Development of DNA Reference Materials of Citrus Huanglongbing Candidatus Liberibacter asiaticus

Citrus Huanglongbing (HLB) is a devastating disease within the Citrus industry. Candidatus Liberibacter asiaticus (CLas) is one of the most prevalent HLB-associated strains that has not been cultured in vitro. To ensure the accuracy and comparability of the molecular diagnostic method for HLB detection, certified reference materials urgently need to be developed for CLas detection. Here, we developed a series of DNA reference materials of CLas using 16S rDNA as the target gene and the SAND gene as the Citrus reference gene. The 16S rDNA gene fragment cloned by the NCBI sequence and Citrus DNA extracted by healthy Citrus leaves are thoroughly mixed for preparation. Droplet digital PCR (ddPCR) was used as an accurate quantification method for 16S rDNA, and the SAND was established and optimized through this study. Nine laboratories collaborated in determining these two parameters, and the homogeneity and stability were adequate. The quantification results demonstrated that the copy number certified values and expanded uncertainty of 16S rDNA and SAND in the high-concentration reference material were (3.86 ± 0.34) × 10³ and (4.43 ± 0.39) × 10³ cp/μL, respectively. The copy number certified values and expanded uncertainty of 16S rDNA and SAND in the low-concentration reference material were (3.98 ± 0.36) × 10² and (4.34 ± 0.37) × 10³ cp/μL, respectively. In addition, this certified reference material will provide reliable quality control for detecting CLas.