Influence of the DNA extraction protocol on Aspergillus PCR performancea

Extraction protocole10 conidia/mld33 conidia/mld
Aspergillus PCRInternal control PCRAspergillus PCRInternal control PCR
Detection rate (%)Mean CT (range)Difference from EAPCRI valueReferenced to PCR standard (cycles)bDifference from EAPCRI valueDetection rate (%)Mean CT (range)Difference from EAPCRI valueReferenced to PCR standard (cycles)bDifference from EAPCRI value
EAPCRI method10037.2 (35.3-39.8)NA−0.18NA10035.0 (33.3-36.8)NA0.66NA
Modified EAPCRI method
    Plus NaOH10036.3 (35.6-37.1)− (32.3-36.9)−0.60.680.02
    Minus one RCL10037.2 (36.4-38.2) (34.0-35.5)−
    No RCL9039.1 (37.7-43.0)1.9*0.76c0.94c10035.8 (33.9-36.9)0.8−0.020.68*
    WCL replaced with NaOH60*40.1 (36.3-44.5)2.9**0.740.92**10038.9 (36.9-41.8)3.9***2.742.08***
    Using 100-μl elution vol50*40.0 (38.1-45.1)2.8*−0.120.06
    No WCL40*40.6 (39.9-41.3)3.4**1.92.08**10034.8 (33.7-35.6)−***
    Using 200-μl elution vol40*41.5 (39.0-43.4)4.3***1.141.32**9038.0 (36.1-39.6)3.0***2.942.28**
    Using 2 ml of sample30**38.8 (37.5-41.3)1.6−0.560.38
    Using 1 ml of sample0***NANA−0.38−0.2010037.4 (35.9-38.7)2.4***1.080.42
    No bead beating30**38.8 (37.1-40.2)3.8**1.080.42
  • a Five replicate 3-ml EDTA-blood specimens were spiked with each fungal burden, and Aspergillus PCR was performed in duplicate.

  • b The results shown represent the mean differences between the internal control PCR CT value for the five replicates and that for the positive PCR standard used to calibrate the individual PCR experiments.

  • c The mean difference is influenced by a single outlying value of 3.1 cycles. The other 4 replicates had a difference in CT of 0.1 to 0.2 cycles. Consequently, the difference between the mean IC PCR CT values for this method and the EAPCRI method is not statistically significant.

  • d NA, not applicable; —, not performed; *, 0.01 < P value < 0.05; **, 0.001 < P value < 0.01; ***, P value of <0.001 (measuring the difference between the values with the asterisks and the value for the unmodified EAPCRI method).

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