Received on July 25, 2007
Accepted on October 30, 2007

Molecular Diagnostics and Genetics

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

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 Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, and Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah

^* To whom correspondence should be addressed. 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.