A specific diagnostic method for differentiation of Citrobacter freundii based on target amplification

Background and Aim : Citrobacter freundii is able to cause dangerous diseases such as neonatal meningitis associated with brain abscesses, diarrhea, sepsis, and nosocomial infections in humans. Therefore, rapid identification is essential for effective treatment. In this study, we detected bacteria using a specific molecular polymerase-based method. Methods : The bacterial cells were cultured in tryptic soy broth medium followed by genomic DNA extraction. We designed specific oligonucleotides targeting the gene and amplified the region with the polymerase chain reaction. The target gene was a protein coding of YdcF family with unique sequence in Citrobacter freundii. The assay was tested by analyzing different bacteria from various genera and species. The products are visualized and confirmed by gel electrophoresis. The specificity and limit of detection (LOD) were analyzed.Results : DNA extraction has been performed and the quality of the extracted DNA was evaluated by spectroscopy and ۲% agarose gel electrophoresis. The target gene was amplified and a ۲۶۶ bp product was expected. The specificity was analyzed using different bacteria namely Yersinia enterocolitica, Morganella morganii, Serratia marcescens and Shigella sonnei. A unique amplicon band was detected for Citrobacter freundii while there was no observable amplification for other bacteria. For evaluating the sensitivity of the method, the extracted genomic DNA of Citrobacter freundii was subjected to serial dilution. Amplification occurred at up to ۱۰^(-۵) dilution of initial concentration. Conclusion : As a diagnostic system, the used technique could identify target microorganism efficiently with high sensitivity in a few hours. By making the identification process easier, faster, and more affordable, the difficulties associated with the late diagnosis of infectious diseases could be facilitated.

Keywords:

Citrobacter freundii , Molecular Diagnostics , Pathogen , Nucleobiomarker , PCR