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DNA extraction methods for the diagnosis of Acanthamoeba
  1. Pablo Goldschmidt (pablogol{at}aol.com),
  2. Sandrine Degorge,
  3. Cathy Saint-Jean,
  4. Hélène Yera,
  5. Fouad Zekhnini,
  6. Laurence Batellier,
  7. Laurent Laroche,
  8. Christine Chaumeil
  1. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  2. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  3. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  4. Laboratoire de Parasitologie et Mycologie Hopital Cochin Université René Descartes, Paris, France
  5. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  6. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France
  7. Service 5- Centre National d'Ophtalmologie des Quinze-Vingts, France
  8. Laboratoire du Centre National d'Ophtalmologie des Quinze-Vingts, France

    Abstract

    Aims: Sensitive diagnosis of Acanthamoeba infections may prevent the clinical condition from becoming worse. In order to improve the diagnosis tool performances the implication of the DNA extraction procedures on the detection of Acanthamoeba by real time PCR was studied. Methods: Acanthamoeba cysts mixed with a tag virus were processed according to different DNA preparation procedures: heat, Proteinase K (ProtK), alkali lysis, QIAmp kit®, MagNA Pure (DNA Mini kit, MagNA Pure® Nucleic Acid isolation kit), ProtK + QIAmp and ProtK + MagNA Pure. Parasite-DNA loads were assessed by real-time PCR.

    Results: The results show that the structures of Acanthamoeba cysts are resistant to reagents releasing the DNA from other cells and viruses. Heat, NaOH or ProtK did not allow to asses the DNA extraction yields or to eliminate the inhibitors. The QIAmp and the MagNA Pure partially improved the sensitivity of the PCR and eliminated the inhibitors. Significant increase in positive results was obtained with a ProtK treatment before commercial extraction kits. ProtK + MagNA Pure yielded the highest rates of positivity.

    Conclusion: To minimize false negative results the nucleic-acid based Acanthamoeba diagnosis requires, first: the efficient lysis of cysts (without affecting the DNA) to make the DNA available for extraction and amplification, and second: the elimination of PCR inhibitors. Significant increase in the detection rates are obtained by adding a ProtK treatment (10min at 56°C) before the commercial procedures. ProtK + MagNA Pure yielded in 30 min the best results followed by ProtK + QIAmp (150 min).

    • Cornea
    • Public health
    • Acanthamoeba
    • DNA-extraction
    • PCR
    • keratitis

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