Abstract：
Bacillus cereus (B. cereus) is a foodborne pathogen that can produce tripartite enterotoxins, which can cause a variety of diseases after infection. It is critical to rapidly and accurately detect strains with enteropathogenic potential to safeguard human health. In this study, a dual-signal visualized detection platform with fluorescence assay and paper-based lateral flow assay (LFA) based on recombinase polymerase amplification (RPA), CRISPR/Cas12a system, and self-developed CRISPR nucleic acid test strips was constructed for enterotoxigenic B. cereus. The genes that encode two tripartite enterotoxins─nheA, nheB, and nheC for nonhemolytic enterotoxin and hblA, hblC, and hblD for hemolysin BL─were utilized as detection targets. The platform was capable of detecting six enterotoxin genes at the same genomic DNA level. The limits of detection for each gene were 10-3 ng/μL in fluorescence assay and 10-4 ng/μL in LFA. Furthermore, 101-102 CFU/mL of B. cereus in pure culture was detected. Additionally, a smartphone miniprogram could assist in evaluating the results in LFA. The platform demonstrated good utility by detecting B. cereus in food samples, including milk and rice. The results indicate that our RPA-CRISPR/Cas12a dual-signal visualized detection platform can quickly and easily detect B. cereus with three-component enterotoxin-producing potentials. The whole analytic process took less than 60 min without complex operation or expensive equipment.
摘要：
蜡样芽孢杆菌(B.蜡质）是一种食源性病原体，可以产生三方肠毒素，感染后可引起多种疾病。快速准确地检测具有肠致病性的菌株对保护人类健康至关重要。在这项研究中,基于重组酶聚合酶扩增（RPA）的双信号可视化检测平台，具有荧光测定和纸基侧流测定（LFA），CRISPR/Cas12a系统,并构建了自行开发的CRISPR核酸试纸，用于肠产毒性蜡样芽孢杆菌。编码两种三方肠毒素的基因─nheA，nheB,nheC用于非溶血性肠毒素和hblA，HBLC,和溶血素BL的hblD-被用作检测目标。该平台能够在相同的基因组DNA水平上检测六个肠毒素基因。每个基因的检测限在荧光测定中为10-3ng/μL，在LFA中为10-4ng/μL。此外，在纯培养物中检测到101-102CFU/mL的蜡状芽孢杆菌。此外,智能手机小程序可以帮助评估LFA的结果.该平台通过检测食品样品中的蜡状芽孢杆菌证明了良好的实用性，包括牛奶和米饭。结果表明，我们的RPA-CRISPR/Cas12a双信号可视化检测平台可以快速，轻松地检测具有三组分肠毒素产生潜力的蜡样芽孢杆菌。整个分析过程不到60分钟，无需复杂的操作或昂贵的设备。
