Preparation of plasmid DNA reference material for Listeria monocytogenes

Abstract

ObjectiveTo develop a plasmid DNA reference material for rapid and comprehensive identification of Listeria monocytogenes.MethodsThe 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.ConclusionThe 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.

Keywords

• listeria monocytogenes
• synthetic plasmid standard
• real time fluorescent quantitative pcr