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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Publications at this Location » Publication #377033
Research Project: Mitigating High Consequence Domestic, Exotic, and Emerging Diseases of Fruits, Vegetables, and Ornamentals

Location: Subtropical Plant Pathology Research

Title: Highly sensitive and rapid detection of citrus huanglongbing pathogen (Candidatas Lliberibacter asiacticus) using Cas12a-based methods

Author
item WHEATLEY, MATTHEW - Pennsylvania State University
item Duan, Ping
item YANG, YINONG - Pennsylvania State University

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2021
Publication Date: 5/4/2021
Citation: Wheatley, M.S., Duan, Y., Yang, Y. 2021. Highly sensitive and rapid detection of citrus huanglongbing pathogen (Candidatas Lliberibacter asiacticus) using Cas12a-based methods. Phytopathology. https://doi.org/10.1094/PHYTO-09-20-0443-R.
DOI: https://doi.org/10.1094/PHYTO-09-20-0443-R

Interpretive Summary: Citrus Huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of the HLB causal agent, Candidatus Liberibacter asiaticus (CLas) is critical for early diagnosis and successful management of HLB. Recently, the CRISPR-Cas (Clustered Regularly Interspersed Short Palindromic Repeats-CRISPR associated protein) system has been adapted as a molecular diagnostic tool outside of its conventional genome editing applications. These CRISPR-based detection strategies, such as DETECTR have been demonstrated as point of care diagnostic tools for highly sensitive and specific detection of pathogen nucleic acids in biomedical samples. These CRISPR/Cas-based diagnostic method rely on the direction of Cas12a or Cas13a by CRISPR RNA (crRNA) to target nucleic acids and the subsequent degradation of fluorescent reporter oligonucleotides. When coupled with isothermal amplification strategies, CRISPR/Cas-based detection strategies resulted in rapid (1-2 hours) and robust detection of nucleic acids (RNA or DNA) at the attomolar and zeptomolar sensitivity with single nucleotide specificity. Similarly, the adoption of CRISPR/Cas-based detection for phytopathogen nucleic acid should enable the highly sensitive, specific, and rapid diagnostics of various plant diseases including HLB. In this study we employed Lachnospiraceae bacterium (Lba) Cas12a, also known as Cpf1, for highly specific and sensitive detection of CLas nucleic acids. By targeting the higher copy number of an CLas gene, we developed a highly sensitive and specific DETECTR assay for detection of CLas in various citrus and insect samples. The CLas DETECTR assay was capable of detecting the nrdB target down to the attomolar level in both the microplate- and lateral flow-based assays. In addition, we have shown that CLas DETECTR assay has the capability of being used in-field to provide real-time diagnostics of CLas from infected samples with the use of lateral flow strips and portable endpoint fluorometers. This new technology provides new tools for early and high throughput detections of CLas infection, and potential for commercialization.

Technical Abstract: Citrus Huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of Candidatus Liberibacter asiaticus (CLas) is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue, and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay that targets the five-copy nrdB gene specific to CLas couples isothermal amplification and Cas12a trans-cleavage of fluorescent reporter oligos for detection of CLas nucleic acids at the attomolar level. The DETECTR assay was able to accurately detect the presence of CLas across different infected citrus, periwinkle and psyllid samples, and shown to be compatible with lateral flow assay technology for potentially field-deployable diagnosis. The improvement in detection sensitivity and flexibility of the DETECTR technology position the DETECTR assay as a suitable tool for early detection of CLas in infected regions.