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Updated: Jul 9, 2026
EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR
Published on: January 16, 2016
Description and validation of a novel real-time RT-PCR enterovirus assay
Weston C Hymas1, Wade K Aldous, Edward W Taggart
1Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA. hymasw@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.
Insights
A new real-time reverse transcription-polymerase chain reaction (RT-PCR) assay effectively detects enteroviruses, a common cause of aseptic meningitis. This sensitive assay is suitable for clinical use, though single nucleotide polymorphisms may impact results.
Area of Science:
- Virology
- Molecular Biology
- Clinical Diagnostics
Background:
- Enteroviruses are a primary cause of aseptic meningitis in children and adults.
- Current diagnostic methods may have limitations in detection sensitivity.
- Developing a more sensitive and reliable diagnostic tool is crucial for patient care.
Purpose of the Study:
- To develop and evaluate a novel real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for enterovirus detection.
- To compare the performance of the new assay against a commercial RT-PCR kit.
- To assess the assay's suitability for routine clinical use.
Main Methods:
- A real-time RT-PCR assay was designed to target a specific region of the enterovirus genome.
- The assay incorporated an RNA internal control and modified nucleotide chemistry.
- Performance was evaluated using blinded parallel testing on 778 clinical samples, compared to a commercial assay.
Main Results:
- The novel real-time assay demonstrated high sensitivity, with a limit of detection of approximately 430 copies/mL in cerebrospinal fluid.
- Out of 14 discrepant samples, the real-time assay correctly identified 6 missed by the commercial kit, confirmed by sequencing.
- Sequence analysis revealed single nucleotide polymorphisms (SNPs) in the target region, potentially affecting assay performance.
Conclusions:
- The developed real-time enterovirus assay is sensitive and appropriate for routine clinical diagnostics.
- The presence of SNPs can influence the accuracy of real-time PCR assays.
- This assay offers a valuable tool for diagnosing enteroviral infections, aiding in timely patient management.
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