No False Positives with New Enterovirus D68 PCR-Based Assay

A newly developed PCR-based assay specifically detects current variants of Enterovirus D68 with no false positive reactions with other enteroviruses or with rhinoviruses.

Investigators at Washington University School of Medicine (St. Louis, MO, USA) used an Applied Biosystems (Foster City, CA, USA) 7500 real-time PCR system to develop and evaluate a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human Enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness.


The investigators mapped the genome sequence of the EV-D68 virus circulating in St. Louis (MO, USA). This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplified a segment of the VP1 gene, with an analytic limit of detection of four copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the [US] Food and Drug Administration. The new assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68,

"Commercial tests for respiratory viral infections typically do not distinguish between rhinoviruses, which cause the common cold, and enteroviruses, and within each of those groups there are many different types," said senior author Dr. Gregory A. Storch, professor of pediatrics at Washington University School of Medicine. "Having a tool to identify which cases of respiratory illness are actually EV-D68 is an advantage for public health. These kinds of tests help treatment decisions because it is important to know that the patient does not have influenza or another disease that might require a specific treatment. It is also important in a hospital for preventing infections because doctors take patients with one particular virus and keep them apart from patients infected with other infectious agents."

"There are a range of D68 viruses, and our assay was designed to detect them all," said Dr. Storch. "We received many samples of enterovirus from other hospitals and ran the test blindly on all of them. In the viruses we looked at, the test worked 100% of the time. It only detected EV-D68 strains, and it did detect all of them; the test did not detect any of the other enteroviruses or rhinoviruses."

Details of the EV-D68 assay were published in the June 10, 2015, online edition of the Journal of Clinical Microbiology.

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Washington University School of Medicine
Applied Biosystems