and pseudoviruses, and the specificity was evaluated using other viruses that may cause febrile respiratory syndrome. The consistency between the results of the one-step detection method and those of RT-qPCR detection was evaluated by testing real samples.
A one-step detection method based on RPA-CRISPR/Cas12a was successfully established. The optimal reaction conditions included a reaction temperature of 37 ℃, a Cas12a/crRNA concentation ratio of 1∶1, a Cas12a concentration of 120 nmol/L, a single-stranded DNA (ssDNA) probe concentration of 300 nmol/L, and a primer concentration of 480 nmol/L. The method could detect standard plasmid DNA as low as 2.8 copies/μL within 25 minutes and pseudoviruses as low as 2.77 copies/μL within 30 minutes. The testing method showed high specificity, and no cross-reaction was observed with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A (H1N1) virus, or respiratory syncytial virus subgroup A. When testing clinical samples, the sensitivity and the specificity for examining clinical samples were 93.33% and 100%, respectively, and consistency with RT-qPCR results was 97.14%.
With the one-step detection method based on RPA-CRISPR/Cas12a established in this study, the whole sample detection process, including nucleic acid release, reverse transcription, isothermal amplification, CRISPR/Cas12a system cleavage, and fluorescence signal output, can be completed within 30 minutes. Its high sensitivity, specificity, and successful application in clinical samples highlight its potential for rapid point-of-care testing in clinical settings.
建立一种基于重组酶聚合酶扩增（RPA）和CRISPR/Cas12a蛋白的一步法检测方法，用于快速、灵敏地检测人乙型流感病毒。
根据人乙型流感病毒（维多利亚系）的保守基因设计RPA扩增引物。以标准质粒为模板建立反应体系。首先，将反应体系在37℃孵育15分钟进行RPA扩增。然后，通过快速离心将管帽上的CRISPR/Cas12a系统与管底的RPA扩增产物充分混合，并在37℃进行实时荧光检测。对反应条件进行优化，建立人乙型流感病毒的一步法RPA-CRISPR/Cas12a检测方法。使用标准质粒和假病毒评估检测方法的灵敏度，使用可能引起发热性呼吸道综合征的其他病毒评估特异性。通过检测实际样本评估一步法检测结果与RT-qPCR检测结果之间的一致性。
成功建立了基于RPA-CRISPR/Cas12a的一步法检测方法。最佳反应条件包括反应温度37℃、Cas12a/crRNA浓度比1∶1、Cas12a浓度120 nmol/L、单链DNA（ssDNA）探针浓度300 nmol/L、引物浓度480 nmol/L。该方法可在25分钟内检测低至2.8拷贝/μL的标准质粒DNA，在30分钟内检测低至2.77拷贝/μL的假病毒。该检测方法具有高特异性，未观察到与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）、甲型流感（H1N1）病毒或呼吸道合胞病毒A亚组的交叉反应。检测临床样本时，检测临床样本的灵敏度和特异性分别为93.33%和100%，与RT-qPCR结果的一致性为97.14%。
本研究建立的基于RPA-CRISPR/Cas12a的一步法检测方法，可在30分钟内完成包括核酸释放、逆转录、等温扩增、CRISPR/Cas12a系统切割和荧光信号输出在内的全样本检测过程。其高灵敏度、特异性以及在临床样本中的成功应用突出了其在临床即时检测中的应用潜力。
