单核细胞增生李斯特菌质粒DNA参考物质的研制

Preparation of plasmid DNA reference material for Listeria monocytogenes

目的研制一种能够用于快速鉴定单核细胞增生李斯特菌(以下简称单增李斯特菌)的质粒DNA标准物质。方法设计一种质粒DNA包含目前常用于单增李斯特菌检测的hlyA、plcB、inlA基因,并对其稳定性、均匀性和量值可追溯性进行评价。评估其在实时定量聚合酶链式反应(PCR)检测中的适用性。结果质粒DNA参考物质的最终定值结果为29.85μg/mL。该质粒DNA参考物质量值可靠,均匀性和稳定性良好,可以在-20℃下保存1年以上。质粒DNA参考品在实时荧光PCR检测中的适用性证明,可以保证实时定量PCR检测结果与单增李斯特菌基因组DNA参考品具有可比性。结论研制的质粒DNA标准物质可以替代单增李斯特菌基因组DNA对单增李斯特菌进行质量监控,满足单增李斯特菌检测在实验室质量的有效控制和试验方法验证的需要。

Objective To develop a plasmid DNA reference material for rapid and comprehensive identification of Listeria monocytogenes. Methods The synthetic DNA fragment contained the hlyA, plcB and inlA, which were currently used for the detection of Listeria monocytogenes, was cloned into vector PUC57 to construct the plasmid reference material(pDNA Listeria). The quantity, homogeneity and stability of pDNA Listeria were evaluated by ultraviolet spectrophotometry(UV). real time quantitative polymerase chain reaction(qPCR) was used to evaluate the applicability of pDNA Listeria.ResultsThe results showed that the fixed value of pDNA Listeria was 29.85 μg/mL,which had traceable values, reliable homogeneity and good stability.The pDNA Listeria could be staored at-20 ℃ for more than one year. The pDNA Listeria also provided comparable sensitivity and reliability to the genomic references material. Conclusion The pDNA Listeria could be used not only to identify Listeria monocytogenes but also to describe the biological characteristics such as virulence, pathogenicity, and invasiveness of Listeria monocytogenes at the same time. Importantly, the study proved the application of rapidly synthesized multiple targets plasmid serving as qPCR standard for pathogen identification.