Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus"

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，柑橘溃疡病）是一种对全球柑橘生产造成严重影响的毁灭性柑橘病害。该病与“亚洲黄龙病菌”（Candidatus Liberibacter asiaticus，CLas）相关，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、在感染植物中的分布不均，以及采样问题和抑制剂的共存，利用qPCR检测CLas具有挑战性。本研究评估了使用多拷贝基因靶标（16S和RNR）的双重滴定数字聚合酶链反应（ddPCR），以在同一样本中同时检测CLas DNA靶标，从而实现对来自柑橘叶片和ACP的DNA提取物中HLB病原体的明确检测。使用质粒、柑橘叶片和ACP DNA的十倍稀释系列进行的标准曲线分析表明，双重ddPCR和qPCR在双重实验中均表现出良好的线性度和效率。使用CLas感染低滴度样本验证双重ddPCR和qPCR的性能，结果表明，在双重实验中同时使用16S和RNR引物时，检测率更高。然而，受试者工作特征分析表明，与16S引物相比，RNR引物的曲线下面积在低靶标滴度下检测CLas时显著更宽。两种引物集在可变滴度下对CLas的绝对定量均具有可重复性和可重复性，并且ddPCR对柑橘叶片和ACP提取物中的PCR抑制剂具有更高的抵抗力。因此，所得的双重ddPCR实验结果显著提高了在病原体滴度低的样本中诊断CLas的检测平台。