title: Development of an ethidium monoazide-enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates.
abstract: BACKGROUND
  Bacterial contamination of platelet (PLT) concentrates remains a problem for blood transfusion services. Culture-based bacterial screening techniques are available but offer inadequate speed and sensitivity. Alternative techniques based on polymerase chain reaction (PCR) amplification have been described but their performance is often compromised by traces of bacterial DNA in reagents.
  
  
  METHODS
  Universal 16S rDNA primers were used to develop a real-time PCR assay (TaqMan, Applied Biosystems) and various reagent decontamination strategies were explored. Detection sensitivity was assessed by spiking PLT concentrates with known concentrations of 13 different organisms.
  
  
  RESULTS
  Restriction enzyme digestion, master mix ultrafiltration, and use of alternative Taq polymerases all reduced the level of reagent DNA contamination to some extent but all proved unreliable. In contrast, ethidium monoazide (EMA) treatment of the PCR master mix followed by photoactivation was reliable and effective, permitting a full 40 amplification cycles, and totally eliminated contamination without compromising assay sensitivity. All 13 organisms were efficiently detected and the limit of detection for Escherichia coli-spiked PLTs was approximately 1 colony-forming unit/mL. Coamplification of human mitochondrial DNA served to confirm efficient nucleic acid extraction and the absence of PCR inhibition in each sample. One of five automated extraction platforms evaluated was found to be contamination free and capable of high-throughput processing.
  
  
  CONCLUSIONS
  Cross-linking of EMA to DNA via photoactivation solved the previously intractable problem of reagent contamination and permitted the development of a high-sensitivity universal bacterial detection system. Trials are ongoing to assess the suitability of the system for high-throughput screening of PLT concentrates.
authors.affiliation: National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale, London, UK.
authors.forename: Poorvi
authors.initials: P
authors.lastname: Patel
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Garson
authors.forename: Kate I
authors.initials: KI
authors.lastname: Tettmar
authors.forename: Siobhan
authors.initials: S
authors.lastname: Ancliff
authors.forename: Carl
authors.initials: C
authors.lastname: McDonald
authors.forename: Tyrone
authors.initials: T
authors.lastname: Pitt
authors.forename: Juliana
authors.initials: J
authors.lastname: Coelho
authors.forename: Richard S
authors.initials: RS
authors.lastname: Tedder
mesh_terms: D001386:Azides / Q000737:chemistry*; D001419:Bacteria / Q000235:genetics*; D001792:Blood Platelets / Q000382:microbiology*; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics*; D004275:DNA, Ribosomal / Q000097:blood / Q000235:genetics*; D006801:Humans; D017713:Platelet Transfusion; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000097:blood / Q000235:genetics*
chemical_list: D001386:Azides; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; C014535:8-azidoethidium