Citrus concave-gum associated virus (CCGaV) and citrus virus A (CiVA) are two negative-stranded RNA viruses infecting citrus. Both viruses have been identified by high-throughput sequencing in Italy and their simultaneous presence in the same host documented. While CCGaV has been associated with citrus concave gum disease, the association of CiVA with symptoms is still unclear. Since the two virus genomes share about 75% sequence identity, their identification by RT-PCR is not obvious. Here, a multiplex RT-PCR to simultaneously detect and identify them is reported. Amplification of the citrus cytochrome oxidase (COX) gene transcript was included as internal control to minimize the risk of false negatives. Specificity and sensitivity of multiplex RT-PCR were comparable to that of conventional uniplex RT-PCR set up in previous studies for the independent detection of the two viruses. The method was validated by a field survey performed testing almost 300 trees grown in different areas of southern Italy. The assay revealed infection by CCGaV and CiVA in 17.7% and 18.4% of the tested trees, respectively, with mixed infection recorded in 5.0% of the samples. The novel mRT-PCR method saves time and costs and has the potential to be used for large-scale surveys and certification programs.

中文翻译：

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通过多重 RT-PCR 同时检测柑橘凹胶相关病毒 (CCGaV) 和柑橘病毒 A (CiVA)

柑橘凹胶相关病毒（CCGaV）和柑橘病毒A（CiVA）是两种感染柑橘的负链RNA病毒。这两种病毒都已在意大利通过高通量测序进行鉴定，并记录了它们在同一宿主中的同时存在。虽然 CCGaV 与柑橘凹牙龈疾病有关，但 CiVA 与症状的关联仍不清楚。由于两种病毒基因组的序列同一性约为 75%，因此通过 RT-PCR 对其进行鉴定并不明显。在这里，报告了同时检测和识别它们的多重 RT-PCR。柑橘细胞色素氧化酶 (COX) 基因转录本的扩增被包括在内作为内部对照，以尽量减少假阴性的风险。多重 RT-PCR 的特异性和灵敏度与先前研究中建立的用于独立检测两种病毒的常规单重 RT-PCR 的特异性和灵敏度相当。该方法通过对意大利南部不同地区种植的近 300 棵树进行的实地调查验证。检测结果显示，CCGaV 和 CiVA 分别感染了 17.7% 和 18.4% 的测试树木，5.0% 的样本记录了混合感染。新的 mRT-PCR 方法节省了时间和成本，并有可能用于大规模调查和认证计划。混合感染记录在 5.0% 的样本中。新的 mRT-PCR 方法节省了时间和成本，并有可能用于大规模调查和认证计划。混合感染记录在 5.0% 的样本中。新的 mRT-PCR 方法节省了时间和成本，并有可能用于大规模调查和认证计划。