Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan.
Division of Forest Protection, Taiwan Forestry Research Institute, Taipei, Taiwan.
PLoS One. 2023 Jun 23;18(6):e0287699. doi: 10.1371/journal.pone.0287699. eCollection 2023.
Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus plants caused by the obligate and phloem-limiting bacterium Candidatus Liberibacter asiaticus (Las). Reliable detection methods are important for successful control of the disease. This study was aimed to develop a rapid and user-friendly on-site detection system for Las using the TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay. The Las-specific on-site detection system could be completed within one hour by simple DNA extraction coupled with a portable POCKIT device, which can perform PCR amplification and automatically provide qualitative results derived from fluorescence signals. The sensitivity of the TaqMan probe-iiPCR assay could be as low as single copy of Las, comparable to a real-time PCR method. Further testing of the field citrus samples showed 100% agreement between the TaqMan probe-iiPCR assay and the real-time PCR method, and the on-site detection system also demonstrated a great performance of Las detection. With high specificity and sensitivity, the on-site detection system developed in this study becomes a simple, rapid and powerful tool for detecting Las in fields.
柑橘黄龙病（HLB）是由韧皮部限养的专性细菌柑橘黄龙病菌（Candidatus Liberibacter asiaticus，Las）引起的柑橘类植物最具破坏性的疾病之一。可靠的检测方法对于成功控制该疾病至关重要。本研究旨在利用 TaqMan 探针基于隔热等温聚合酶链反应（iiPCR）检测法，开发一种用于 Las 的快速、用户友好的现场检测系统。Las 特异性现场检测系统通过简单的 DNA 提取与便携式 POCKIT 设备结合，在一小时内即可完成，该设备可进行 PCR 扩增，并自动提供源自荧光信号的定性结果。TaqMan 探针-iiPCR 检测法的灵敏度可低至 Las 的单个拷贝，与实时 PCR 方法相当。对田间柑橘样本的进一步测试表明，TaqMan 探针-iiPCR 检测法与实时 PCR 方法之间的一致性达到 100%，现场检测系统还展示了 Las 检测的出色性能。该研究开发的现场检测系统具有高度特异性和灵敏度，成为田间检测 Las 的一种简单、快速和强大的工具。
