Project

Developing an RT-PCR Assay Using Peptide Nucliec Acid Capture Probes to Identify the 16s RRNA Genes in Bacteria Causing Sepsis

Although many advances in research for the identification of infections have been made, Sepsis stands as one of the leading causes of death in the United States3. Sepsis is life threatening since it causes organ dysfunction caused by a deregulated immune response to infection. Bacterial infections are a common cause of Sepsis. As clinical intervention is paramount for the survival of Septic patients; early detection of Sepsis2 with well-timed and appropriate treatment greatly increases the chance of survival. Blood culture tests still stand as the basic method of detection of bacterial infection. A more conventional method is to develop a molecular diagnostic test that detects the organism causing Sepsis. An RT-PCR based assay for detecting the highly conserved 16s rRNA genes of the bacteria, Klebsiella pnuemoniae has been developed. The assay was tested on the IRIDICA system and confirmed that RT-PCR can be used with the BAC-BSI strip to generate amplicons. To enhance the sensitivity of the Ibis BAC assay, peptide nucleic acid capture probes (PNA) were used. By implementing this method capturing the bacterial RNA was possible. Blood samples treated with PNA capture probes were well detected on the Mass spectrometer compared to samples without PNA. However, further optimization of the peptide nucleic acid capture probes could eventually lead to further increases in sensitivity and the technique could be used on the Ibis IRIDICA diagnostic assay.

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