背景:假单胞菌是一种对水果和蔬菜行业产生负面影响的检疫性害虫。将B.correcta与相似物种区分开来,尤其是在非成人阶段,仍然具有挑战性。快速分子鉴定技术,如重组酶聚合酶扩增(RPA)结合CRISPR/Cas12a和多酶等温快速扩增与侧流试纸(MIRA-LFD),在早期监测和保障农业生产方面发挥着至关重要的作用。我们的研究介绍了两种快速视觉识别B.correcta的方法。
结果:B.正确的RPA特异性引物,crRNA,并基于cox1基因设计了LFD探针。优化RPA反应条件(在37°C持续8分钟)以用于有效的模板DNA扩增。建立了两种核酸检测方法来可视化RPA扩增。在RPA-CRISPR/Cas12a系统中,最佳LbCas12a:crRNA浓度比为200:400nM。通过在37°C下孵育15分钟后是否存在绿色荧光来确定成功的扩增。MIRA-LFD系统在37°C下在4分钟内实现了目标物种的精确识别。两种方法均表现出较高的特异性和敏感性,允许检测1.0×10-1ng/μL的DNA。结合DNA快速提取,在不同的发育阶段实现了对个体B.correcta的快速识别,提高了所建立方法的实用性和便捷性。
结论:我们的研究结果表明,用于B.correcta检测的RPA-CRISPR/Cas12a和MIRA-LFD方法都是准确和快速的(在30分钟和10分钟内,分别),在37°C我们的方法不依赖于昂贵的设备,因此具有很高的实用价值,为港口检疫害虫和现场应用提供改进的识别解决方案。本文受版权保护。保留所有权利。
BACKGROUND: Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta.
RESULTS: Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng μL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods.
CONCLUSIONS: Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.