TABLE 1.

Extraction variables assessed for their influence on fungal DNA extraction efficiencya

Extraction protocolgDescriptionTheoretical maximum eluate DNA concn (rRNA copies/μl)b
10 conidia/ml blood33 conidia/ml blood
EAPCRI methodDNA extraction as described by the EAPCRIc26.588.3
Modified EAPCRI method
    Plus NaOHDNA extraction as described by the EAPCRIc but with the additional step of 95°C incubation in 50 mM NaOHd26.588.3
    Using 1 ml of 3-ml sampleDNA extraction as described by the EAPCRIc but using 1 ml of a 3-ml sample8.8 (8.2-9.5e)29.4
    Using 2 ml of 3-ml sampleDNA extraction as described by the EAPCRIc but using 2 ml of a 3-ml sample17.7 (17.0-18.2f)58.8
    Minus one RCLDNA extraction as described by the EAPCRIc but using only one rather than two red cell lysis steps26.588.3
    No RCLDNA extraction as described by the EAPCRIc but without red cell lysis26.588.3
    No WCLDNA extraction as described by the EAPCRIc but without white cell lysis26.588.3
    No bead beatingDNA extraction as described by the EAPCRIc but without bead beating26.588.3
    WCL replaced with NaOHDNA extraction as described by the EAPCRIc but with white cell lysis buffer replaced with a 50 mM NaOH incubation26.588.3
    Using 100-μl elution volDNA extraction as described by the EAPCRIc but with DNA eluted in 100 μl15.953.0
    Using 200-μl elution volDNA extraction as described by the EAPCRIc but with DNA eluted in 200 μl8.026.5
  • a DNA eluted in 60 μl unless otherwise stated. The theoretical maximum eluate DNA concentrations are shown for both fungal burdens and show the influence of using smaller sample volumes and eluting in larger volumes.

  • b Assuming 100% extraction efficiency, 1 genome/conidium, and 53 rRNA copies/genome (per reference 2).

  • c See reference 5.

  • d See reference 3.

  • e 95% CI for using one-third of the specimen.

  • f 95% CI for using two-thirds of the specimen.

  • g RCL, red cell lysis; WCL, white cell lysis.