San Joaquin Valley Agricultural Sciences Center, Agricultural Research Service, United States Department of Agriculture, Parlier, California, United States of America.
Contained Research Facility, University of California, Davis, Davis, California, United States of America.
PLoS One. 2021 Mar 17;16(3):e0242392. doi: 10.1371/journal.pone.0242392. eCollection 2021.
"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.
“亚洲韧皮杆菌”（CLas）和柑橘僵化螺旋体（Spiroplasma citri）是两种局限于韧皮部的细菌，感染柑橘并通过昆虫媒介传播。S. citri 引起柑橘顽固病（CSD），在加利福尼亚州由桃蚜传播。CLas 与毁灭性的柑橘黄龙病（HLB）有关，由亚洲柑橘木虱传播。CLas 在加利福尼亚州南部的居民区柑橘上传播，是一种即将传播到商业柑橘种植园的威胁。CSD 在加利福尼亚州流行，其柑橘症状容易与 HLB 混淆。因此，本研究的目的是开发一种多重 qPCR 和双duplex 数字 PCR（ddPCR）检测方法，用于同时检测可能共存的 CLas 和 S. citri。多重 qPCR 检测方法设计用于分别检测 CLas-RNR（5 个拷贝）和 S. citri-SPV1 ORF1（13 个拷贝）的多拷贝基因，以及柑橘细胞色素氧化酶（COX）作为内部阳性对照。通过 duplex ddPCR 进行绝对定量，作为 qPCR 结果的补充。duplex ddPCR 比 qPCR 更灵敏地检测到 CLas 和 S. citri。ddPCR 对抑制剂的耐受性更高，产生的结果高度可重复。多重 qPCR 检测方法具有降低成本同时检测两种病原体的优势，并可补充加利福尼亚州用于检测 CLas 的官方监管方案。此外，ddPCR 无需病原体滴度标准即可在非常低的浓度下提供 CLas 和 S. citri 的明确绝对检测。
