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

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Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2024-07-29 , DOI: 10.1016/j.jare.2024.07.027
Lin Ding ₁ , Xiaofu Wang ₁ , Xiaoyun Chen ₁ , Xiaoli Xu ₁ , Wei Wei ₁ , Lei Yang ₁ , Yi Ji ₁ , Jian Wu ₂ , Junfeng Xu ₁ , Cheng Peng ₁

Affiliation

Introduction

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.

Objective

In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS 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.

Methods

The RPA reaction used for amplification CaMV35S and NOS 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 CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically.

Results

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.

Conclusion

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.

中文翻译：

开发用于转基因作物的新型 Cas13a/Cas12a 介导的“一锅”双重检测方法

介绍

转基因（GM）作物已在世界范围内广泛种植，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体的监管至关重要。

目的

在这项研究中，我们开发了一种新型的单罐系统，称为 MR-DCA（多重 RPA 和双 CRISPR 检测），用于同时检测转基因作物中的 CaMV35S 和 NOS 遗传靶标。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序列）检测技术相结合，为转基因作物检测提供了一种简化且高效的方法。

方法

用于扩增 CaMV35S 和 NOS 靶标的 RPA 反应包含在管基中，而双 CRISPR 酶置于管帽中。离心后，启动双 CRISPR （Cas13a/Cas12a）检测系统。荧光可视化用于通过 FAM 通道测量 CaMV35S，通过 HEX 通道测量 NOS。当使用侧向层析试纸条时，使用兔抗地高辛（蓝线）检测 CaMV35S，而使用抗小鼠 FITC（红线）检测 NOS。使用图像 J 量化线强度并以图形方式描述。