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Virology

Comparison of Six Commercial DNA Extraction Kits for Recovery of Cytomegalovirus DNA from Spiked Human Specimens

Gary A. Fahle, Steven H. Fischer
Gary A. Fahle
Microbiology Service, Clinical Pathology Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1508
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Steven H. Fischer
Microbiology Service, Clinical Pathology Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1508
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DOI: 10.1128/JCM.38.10.3860-3863.2000
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Tables

  • Table 1.

    Comparison of DNA extraction kit sensitivity from spiked specimens as determined by mean time-resolved fluorescence valuesa

    Specimen (concn [PFU/ml])NSPGQIAMPIQGCC
    Mean valueNo. posbMean valueNo. posbMean valueNo. posbMean valueNo. posbMean valueNo. posbMean valueNo. posb
    BAL (4)293,7686/6242,6406/6123,2266/6375,4055/697,3254/614,3002/6
    BLD (4)292,3756/679,8085/6587,6796/642,1764/663,4024/6220,7226/6
    Plasma (4)359,9986/6296,3046/6123,1736/6303,1366/647,8822/68,8941/6
    CSF
     4662,4336/6527,4605/665,4693/697,5732/631,7872/61,4860/6
     272,0425/664,0694/6
     0.835,5425/625,8473/6
     0.428,9433/68,1931/6
    • ↵a Six replicates from each sample. pos, positive.

    • ↵b Number of replicates out of six with a DELFIA fluorescence value of >15,000.

  • Table 2.

    Comparison of the NucliSens isolation kit sensitivity to other extraction methods from spiked specimens as determined by mean time-resolved fluorescence valuesa

    Specimen (concn [PFU/ml])Mean time-resolved fluorescence values (P value) for paired tests:
    NS/PGNS/QIANS/MPNS/IQNS/GCC
    BAL (4)293,768/242,640 (0.3807b)293,768/123,226 (0.0255)293,768/375,405 (0.3637b)293,768/97,325 (0.0654b)293,768/14,300 (0.0009)
    BLD (4)292,375/79,808 (0.0265)292,375/587,679 (0.5139b)292,375/42,176 (0.0235)292,375/63,402 (0.0087)292,375/220,722 (0.4887b)
    Plasma (4)359,998/296,304 (0.6317b)359,998/123,173 (0.0105)359,998/303,136 (0.3558b)359,998/47,882 (0.0022)359,998/8,894 (0.0027)
    CSF
     4662,433/527,460 (0.4165b)662,433/65,469 (0.0121)662,433/97,573 (0.0416)662,433/31,787 (0.0085)662,433/1,486 (0.0076)
     272,042/64,069 (0.7741b)
     0.835,542/25,847 (0.4816b)
     0.428,943/8,193 (0.1353b)
    • ↵a Six replicates were performed from each sample. Results are presented as pairwise comparisons and Pvalues (by paired t test; calculated with StatView).

    • ↵b Not significant (P > 0.05).

  • Table 3.

    Comparison of cost, processing time, and other miscellaneous aspects between the evaluated DNA extraction kits

    Extraction methodCost/testaProcessing timeb (h:min)Maximum sample vol (μl)Recovered extraction vol (μl)Required reagents not included in kitSpecial equipmente
    Hands-oncTotald
    NSf$4.0002:2803:08200g5070% ethanolVacuum aspirator
    Acetone
    PG0.2301:4904:3910020Proteinase K
    100% isopropanol
    70% ethanol
    Glycogen
    QIA1.1001:3501:55200200100% ethanol
    MP0.6901:0401:59150 (BAL, CSF)35100% isopropanolRefrigerated centrifuge
    50 (PLM)75% ethanol
    12.5 (BLD)
    IQ0.8401:5302:38100100100% isopropanol
    70% ethanol
    GCC1.0800:4500:55200200Fitted heat blockh
    • ↵a Based on pricing quote (early 1999) from each manufacturer for large-volume orders.

    • ↵b Determined for an 18-sample run. Timing began with aliquoting of first reagent or sample and concluded with recovery of extracted DNA.

    • ↵c Time required to perform manual manipulation of tubes, reagents, and samples.

    • ↵d Time required to obtain extracted DNA, including hands-on time and incubation periods.

    • ↵e Excluding standard laboratory equipment.

    • ↵f An automated extractor is available for laboratories with high sample volumes.

    • ↵g Kit available for larger-sample-volume processing.

    • ↵h Bore size must be large enough to completely contain capture column. Manufacturer recommends specific models.

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Comparison of Six Commercial DNA Extraction Kits for Recovery of Cytomegalovirus DNA from Spiked Human Specimens
Gary A. Fahle, Steven H. Fischer
Journal of Clinical Microbiology Oct 2000, 38 (10) 3860-3863; DOI: 10.1128/JCM.38.10.3860-3863.2000

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Comparison of Six Commercial DNA Extraction Kits for Recovery of Cytomegalovirus DNA from Spiked Human Specimens
Gary A. Fahle, Steven H. Fischer
Journal of Clinical Microbiology Oct 2000, 38 (10) 3860-3863; DOI: 10.1128/JCM.38.10.3860-3863.2000
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KEYWORDS

cytomegalovirus
DNA, Viral
Reagent Kits, Diagnostic

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