Fahle GA, Fischer SH; American Society for Microbiology. General Meeting.
Abstr Gen Meet Am Soc Microbiol. 1999 May 30-Jun 3; 99: 173 (abstract no. C-334).
NIH, Bethesda, MD.
Six commercially available DNA extraction kits were tested for their ability to recover three different dilutions of cytomegalovirus (CMV) DNA for PCR amplification. The kits included in the comparison were the Puregene(R) DNA Isolation Kit (PG) (Gentra Systems, Inc.), Generation(TM) Capture Column(R) Kit (GCC) (Gentra Systems, Inc.), MasterPure(R) DNA Purification Kit (MP) (Epicentre Technologies), IsoQuick(R) Nucleic Acid Extraction Kit (IQ) (MicroProbe Corp.), QIAamp(R) Blood Kit (QIA) (QIAGEN(R), Inc.), and NucliSens(R) Isolation Kit (NS) (Organon Teknika Corp.). Pooled cerebral spinal fluid (CSF), pooled bronchoalveolar lavage (BAL), serum (SRM), and whole blood (BLD) were spiked with CMV AD169 at concentrations of 10 pfu/microliter, 0.2 pfu/microliter, and 0.004 pfu/microliter. Six aliquots from each dilution of the four specimen types were processed according to the various manufacturer's recommendations and the purified extracts were used for PCR amplification with primers directed against the CMV glycoprotein B gene. In addition, an internal control sequence was co-amplified in each reaction to detect instances of PCR inhibition. The resulting PCR products (amplicons) were measured utilizing the DELFIA(R) time-resolved fluorometer (Wallac Oy) and the fluorescence values were compared. Processing time and cost for each method were also evaluated. At the 10 pfu/microliter and 0.2 pfu/microliter concentrations, all six methods were able to effectively recover sufficient DNA from all specimen types to produce clearly positive fluorescence values. However, at the lowest concentration of CMV, a difference in viral DNA recovery was observed. In general, the NS method resulted in higher and more consistent fluorescence values for all replicates tested of each sample type with the 0.004 pfu/microliter concentration. For the four specimen types, a paired t-test comparing the resulting fluorescence values from the NS to the other five methods was performed. Statistically significant differences with NS (P <0.05) were obtained in only one of four specimen types when compared to PG, but in two of four when compared to MP, and in three of four when compared to GCC, IQ, and QIA. The time required to process an 18-sample run varied widely between the methods and ranged from 55 min for the GCC up to 4 h 39 min for the PG. Likewise, the cost comparison between the methods ranged from as little as $0.23 per sample for the PG up to $4.00 per sample for the NS.
Publication Types:
Keywords:
- Animals
- Base Sequence
- Cytomegalovirus
- Cytomegalovirus Infections
- DNA Primers
- DNA, Complementary
- DNA, Viral
- Humans
- Polymerase Chain Reaction
- genetics
- methods
Other ID:
UI: 102195616
From Meeting Abstracts