Identification of an Isolate of Citrus Tristeza Virus by Nanopore Sequencing in Korea and Development of a CRISPR/Cas12a-based Assay for Rapid Visual Detection of the Virus

Author:

Kim Hae-Jun1,Cho In-Sook2,Choi Se-Ryung3,Jeong Rae-Dong4

Affiliation:

1. Chonnam National University, 34931, Department of Applied Biology, Gwangju, Jeollanam-do, Korea (the Republic of);

2. Rural Development Administration, 54670, Horticultural and Herbal Crop Environment Division, National institute of horticultural and herbal science, Wanju, Jeollabuk-do, Korea (the Republic of);

3. National Institute of Horticultural and Herbal Science, 588019, Wanju-gun, Jeollabuk-do, Korea (the Republic of);

4. Chonnam National University, 34931, Department of Applied Biology, 77 Yongbong-ro, Buk-gu, Gwangju, Korea (the Republic of), 61186;

Abstract

Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. The disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing (ONT) to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid on-site detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.

Publisher

Scientific Societies

Subject

Plant Science,Agronomy and Crop Science

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