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.