Highly Sensitive and Rapid Detection of Citrus Huanglongbing Pathogen ('Candidatus Liberibacter asiaticus') Using Cas12a-Based Methods.

Abstract

Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, '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 DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.