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Virus detection is a highly important measure in epidemic prevention and control, enabling early detection of outbreaks, accurately monitoring epidemic dynamics, and timely implementing preventive measures to effectively restrict the spread of the disease. However, low-cost, highly accurate, and deployable virus detection methods with high sensitivity are still limited. Cas12a, a type II V-A CRISPR system, has widely been used in genetic editing due to its ability to target AT-rich regions. Here we describe the establishment of a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)-Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). In this protocol, we provide a step-by-step guide for the preparation and purification of huLbCas12a. Detailed procedures guide ones all throughout basic steps, including verification of huLbCas12a protein activity, sgRNA design, optimization of the LAMP-CRISPR detection system, and actual sample detection.

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CRISPR-Cas12-Based PCV2 Viral Detection

  • 实验方案

  • 发表日期：

    2025年

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  • Lei Lei,
  • Xiaojiu Huang,
  • Xiaoye Peng,
  • Kaixin Wang,
  • Yifan Guo,
  • Yang Zhan,
  • Lei Tan,
  • Yuge Wang,
  • Yi Yang,
  • Deyong Duan,
  • Aibing Wang

摘要

Virus detection is a highly important measure in epidemic prevention and control, enabling early detection of outbreaks, accurately monitoring epidemic dynamics, and timely implementing preventive measures to effectively restrict the spread of the disease. However, low-cost, highly accurate, and deployable virus detection methods with high sensitivity are still limited. Cas12a, a type II V-A CRISPR system, has widely been used in genetic editing due to its ability to target AT-rich regions. Here we describe the establishment of a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)-Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). In this protocol, we provide a step-by-step guide for the preparation and purification of huLbCas12a. Detailed procedures guide ones all throughout basic steps, including verification of huLbCas12a protein activity, sgRNA design, optimization of the LAMP-CRISPR detection system, and actual sample detection.