Development of PCR-based method for detection of Enterobacteriaceae in septicemia

Abstract

• Objective: Sepsis is a systemic inflammatory response associated with high mortality rates in the clinical setting. A multiplex end point PCR based assay for rapid detection Enterobacteriaceae involvement in septicemia, which included Internal Control and16S rDNA is present. To develop a panel of primers and evaluate analytical sensitivity and specificity for DNA fragments of 16S rDNA, Enterobacteriaceae and internal control.
• Materials and Methods: Primers for amplification of Enterobacteriacea IC and16S rDNA were designed, and then PCR was performed. Minimal analytical sensitivity was determined by Cloning and colony PCR and specificity tested on the basis of their respective standard strains. This study is a cross-sectional kind.
• Results: Our results showed the rpoB gene as the most promising target for detection of Enterobacteriaceae by PCR amplification. Specificity and sensitivity of end point PCR were 100%, 100% and 100%, 10, 1 and 100 copies/reaction for Enterobacteriaceae, IC and 16S rDNA in analytical step by standard strains, respectively.
• Conclusion: The molecular panel of presented offers the advantage of an easy, reliable and cost-effective system when compared to other molecular detection methods. However, further evaluation is needed, our assay holds promising for more rapid pathogens related in clinical sepsis.
• Key words: Bacteria pathogens, Blood culture, DNA primers/diagnosis, ICU, PCR, Sepsis