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Biology
Freilandfähiger Candidatus liberibacter asiaticus-Nachweis mittels Rekombinase-Polymeras...
Freilandfähiger Candidatus liberibacter asiaticus-Nachweis mittels Rekombinase-Polymeras...
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JoVE Journal Biology
Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

Freilandfähiger Candidatus liberibacter asiaticus-Nachweis mittels Rekombinase-Polymerase-Amplifikation in Kombination mit CRISPR-Cas12a

Full Text
3,230 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.

In dieser Arbeit wurde eine schnelle, empfindliche und tragbare Nachweismethode für Candidatus Liberibacter asiaticus entwickelt, die auf Rekombinase-Polymerase-Amplifikation in Kombination mit CRISPR-Cas12a basiert.

Huang long bing, abgekürzt als HLB. Es ist eine verheerende Zitruskrankheit weltweit. In den meisten Zitrusländern wie China und den USA wird HLB durch das phloembeschränkte Bakterium Candidatus Liberibacter asiaticus, abgekürzt CLas, verursacht.

Die Früherkennung von CLas war wichtig für Züchter, um frühzeitig eingreifen und die Ausbreitung von Krankheiten zu verhindern. Hier haben wir eine neue Methode für die schnelle und portable HLB-Diagnose entwickelt, die mit der Rekombinase-Polymerase-Amplifikation und dem CRISPR-Cas12a-System kombiniert wird. Wir nannten es CLas-DETECTR.

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