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Clinical Chemistry 54: 406-413, 2008. First published November 26, 2007; 10.1373/clinchem.2007.095414
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Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2008;54:406-413.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

Weston C. Hymas1,a, Wade K. Aldous2, Edward W. Taggart1, Jeffery B. Stevenson1 and David R. Hillyard1,3

1 Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, Salt Lake City, Utah;
2 Brooke Army Medical Center, San Antonio, Texas;
3 Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah.

aAddress correspondence to this author at: ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108. Fax (801) 584-5109; e-mail hymasw{at}aruplab.com.

Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry.

Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection.

Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid.

Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.






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