Pilot Production and Intralaboratory Characterization of a Candidate Genomic DNA Reference Material from Mycobacterium bovis for PCR-Based Molecular Assays

Accurate and reliable PCR-based detection of Mycobacterium bovis requires quality-control materials with well-defined properties to support molecular measurements. In this study, a candidate genomic DNA reference material (RM) from M. bovis was produced and characterized under intralaboratory conditions. An optimized extraction and pilot-production workflow yielded high-molecular-weight genomic DNA suitable for quantitative molecular applications. qPCR and dPCR measurement procedures targeting the Mb3145 and CysA3 targets were analytically evaluated and subsequently used for material characterization, with dPCR providing the measurements for copy-number assignment. The qPCR measurement procedures showed amplification efficiencies of 89–103% and coefficients of determination ≥0.98, whereas dPCR enabled detection of approximately 3 copies per reaction under the evaluated conditions. Two candidate RM concentration levels were produced, with assigned copy-number concentrations of 62,922 and 426 copies/µL and a relative expanded uncertainties of 8.5% and 15.9% (k = 2), respectively. Between-unit homogeneity contributed relative to standard uncertainties of 0.8% and 1.2%. Both concentration levels remained stable for at least eight weeks at 4 °C; the high-concentration material showed no significant trend over 12 months at 4 °C, whereas the low-concentration material showed significant long-term change under the same conditions. Overall, the results support the intended analytical use of the pilot material as a candidate for genomic DNA RM for quality control, method verification, and analytical performance assessment of PCR-based measurement procedures involving M. bovis genomic DNA. Further interlaboratory evaluation is required to establish its broader applicability and comparability across laboratories.