title: A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood.
abstract: BACKGROUND
  The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR .
  
  
  RESULTS
  Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 10(1) CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi.
  
  
  CONCLUSIONS
  Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours.
authors.affiliation: Department of Bacteriology, Microbial Ecology and Parasitology, Chair of Microbiology Jagiellonian University Medical College, Czysta 18 Str, 31-121 Kraków, Poland. tomasz.gosiewski@uj.edu.pl.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Gosiewski
authors.forename: Danuta
authors.initials: D
authors.lastname: Jurkiewicz-Badacz
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Sroka
authors.forename: Monika
authors.initials: M
authors.lastname: Brzychczy-Włoch
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Bulanda
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001769:Blood / Q000382:microbiology*; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018805:Sepsis / Q000175:diagnosis* / Q000382:microbiology