Department of Plant Pathology and Environmental Microbiology and Huck Institute of the Life Sciences, The Pennsylvania State University, University Park, PA 16802.
USDA/ARS U.S. Horticultural Research Laboratory, Fort Pierce, FL 34945.
Phytopathology. 2021 Dec;111(12):2375-2382. doi: 10.1094/PHYTO-09-20-0443-R. Epub 2021 Dec 6.
Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, ' 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 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.
黄龙病（HLB）又称绿症，是全球柑橘类水果最具破坏性的疾病之一。对其致病因子“亚洲韧皮杆菌”（CLas）的敏感检测对 HLB 的早期诊断和成功管理至关重要。然而，目前基于核酸的检测方法通常不足以从无症状组织中早期检测到 CLas，也不适合 HLB 的高通量和现场诊断。在这里，我们报告了基于 Cas12a 的 DNA 内切酶靶向 CRISPR 报告基因检测（DETECTR）检测方法的开发，用于从感染样本中高度特异性和灵敏地检测 CLas 核酸。DETECTR 检测方法针对 CLas 特异性的 5 拷贝基因，将等温扩增与 Cas12a 对荧光报告寡核苷酸的切割相结合，能够在飞摩尔水平检测 CLas 核酸。DETECTR 检测方法能够特异性地检测不同感染的柑橘、长春花和木虱样本中 CLas 的存在，并且与侧向流动检测技术兼容，具有潜在的现场诊断应用。DETECTR 技术在检测灵敏度和灵活性方面的改进使该检测方法成为感染区域中 CLas 早期检测的潜在合适工具。
