Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA

David Palfrey, P.J. Cook, J.A. Smythe, G.Y. Lip, Anna V. Hine

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)289-294
Number of pages6
JournalMolecular Pathology
Volume52
Issue number5
DOIs
Publication statusPublished - Oct 1999

Keywords

  • polymerase chain reaction
  • sample preparation
  • chlamydia pneumoniae

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