Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA.
OBJECTIVE
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.
Palfrey
D
D
Pharmaceutical Sciences Research Institute, Aston University, Birmingham, UK.
Cook
P J
PJ
Smythe
J A
JA
Lip
G Y
GY
Hine
A V
AV
D001158:Arteries / Q000382:microbiology*; D001161:Arteriosclerosis / Q000382:microbiology*; D016993:Chlamydophila pneumoniae / Q000302:isolation & purification*; D004247:DNA / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis*; D005069:Evaluation Studies as Topic; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling / Q000379:methods
D004269:DNA, Bacterial; D004247:DNA