Huanglongbing disease affects the Rutaceae family and is associated with three phloem-limited bacterium species: Candidatus Liberibacter asiaticus, africanus and americanus. These species are considered quarantine pathogens in the world, and pose major risks for citrus production and industry.

Due to the low titer and the uneven distribution of the bacteria within its host plant, conventional PCR detection protocols can lead to false negative results, especially for early detection. Herein, three real-time PCR diagnostic methods recommended by the EPPO and FAO for asiaticus and africanus species detection were evaluated for their performance and compared with a conventional duplex PCR. Assessments were done as part of an international cooperative project under the EUPHRESCO guidance. Intra-laboratory assessment of the analytical specificity and analytical sensitivity was performed on 33 target or non-target DNA samples and seven target DNA samples were used to determine the sensitivity. Thereafter, repeatability, reproducibility, and concordance odds ratio were assessed on 20 target or non-target DNA samples through a collaborative test performance study organized among eight international laboratories. Results showed that the Li protocol proved to be the best method for asiaticus and africanus species detection, along with the conventional duplex PCR; whereas the Morgan protocol showed high performance only for asiaticus species. Interlaboratory reproducibility was high, suggesting that these real-time PCR methods can be readily transferred to diagnostic laboratories.

黄龙病影响芸香科，并有三个韧皮部限制细菌种类有关：暂定Liberibacter胭脂，非洲种和美洲。这些物种在世界范围内被视为隔离病原体，对柑橘的生产和工业构成重大风险。

由于其宿主植物中的滴度低且细菌分布不均，常规PCR检测方案可能导致假阴性结果，尤其是对于早期检测。在此，评估了EPPO和FAO推荐的三种实时PCR诊断方法对亚洲和非洲物种的检测性能，并与常规双工PCR进行了比较。评估是在EUPHRESCO指导下的国际合作项目的一部分。在实验室内对33个目标或非目标DNA样品进行了分析特异性和分析灵敏度的评估，并使用7个目标DNA样品确定了灵敏度。此后，重复性，再现性，通过在八个国际实验室之间组织的协作测试性能研究，对20个靶标或非靶标DNA样品进行了评估和一致性比值比。结果表明，与常规的双链PCR相比，Li方案被证明是检测积雪草和非洲种的最佳方法。而Morgan协议仅对积雪草物种显示出高性能。实验室间的可重复性很高，表明这些实时PCR方法可以很容易地转移到诊断实验室。