PCR-based detection of Pythium and Lagenidium DNA in frozen and ethanol-fixed animal tissues

Authors

  • Nadine R. Znajda,

    1. Department of Small Animal Clinical Sciences, PO Box 100126, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610–0126, USA
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      Dr Znajda’s current address is Florida Veterinary Specialists, 3000 Busch Lake Blvd, Tampa, FL 33614, USA.
      Fax: +1 (813) 936 9595;
      E-mail: Znajdan@mail.vetmed.ufl.edu.
      This project was conducted while Dr Znajda was a visiting scientist in the Department of Veterinary Clinical Sciences, Louisiana State University.
  • Amy M. Grooters,

    Corresponding author
    1. Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
      Dr Amy Grooters,
      Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
      Fax: +1 (225) 578 9559;
      E-mail: agrooters@vetmed.lsu.edu
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  • Rosanna Marsella

    1. Department of Small Animal Clinical Sciences, PO Box 100126, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610–0126, USA
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Dr Amy Grooters,
Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Fax: +1 (225) 578 9559;
E-mail: agrooters@vetmed.lsu.edu

Abstract

Abstract  The purpose of this study was to evaluate the application of previously described Pythium insidiosum- and Lagenidium-specific nested PCR assays to the detection of oomycete DNA in animal tissues. DNA was extracted from 15 frozen and 10 ethanol-fixed tissues obtained from six animals with pythiosis, five animals with lagenidiosis, one animal with nonoomycotic skin disease and two animals without skin disease. First-round PCR, which utilized universal fungal primers ITS1 and ITS2P, amplified a single product of the expected size for each of the P. insidiosum- and Lagenidium-infected tissues, but not for tissues obtained from animals without fungal disease. Second-round PCR using the P. insidiosum-specific primers PI1 and PI2 produced a single 105-bp product for the P. insidiosum-infected tissues, but not for any of the other tissues. Second-round PCR using the Lagenidium-specific primers LAG1 and LAG2 produced a single 76-bp product for the Lagenidium-infected tissues, but not for any of the other tissues.

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