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

Aims-To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most sim ple techniques for routine use in the laboratory. Methods-Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Ge ne Clean(TM), lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. Results-Treatment with either GeneClean or lectin and the Fe3+ specific che lator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplificati on of C pneumoniae DNA from within a section of atherosclerotic carotid art ery, implying that C pneumoniae elementary bodies had been disrupted. In ei ght further clinical samples from patients not known to have C pneumoniae i nfection, human DNA was amplified and no cross contamination was observed b etween samples. These samples contained no evidence of C pneumoniae by PCR. Conclusions-A simple preparation of solid tissue for PCR analysis, involvin g homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA.