等温扩增允许核酸的高灵敏度检测，绕过仪器热循环的使用。这项工作旨在对四种最有前途的技术进行实验比较：重组酶聚合酶扩增（RPA）和环介导等温扩增（LAMP）与侧流测试耦合或与基于CRISPR/Cas12a的额外扩增耦合由Cas12a切割探针的荧光产生。为了比较四种扩增技术，我们选择了细菌性植物病原体淀粉样欧文氏菌（火疫病的病原体），在许多国家具有检疫意义，对农业具有严重威胁。选择三个基因作为靶标,并为每个选择引物(两个用于RPA,六个用于LAMP)。它们被标记(生物素，荧光素）在5'末端用于LFT识别扩增子。因此,我们开发了LAMP-LFT,LAMP-CRISPR/Cas,RPA-LFT,和RPA-CRISPR/Cas用于淀粉样肠球菌的检测。LAMP-LFT的检出限为104CFU/mL，LAMP-CRISPR/Cas为103CFU/mL，RPA-LFT和RPA-CRISPR/Cas为102CFU/mL。在一组真实样品上通过qPCR验证了四个开发的测试系统的结果。基于RPA开发的测定法，LAMP,CRISPR/Cas12a,LFT快速（30-55分钟），用户友好，对淀粉芽孢杆菌的检测高度敏感。所有提出的检测方法均可应用于火灾疫病的诊断和有效管理。
Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5\' ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 104 CFU/mL for LAMP-LFT, 103 CFU/mL for LAMP-CRISPR/Cas, and 102 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30-55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.
