A Candidate Genomic DNA Reference Material for PCR-Based Detection of Brucella spp.: Intralaboratory Characterization and Preliminary Application in LAMP Assays

Analytical standardization of Brucella molecular assays requires reference materials with assigned values and defined uncertainty. We produced and characterized intralaboratory a candidate genomic DNA reference material from Brucella abortus RB51 for PCR-based detection of Brucella spp., and evaluated its preliminary use in fluorescence-based and colorimetric LAMP assays. DNA was extracted, assessed by qPCR and dPCR, and diluted to two levels. Homogeneity and stability were evaluated by duplex qPCR targeting ctrA and nikR, whereas property values were assigned by duplex dPCR. qPCR efficiencies were 95.3% for ctrA and 92.1% for nikR. The material fulfilled the homogeneity criterion, with relative homogeneity uncertainties between 0.37% and 1.41%. Assigned values (levels 1 and 2) were 32 989 ± 5 475 and 285 ± 47 copies/µL for ctrA, and 29 722 ± 3 263 and 286 ± 42 copies/µL for nikR. Short-term stability at 4 °C was supported for 8 weeks and was included in the uncertainty budget. ctrA-based LAMP assays were optimized at 67 °C for 45 min and amplified reproducibly in both formats. Amplification was consistent from 2411 to 6.34 copies/µL. This material provides a metrologically characterized tool for quality control, method verification, and optimization of PCR- and LAMP-based assays for Brucella spp.