Preparation and Application of Plasmid DNA Reference Material in the Detection of Cronobacter in Infant Formula

Abstract

The housekeeping genes atpD, gyrB, infB, fusA and glnS in Cronobacter sakazakii were selected as target sequences to construct plasmid standards for rapidly detecting C. sakazakii. The constructed recombinant plasmids was verified by colony polymerase chain reaction (PCR) and sequencing and was checked for stability after 15 passages. The atpD, gyrB, infB, fusA and glnS plasmid standards were qualitatively tested and evaluated in terms of limit of detection (LOD) and stability. Then, these plasmid standards were used to detect C. sakazakii in infant milk powder. The results of colony PCR and sequencing showed successful construction of the atpD, gyrB, infB, fusA and glnS plasmid standards. The PCR system was specific for the detection of the plasmid standards. The detection limits of atpD, gyrB, infB, fusA and glnS were 1.14 × 106, 1.07 × 107, 1.09 × 107, 1.73 × 105 and 7.54 × 105 copies/μL, respectively. The lyophilized plasmids could be stored stably at −20, 4 and 25 ℃ for 90 days. The plasmid standards were used to detect 23 samples of infant milk powder, and one of them was detected positive for gyrB. However, C. sakazakii was undetectable by the traditional national standard method (GB 4789.40-2016). Compared with the traditional method, the application of the plasmid standards greatly improved the detection efficiency of samples.

Keywords

• cronobacter; house-keeping genes; plasmid standards; rapid detection