Efforts to improve diagnostic performance of PCR assays cannot rely on improved PCR
detection limits alone because we have already achieved what approaches the maximum
analytical sensitivity with PCR technology, namely single molecule detection. Rather,
improvements in PCR assay performance will rest on a foundation of improvements in sample
manipulation, DNA extraction and target concentration methods prior to the PCR step. This
area of investigation has received little attention despite the great potential to impact diagnostic
performance. For example, a major limitation of fungal PCR is the small volume of DNA added
to each reaction, usually 1–20 μl, and the high proportion of human DNA, leading to limited
sampling of tissues for fungal nucleic acids. If a fungal organism is present at one to two
genomes per ml of blood then it is unlikely to be detected in most PCR assays, but this low
level of fungal DNA may be most informative diagnostically.
Efforts to improve diagnostic performance of PCR assays cannot rely on improved PCRdetection limits alone because we have already achieved what approaches the maximumanalytical sensitivity with PCR technology, namely single molecule detection. Rather,improvements in PCR assay performance will rest on a foundation of improvements in samplemanipulation, DNA extraction and target concentration methods prior to the PCR step. Thisarea of investigation has received little attention despite the great potential to impact diagnosticperformance. For example, a major limitation of fungal PCR is the small volume of DNA addedto each reaction, usually 1–20 μl, and the high proportion of human DNA, leading to limitedsampling of tissues for fungal nucleic acids. If a fungal organism is present at one to twogenomes per ml of blood then it is unlikely to be detected in most PCR assays, but this lowlevel of fungal DNA may be most informative diagnostically.
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