title: Nuclease-assisted suppression of human DNA background in sepsis.
abstract: Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.
authors: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.
authors: Yajing
authors: Y
authors: Song
authors: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden; Karolinska University Hospital, Department of Clinical Microbiology, Stockholm, Sweden.
authors: Christian G
authors: CG
authors: Giske
authors: Division of Infectious Diseases, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors: Patrik
authors: P
authors: Gille-Johnson
authors: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.
authors: Olof
authors: O
authors: Emanuelsson
authors: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.
authors: Joakim
authors: J
authors: Lundeberg
authors: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden; Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden; Karolinska University Hospital, Department of Clinical Microbiology, Stockholm, Sweden.
authors: Peter
authors: P
authors: Gyarmati
mesh_terms: D002138:Calibration; D004247:DNA / Q000097:blood* / Q000737:chemistry; D003851:Deoxyribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D004927:Escherichia coli Infections / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D005798:Genes, Bacterial; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D018746:Systemic Inflammatory Response Syndrome / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004247:DNA; D003851:Deoxyribonucleases