Sepsis claims up to 120,000 lives per year in the U.S., is a leading cause of death in hospitalized patients, and is estimated to cost $17 billion annually in healthcare. Yet, at present there is no straightforward diagnostic test available. Blood cultures are in routine use, yet typically require 24-48 hours for results and are negative in a substantial percentage of cases. Identification of the causative organism(s) is critical to effective diagnosis and treatment. Matching the antimicrobial to the infectious agent significantly enhances the probability of successful treatment and outcome. In particular, multiplexed detection methods which would enable simultaneous probing for a range of organisms may potentially substitute for culture. ? ? The aim of this project is development of a Reverse Line Blot assay for multiple bacterial and fungal agents of sepsis. In the Reverse Line Blot (RLE) approach, separate probes are attached to a membrane in discrete bands. Extracted bacterial DNA is then incubated with the membrane permitting complementary sequences to hybridize with the corresponding probes. The advantages of this method are that (1) typing of organisms can be accomplished via a matrix of probes, with hybridization occurring independently to each probe; (2) the source sample can be amplified by PCR prior to hybridization; (3) the matrix is macroscopic, easily analyzed without expensive instrumentation, and is suitable for analysis of tens to hundreds of probe hybridizations. Applicants intend to further improve this methodology by converting it to a flow-through membrane format, in order to significantly reduce hybridization times. In the proposed approach, bacterial DNA will be extracted directly from a blood sample to eliminate the delay associated with blood culture, and subject to PCR amplification using universal degenerate primers for a region of the chaperonin 60 gene (Cpn60 or HSP60) widely conserved among prokaryotic species. Cpn60 has been shown to contain species-specific sequences useful as probes for identification of bacterial species. The final product will be a diagnostic test kit for identification of bacterial and fungal agents of sepsis, comprising the flow-through hybridization unit, a Reverse Line Blot membrane containing Cpn60-derived probes to multiple pathogens, and primers necessary for PCR amplification of the Cpn60 sequence in pathogens present in blood samples. ? ? ?
