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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

Authors

  • Poorvi Patel,

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • Jeremy A. Garson,

    Corresponding author
    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
      Jeremy A. Garson, Clinical Microbiology and Virology, UCLH NHS Foundation Trust, 60 Whitfield Street, London, W1T 4EU, UK; e-mail j.garson@ucl.ac.uk.
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  • Kate I. Tettmar,

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • Siobhan Ancliff,

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • Carl McDonald,

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • Tyrone Pitt,

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • Juliana Coelho,

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • Richard S. Tedder

    1. From the National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale; the Research Department of Infection, Division of Infection and Immunity, University College London; and the Blood Borne Virus Unit, Viral Reference Department, Centre for Infections, Health Protection Agency, Colindale, London, UK.
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  • PP and JAG contributed equally to this paper.

  • Supported by the NHS Blood and Transplant research fund.

Jeremy A. Garson, Clinical Microbiology and Virology, UCLH NHS Foundation Trust, 60 Whitfield Street, London, W1T 4EU, UK; e-mail j.garson@ucl.ac.uk.

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.

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.

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