DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

Changyue Liu; Zhengyang Lei; Lijin Lian; Likun Zhang; Zhicheng Du; Peiwu Qin

doi:10.3791/64833

JoVE Journal
Biology
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JoVE Journal Biology

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

基于RPA-CRISPR/Cas12a-SPM和深度学习的DNA病毒检测系统

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04:17 min

May 10, 2024

DOI: 10.3791/64833-v

Changyue Liu*^1,2, Zhengyang Lei*^1,2, Lijin Lian², Likun Zhang^1,2, Zhicheng Du^1,2, Peiwu Qin^1,2

¹Center of Precision Medicine and Healthcare,Tsinghua-Berkeley Shenzhen Institute, ²Institute of Biopharmaceutics and Health Engineering,Tsinghua Shenzhen International Graduate School

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Overview

This study presents a rapid and sensitive detection system for frog virus 3, emphasizing the integration of recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system to enable point-of-care detection of DNA viruses. This innovative method aims to enhance detection accuracy and efficiency, which is crucial in preventing potential pandemic outbreaks.

Key Study Components

Research Area

Molecular biology
Pathogen detection
Point-of-care diagnostics

Background

The significance of rapid viral detection in preventing pandemics.
Integration of advanced methodologies like RPA and CRISPR technology.
The role of portable systems in field-ready diagnostic assessments.

Methods Used

Recombinase polymerase amplification (RPA)
CRISPR/Cas12a system
Smartphone-based microscopy with AI-assisted classification

Main Results

Significant differentiation between 10 aM FV3 and control samples.
Enhanced detection accuracy through the use of specific primers and CRISPR RNA.
Potential for adapting the method for other DNA viruses by modifying CRISPR RNA.

Conclusions

The study demonstrates an efficient portable detection method for DNA viruses.
This approach is relevant for timely protective measures in the breeding industry, illustrating its importance in biological research and public health.

Frequently Asked Questions

What is the primary application of this detection system?

The system is designed for point-of-care detection of DNA viruses, enabling rapid diagnostic results.

Which virus is primarily studied in this protocol?

Frog virus 3 is the main focus of the detection method outlined in this study.

How does the integration of RPA and CRISPR/Cas12a improve detection?

This integration enhances efficiency and accuracy while minimizing human error in the detection process.

What technological innovations are used in this protocol?

The protocol utilizes a portable smartphone microscope and AI-assisted classification for analyzing results.

Can the methodology be adapted for other viruses?

Yes, the CRISPR RNA can be modified to target other DNA viruses, making the technique versatile.

What is the significance of this study in terms of public health?

Timely detection and effective responses to viral outbreaks can help mitigate economic losses and health risks in breeding industries.

What role does AI play in the detection process?

AI assists in classifying the results obtained from the smartphone microscopy images, thus improving accuracy.

我们提出了一种方案，该方案将重组酶聚合酶扩增与CRISPR / Cas12a系统相结合，用于DNA病毒的痕量检测，并构建了具有人工智能辅助分类的便携式智能手机显微镜，用于即时DNA病毒检测。

我们的方案引入了一种快速且高度灵敏的青蛙病毒 3 检测系统。值得注意的是，这种方法能够对DNA病毒进行即时检测，在预防大流行性疾病的发生方面发挥着至关重要的作用。该技术的主要优势在于它将RPA与CRISPR / Cas12a系统集成，并辅以基于智能手机的便携式显微镜和AI分类。

这种集成显著提高了检测效率和准确性，同时减少了人为因素造成的错误。首先，在反应缓冲液中加入四种关键的RPA酶。然后将预先设计的引物添加到混合物中。

彻底涡旋混合物。向每个 RPA 反应中加入 1 微升从青蛙病毒 3 获得的靶 DNA，并涡旋混合均匀。接下来，吸取 7 微升 100 毫摩尔氯化镁以引发反应。

将混合物在 37 摄氏度下孵育 30 分钟以完成测定。用CRISPR RNA制备Lachnospiraceae细菌CAS12a蛋白，形成功能性复合物。混合细菌蛋白和 10 X CRISPR/Cas12a 反应缓冲液。

现在添加 500 纳摩尔单链 DNA 报告探针。向 CRISPR/Cas12a CRISPR RNA 反应混合物中加入 1 微升 RPA 反应产物。将混合物在 37 摄氏度下孵育 30 分钟。

用酶标仪测量荧光信号。为了使用智能手机显微镜进行检测，首先将准备好的反应混合物移液到退缩的载玻片上。然后在孵育前用盖玻片盖住它。

将载玻片放在智能手机显微镜的载物台上，并调整焦距和清晰度。捕获图像以测量荧光信号。使用 ImageJ 测量每幅图像的平均灰度值和浓度组中平均灰度值的标准差。

采用深度学习模型 AlexNet 33 进行分类。现在使用 Python 将输入图像调整为 224 x 224 乘以三个通道。最后，使用预训练的骨干网络和ImageNet数据集来提取图像特征。

第六对引物提供了最大的扩增效率，并被选中。CRISPR RNA-3被证明是最有效的侧支切割方法。使用具有选定 RPA 引物和 CRISPR RNA 的开发检测系统导致 10 aM FV3 和对照组之间存在显着差异。

要记住的最重要的一点是具体的 CAS 12a 检测步骤。反应的CRISPR RNA可以被修饰或重新设计，以基于CAS 12a检测靶向其他DNA病毒。该方法有助于对目标病毒的数量进行初步评估。

基于此，可以采用qPCR等其他方法来获得更准确的病毒载量。该技术为便携式DNA病毒检测提供了初步尝试。对于本议定书中使用的青蛙病毒3，及时发现可以促使采取有效的保护措施，减少养殖业的损失。

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