Rapid detection and effective treatment of Citrobacter Freundii as a heat-stable and Shiga-like toxin-secreting cause of diarrhea in humans is of particular importance. In this study, the identification of C. freundii was performed by targeting a specific nucleobiomarker using the polymerase chain reaction (PCR) method.

Genomic DNA extraction was performed using overnight culture of the bacteria in tryptic soy broth medium. A unique sequence of a protein-coding gene in C. freundii was targeted for specific primer design. The target region was amplified using a gradient thermocycler. The specificity of the method was investigated using various bacteria from closely related genera and species. PCR products visualized using a natural dye in a colorimetric assay and confirmed by gel electrophoresis.

Concentration of 1-85 ng/µl was considered as appropriate for further analysis. A high molecular weighted band of genomic DNA on agarose gel was determined as quality control of extraction process. A 266 bp band of target gene was observed in C. freundii while no amplicon band was detected for other bacteria including Morganella morganii, Enterobacter aerogenes, Pseudomonas aeruginosa, Yersinia enterocolitica, Shigella sonnei, Serratia marcescens, Burkholderia cepacia, and Klebsiella pneumoniae. As in the colorimetric assay, a color change from purple to green was observed for C. freundii where for negative samples the purple color of the dye changed to blue leaving a distinct color differentiation.

The developed technique could differentiate positive and negative samples in a less than two hours with high specificity facilitating commercialization in diagnostic kits.