Jakomin Tjaša, Žunič Janja Zajc, Kogovšek Polona
Department of Biotechnology and Systems Biology, National Institute of Biology, 1000 Ljubljana, Slovenia.
Plant Protection Department, Agricultural Institute of Slovenia, 1000 Ljubljana, Slovenia.
Pathogens. 2025 Apr 24;14(5):413. doi: 10.3390/pathogens14050413.
Citrus black spot (CBS), caused by the fungus , significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on region, for detecting through a collaborative test performance study (TPS). DNA from the isolates of spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related species, while Pc-TEF1 showed higher specificity for included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of , contributing to the effective management of CBS disease in citrus production and trade.
柑橘黑斑病（CBS）由真菌引起，严重影响柑橘果实的市场适销性，并可能导致果实提前脱落。准确可靠地检测这种检疫性病原菌至关重要，尤其是对于无症状的植物材料。本研究通过一项协作测试性能研究（TPS），评估了两种基于 区域的qPCR检测方法，即欧洲和地中海植物保护组织（EPPO）推荐的检测方法PC和检测方法Pc - TEF1，用于检测 。将 spp. 分离株和其他真菌的DNA添加到柑橘果皮提取物（柠檬、橙子和柚子）中，并分发给13个实验室。尽管长时间储存会影响Pc - TEF1检测方法的性能，但在典型运输条件下样本和qPCR检测方法的稳定性得到了证实。根据灵敏度、特异性、重现性和重复性对检测方法进行了评估。两种检测方法均表现出高性能，重复性和重现性超过95%。正如预期的那样，PC检测方法检测到了不同的相关 物种，而Pc - TEF1仅对TPS中包含的 表现出更高的特异性。此外，在柚子皮样本中特别观察到了抑制作用，表明存在基质依赖性变异性。这项TPS证实了PC和Pc - TEF1 qPCR检测方法都是可靠的。对qPCR检测方法的进一步评估将有助于选择最可靠的检测方法来检测 ，为柑橘生产和贸易中CBS病害的有效管理做出贡献。
