Sources of False Positive Aspergillus DNA by PCR from Normal Human Blood.

Palmeri,J.; FRANCESCON, A.; KASA, M.;WALSH, T J.; ORLE, K A.

Author address: 

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Abstract: 

We have developed a PCR assay for the detection of Aspergillus genus DNA in clinical specimens. DNA is extracted from whole blood and captured on silica, using either QIAGEN DNA columns or silica-coated magnetic particles. Initial studies on normal human blood processed with the QIAGEN protocol revealed that a high percentage (66/360 or 19%) of the samples was positive for Aspergillus DNA. AIM. To determine the cause of the high percentage of Aspergillus DNA positives in normal blood samples. Methods: (1) Determine background level of false-positivity in PCR reagents; (2) determine background contribution from the QIAGEN columns and reagents; (3) determine background contribution from the DNA capture protocol that uses magnetic beads; and (4) sequence Aspergillus false positives to determine species. Results: (1) Aspergillus PCR master mix generated 24/992 (2.4%) false-positives. (2) QIAGEN columns and reagents generated 17/82 (12.5%) false-positives. (3) Our magnetic bead protocol and reagents generated 6/104 (5%) false-positives. Processing normal human blood using our magnetic bead protocol generated 28/182 (13%) Aspergillus positive results. (4) 27 Aspergillus amplicon generated from normal human blood were sequenced, and 12 different Aspergillus species were identified. Conclusions: A 2.4% Aspergillus contamination was present in our PCR master mix. Despite a high level of quality control, Aspergillus DNA from buffers, columns, and other sources may give rise to false positive results in diagnostic PCR assays
2001

abstract No: 

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Full conference title: 

41st ICAAC
    • ICAAC 41st