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. STUDY DESIGN AND 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. CONCLUSION: 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.
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. STUDY DESIGN AND 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. CONCLUSION: 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: Teri M Greiling; Carina Dehner; Xinguo Chen; Kevin Hughes; Alonso J Iñiguez; Marco Boccitto; Daniel Zegarra Ruiz; Stephen C Renfroe; Silvio M Vieira; William E Ruff; Soyeong Sim; Christina Kriegel; Julia Glanternik; Xindi Chen; Michael Girardi; Patrick Degnan; Karen H Costenbader; Andrew L Goodman; Sandra L Wolin; Martin A Kriegel Journal: Sci Transl Med Date: 2018-03-28 Impact factor: 17.956
Authors: Bruce Humphrey; Neil McLeod; Carrie Turner; J Mark Sutton; Paul M Dark; Geoffrey Warhurst Journal: PLoS One Date: 2015-07-14 Impact factor: 3.240
Authors: Susannah J Salter; Michael J Cox; Elena M Turek; Szymon T Calus; William O Cookson; Miriam F Moffatt; Paul Turner; Julian Parkhill; Nicholas J Loman; Alan W Walker Journal: BMC Biol Date: 2014-11-12 Impact factor: 7.431
Authors: Nicole M Davis; Diana M Proctor; Susan P Holmes; David A Relman; Benjamin J Callahan Journal: Microbiome Date: 2018-12-17 Impact factor: 14.650
Authors: Jeremy A Garson; Poorvi Patel; Carl McDonald; Joanne Ball; Gillian Rosenberg; Kate I Tettmar; Susan R Brailsford; Tyrone Pitt; Richard S Tedder Journal: Transfusion Date: 2013-05-23 Impact factor: 3.157