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Literature reviews > Articles for review > Issa et al. Comparison of specimen processing... |
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DNA extraction using the Swab Extraction Tube System was faster, less expensive, and more sensitive than the MagNA Pure LC System for the laboratory diagnosis of HSV using LightCycler PCR. Comparison of specimen processing and nucleic acid extraction by the Swab Extraction Tube System versus the MagNA Pure LC System for laboratory diagnosis of herpes simplex virus infections by LightCycler PCR. Question How does a manual method compare to an automated method for the processing of specimens being tested for HSV DNA by real-time PCR? Design This article describes a head-to-head comparison of two different DNA extraction methods, a manual system and an automated system, for extraction of HSV DNA from dermal and genital swabs. The amount of HSV DNA extracted by each method was determined by real-time PCR amplification. Participants Residual fluid from the collection and transport tubes of 234 dermal and 329 genital swab specimens from adult and pediatric patients suspected of having HSV infections was tested. Description of Tests and Diagnostic Standard Dermal and genital swab collection and transport tubes containing a gel or sponge with remaining fluid were used for the comparison. Two swabs (CultureSwab, Becton Dickinson Microbiology Systems, Cockeysville, MD) were inserted into each collection tube. One swab was then swirled in 2 ml of serum-free medium and processed using the MagNA Pure LC automated extraction instrument (Roche Molecular Biochemistries) and the Total Nucleic Acid isolation extraction reagent kit.The other swab was inserted into the inner tube of the Swab Extraction Tube System (SETS, Roche Applied Science) and the shaft was broken off above the swab. The inner tube was placed inside the outer tube, which contained 200 μl of swab neutralization buffer, and centrifuged for 1 min to extract the fluid from the swab. The inner tube containing the swab was discarded and the outer tube containing the fluid was heated at 100oC for 2 min. Five μl of each DNA sample from each clinical specimen were added to separate real-time HSV PCR amplification reactions performed in a LightCycler instrument using LightCycler herpes simplex virus 1/2 primer/hyb probes (Roche Diagnostics). Amplicons were identified after PCR by melting curve analysis. Main Outcome Measures The positivity rates of specimens processed by the MagNA Pure LC system and SETS for each HSV genotype and specimen source were compared. Main Results The number of HSV real-time PCR positive specimens, by specimen source, for the 563 DNA samples prepared by MagNA Pure and by SETS are shown in the table. There were no specimens positive only by the MagNA Pure method. Among the 157 specimens positive by both methods, the specimens processed by SETS had a mean Ct of 25.9 cycles in the PCR (median = 26.0, range = 19 to 39) compared to a mean Ct of 32.0 cycles for specimens processed by the MagNA Pure method (median = 31.0, range = 23 to 44) (P < 0.0001). There were no HSV genotype discrepancies between specimens prepared by the two methods. To process 30 samples, approximately 30 min of hands on time and 1.5 h to complete the extraction were required by the MagNA Pure LC system at a cost of $5.69 per sample, excluding the cost of the instrument. In comparison, for the SETS, between 25 and 35 min were required to complete the extraction of 30 samples with a cost of $0.50 per sample. Number of real-time HSV PCR positive specimens by specimen source for 234 dermal and 329 genital swabs processed by MagNA Pure LC and by SETS
* P = 0.0001 Authors' Conclusions The SETS extraction method was more sensitive, faster, and less expensive than the MagNA Pure LC system for the detection of HSV DNA by LightCycler PCR amplification. Source of funding: None given For correspondence: T. F. Smith, Division of Clinical Microbiology, Mayo Clinic, 200 First St., SW, Rochester, MN 55905.E-mail address: tfsmith@mayo.edu |
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