Online Resource: Paramagnetic Bead Extraction and Detection of C. albicans DNA from Blood
Specimens
Method
As previously reported, paramagnetic beads with attached biotinylated capture probes were used
to concentrate and purify the DNA [1,2]. To tubes of DNA extracted from blood samples of 500
uL or less, we added a 4.5 µL biotinylated capture probe, mixed briefly by vortexing and
incubated at 95°C for 15 min to obtain single-stranded DNA. Then, incubation at 56°C for 20
minutes allowed the capture probe to anneal to fungal DNA. Each sample was then pulse
minifuged and 5 uL streptavidin-coated Dynal® beads (Cat. No. 653.06) were added. Tubes were
gently mixed and incubated at room temperature for 30 min to allow the biotin moiety of the
capture probe to bind the strepavidin-coated paramagnetic particles. During this annealing
period, tubes were inverted four times at 5 min intervals, pulse minifuged and placed in a
magnetic rack for 10 minutes. Specimens were washed twice by adding 500 µL TE buffer
(Qiagen), placing tubes in the magnetic rack for 10 minutes and removing the separated fluid by
pipette. The residual particles with bound fungal genomic DNA were resuspended in 10 µL TE
buffer.
Results
Conventional real-time PCR was used to evaluate the detection of C. albicans DNA in 500-µL
samples of normal human blood seeded with yeast cells of C. albicans. DNA extraction and
purification with paramagnetic capture particles routinely detected concentrations of 101 to 105
CFU/mL, indicating a limit of detection of 5 CFU per 500-μL sample of blood. Even fewer
yeasts were occasionally detected, but reproducibility was inconsistent (data not shown). To
assess the precision of the assay near the limit of detection, additional tests were conducted using
five 500-µL aliquots of individual samples containing C. albicans at 10 CFU/mL. DNA from
each aliquot was eluted in 10 µL TE buffer and tested in triplicate. As illustrated in the Table
below (Sample 1), only three of the five aliquots were positive (i.e., six of 15 PCRs), which
suggests that blood sample volumes of 500 µL are insufficient to achieve acceptable precision at
this limit of detection. This observation was confirmed by repetitive tests of three clinical
samples that were previously positive for C. albicans DNA (Table, Samples 2, 3 and 4). Because
the volume of these samples was limited, we tested three, five and six 200-μL aliquots of each,
and C. albicans DNA was respectively detected in 1 of 3, 2 of 5, and 1 of 6 aliquots. These
results illustrate the stochastic nature of detecting DNA in small volumes of blood with low
concentrations of C. albicans and suggest that PCR-based assays for candidemia should utilize
blood specimen volumes of at least 1 mL. As described in Methods, increasing the blood volume
necessitated using membrane spin columns to extract the DNA.
References
1. Hua Z, Rouse JL, Eckhardt AE, Srinivasan V, Pamula VK, Schell WA, Benton JL,
Mitchell TG, Pollack MG (2010) Multiplexed real-time polymerase chain reaction on a
digital microfluidic platform. Anal Chem 82:2310-2316
2. Wulff-Burchfield E, Schell WA, Eckhardt AE, Pollack MG, Hua Z, Rouse JL, Pamula VK,
Srinivasan V, Benton JL, Alexander BD, Wilfret DA, Kraft M, Cairns CB, Perfect JR,
Mitchell TG (2010) Microfluidic platform versus conventional real-time polymerase chain
reaction for the detection of Mycoplasma pneumoniae in respiratory specimens. Diagn
Microbiol Infect Dis 67:22-29
Table. Sensitivity of conventional real-time PCR detection of C. albicans DNA in replicate
assays using very low volumes of blood.
No. x
No. PCR-positive
Sample
Total sample
volume of each
no. a
volume (µL)
extracted
No. PCR-positive
replicates (n = total no.
extracted aliquots
replicates)
b
aliquot
1
2,500
5 x 500 µL
6 (n=15)
3
2
600
3 x 200 µL
1 (n= 9)
1
3
1,000
5 x 200 µL
3 (n=15)
2
4
1,200
6 x 200 µL
1 (n=18)
1
a
Sample 1 was normal human blood seeded with 10 CFU/mL C. albicans (i.e., 5 CFU/500-µL
aliquot). Samples 2, 3 and 4 were clinical blood specimens from three patients with cultureproven candidemia. All samples were aliquoted as indicated, and DNA was extracted from each
aliquot using paramagnetic particles.
b
Each DNA extract was tested in triplicate.