便携式激发光源用于转基因筛选

近日浙江省农科院在《Journal of Advanced Research》发表文献《Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops》。文献中使用LUYOR-3415RG便携式双波长荧光蛋白激发光源检测转基因作物。

文献摘要:

开发一种新型 Cas13a/Cas12a 介导的转基因作物“一锅”双重检测方法

转基因（GM）作物已在世界各地广泛种植，开发适合田间部署的快速、超灵敏、视觉多重检测平台对于转基因生物调控至关重要。在这项研究中，我们开发了一种新型一锅法系统，称为 MR-DCA（多重 RPA 和双重 CRISPR 检测），用于同时检测转基因作物中的基因靶标。这种创新方法将多重 RPA（重组酶聚合酶扩增）与双 CRISPR（成簇规则间隔短回文重复）测定技术相结合，为转基因作物检测提供了一种简化且高效的方法。用于扩增和靶标的 RPA 反应包含在管底座中，而双 CRISPR 酶则放置在管盖中。离心后，启动双 CRISPR (Cas13a/Cas12a) 检测系统。使用荧光可视化通过 FAM 通道和 HEX 通道进行测量。使用侧流试纸条时，使用兔抗地高辛（蓝线）进行检测，同时使用抗小鼠 FITC（红线）进行识别。使用 Image J 量化线强度并以图形方式描绘。 35分钟内完成目标检测，检测限低至20个拷贝。此外，还开发了两种分析系统，它们在 MR-DCA 测定中表现良好。在对来自广泛基因组范围的转基因作物的24个盲样进行分析时，MR-DCA给出了与定量PCR方法一致的结果，表明了较高的准确性、适用性和半定量能力。 MR-DCA的发展代表了GM检测领域的重大进步，为可在资源有限的环境中使用的多目标检测提供了快速、灵敏和便携的方法。

Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops

Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of and genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. The RPA reaction used for amplification and targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure through the FAM channel and through the HEX channel. When using lateral flow strips, was detected using rabbit anti-digoxin (blue line), whilst was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

MR-DCA combined the RPA reaction with Cas13a/Cas12a digestion in a one-pot reaction system. The 40 μL one-pot reaction assay consisted of 15 μL multiplexed-

RPA-MIX, 2 μL DNA, 1 μL B buffer, 1×Cas-reaction buffer, 100 nM 13-35S-crRNA, 100 nM 12-NOS-crRNA, 1 μM quenched fluorescent ssRNA reporter, 1 μM quenched

fluorescent ssDNA reporter, 0.1×Transcription buffer (contain rNTP MIX) and 0.5 μL RNase inhibitor, enzyme MIX (100 nM Cas13a, 100 nM Cas12a and 0.5 μL T7 RNA

polymerase) was initially added on the tube cap and add nuclease-free water to 40 μL. After RPA reaction for 15 min, enzyme mix was centrifuged into the reaction solution, then the reaction was performed at 37 °C for 20 min on CFX96 Real-Time PCR system with fluorescence measurements taken every 30s. In System 1, the ChemiDoc Touch Imaging System (Bio Rad, Hercules, CA, USA) was used to observe the green-redyellow fluorescence. For on-site detection, a handheld LUYOR-3415RG instrument (LUYOR, USA) can be equipped with a 494-nm excitation filter for the FAM reporter (green) and a 535-nm excitation filter for the HEX reporter (red).

DOI: 10.1016/j.jare.2024.07.027