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Biology
CRISPR-Cas12aと組み合わせたリコンビナーゼポリメラーゼ増幅を用いたフィールド展開可能な カンデ...
CRISPR-Cas12aと組み合わせたリコンビナーゼポリメラーゼ増幅を用いたフィールド展開可能な カンデ...
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JoVE Journal Biology
Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

CRISPR-Cas12aと組み合わせたリコンビナーゼポリメラーゼ増幅を用いたフィールド展開可能な カンディダトゥス ・リベリバクター・アジアティカス検出(英語)

Full Text
3,221 Views
09:03 min
December 23, 2022

DOI: 10.3791/64070-v

Min Li*1, Hongkun Qin*1, Yunfei Long1, Meiqin Cheng1, Ling Li1, Aijun Huang2, Nian Wang3, Shuo Duan1

1China-USA Citrus Huanglongbing Joint Laboratory, National Navel Orange Engineering Research Center,Gannan Normal University, 2Department of Biological Sciences,Gannan Normal University, 3Citrus Research and Education Center, Department of Microbiology and Cell Science,University of Florida - Institute of Food and Agricultural Sciences

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Overview

This study addresses the need for rapid and accurate detection of Candidatus Liberibacter asiaticus (CLas), the causative agent of Huanglongbing (HLB) in citrus plants. A new method, referred to as CLas-DETECTR, combines recombinase polymerase amplification with CRISPR-Cas12a technology, allowing for portable and sensitive testing in the field.

Key Study Components

Research Area

  • Plant Pathology
  • Diagnostics in Agriculture
  • Microbial Genetics

Background

  • Huanglongbing (HLB) severely impacts citrus production globally.
  • CLas early detection is crucial for preventing disease spread.
  • Current methods like PCR have limitations, requiring improvement for field applicability.

Methods Used

  • Recombinase polymerase amplification combined with CRISPR-Cas12a
  • Field samples of citrus leaves were utilized for testing.
  • Fluorescent detection technology for result visualization.

Main Results

  • CLas-DETECTR showed significantly higher sensitivity than standard PCR.
  • Specific fluorescence was observed exclusively in CLas-positive samples.
  • The method can yield results within 90 minutes under isothermal conditions.

Conclusions

  • The study demonstrates that CLas-DETECTR is a robust and sensitive diagnostic tool for HLB.
  • This method can facilitate timely interventions to manage citrus diseases effectively.

Frequently Asked Questions

What is Huanglongbing (HLB)?
HLB is a destructive disease affecting citrus plants, caused by Candidatus Liberibacter asiaticus.
How does CLas-DETECTR work?
It combines recombinase polymerase amplification with CRISPR-Cas12a for sensitive and rapid detection of CLas.
What advantages does the CLas-DETECTR method offer?
It provides a portable, fast, and highly sensitive approach for detecting CLas in field conditions.
How long does the detection process take?
The complete testing can be performed in about 90 minutes.
What was the specificity of the method?
Only CLas-positive samples showed green fluorescence, indicating high specificity.
Can this method be used in different environmental conditions?
Yes, the isothermal condition makes it applicable in various field situations.

本研究では、CRISPR-Cas12aと組み合わせたリコンビナーゼポリメラーゼ増幅に基づくカン ディダトゥス ・リベリバクター・アジアティカスの迅速、高感度、かつポータブルな検出法を開発しました。

黄龍弓、HLBと略される。それは世界中で壊滅的な柑橘類の病気です。中国や米国などのほとんどの柑橘類生産国では、HLBは師部制限細菌によって引き起こされます カンディダトゥス・リベリバクター・アジアティカス (CLasと略される)。

CLasの早期発見は、早期介入を促進し、病気の蔓延を防ぐ生産者にとって重要でした。そこで、ここでは、リコンビナーゼポリメラーゼ増幅およびCRISPR-Cas12aシステムと組み合わせて、迅速でポータブルなHLB診断のための新しい方法を開発しました。私たちはそれをCLas-DETECTRと呼んでいました。

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