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Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA.
  1. D Palfrey,
  2. P J Cook,
  3. J A Smythe,
  4. G Y Lip,
  5. A V Hine
  1. Pharmaceutical Sciences Research Institute, Aston University, Birmingham, UK.

    Abstract

    AIMS: To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most simple techniques for routine use in the laboratory. METHODS: Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Gene Clean, lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. RESULTS: Treatment with either Gene-Clean or lectin and the Fe3+ specific chelator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplification of C pneumoniae DNA from within a section of atherosclerotic carotid artery, implying that C pneumoniae elementary bodies had been disrupted. In eight further clinical samples from patients not known to have C pneumoniae infection, human DNA was amplified and no cross contamination was observed between samples. These samples contained no evidence of C pneumoniae by PCR. CONCLUSIONS: A simple preparation of solid tissue for PCR analysis, involving homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA.

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