USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, United States of America.
Citrus Pest Detection Program, Central California Tristeza Eradication Agency, Tulare, CA, United States of America.
PLoS One. 2018 May 17;13(5):e0197184. doi: 10.1371/journal.pone.0197184. eCollection 2018.
Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.
黄龙病（HLB，柑橘绿化病）是一种毁灭性的柑橘病害，影响着全球的柑橘生产。它与细菌“亚洲韧皮部杆菌（CLas）”有关，并由亚洲柑橘木虱（ACP）传播。目前，监管样本中 CLas 的诊断基于使用 16S rRNA 基因特异性引物/探针的实时定量聚合酶链反应（qPCR）。由于病原体滴度低且在感染植物中分布不均匀，再加上采样问题和抑制剂的存在，使用 qPCR 检测 CLas 具有挑战性。本研究评估了一种使用多拷贝基因靶标 16S 和 RNR 的双重液滴数字聚合酶链反应（ddPCR），以同时在同一样品中检测 CLas DNA 靶标，从而在柑橘叶片和 ACP 的 DNA 提取物中明确检测 HLB 病原体。用质粒、柑橘叶和 ACP DNA 对十倍稀释系列进行标准曲线分析表明，ddPCR 和 qPCR 在双重检测中均表现出良好的线性和效率。使用 CLas 感染的低滴度样本验证了双重 ddPCR 和 qPCR 的性能，结果表明，在双重检测中使用 16S 和 RNR 引物时，检测率更高。然而，接收者操作特征分析表明，与 16S 引物相比，RNR 引物的曲线下面积在低靶标滴度时用于 CLas 检测的显著更宽。对于可变滴度的 CLas 的绝对定量对于两种引物组都是可重复和可重复的，并且 ddPCR 显示出对柑橘叶和 ACP 提取物中 PCR 抑制剂更高的弹性。因此，所得的双重 ddPCR 检测方法为低病原体滴度样本中 CLas 的诊断提供了一个显著改进的检测平台。
