Fig 1.
PCR amplification of a portion of the 5’-UTR from GenMark eSensor RVP samples.
cDNA synthesized from RNA extracted from de-identified and masked RVP samples was used for traditional PCR amplification of the 5’-UTR. Primers were described by Oberste, et. al., for the sequencing of EV-D68 [2]. Agarose-ethidium bromide electrophoresis demonstrated that 49 of 62 samples analyzed yielded a 396 base pair amplicon compatible with EV-D68. Results shown corresponded to samples 2–4, 6–13, and 15–17, and are representative of the range of band intensities observed for all positive samples. NTC: no template control.
Table 1.
RVP Signal, 5'-UTR PCR, and sequencing results.
Table 2.
Percent 5'-UTR Fragment Sequence Identity.
Fig 2.
Detection of enterovirus in sample 13 using the GeneXpert enterovirus assay.
As independent confirmation of the sequencing results for sample 13, we analyzed extracted nucleic acid on the GeneXpert platform. Sample 13, which had a RVP signal for HRV of 21.3 nA, gave a positive signal (blue line) with a CT of 24.2 and an excellent amplification curve consistent with positive results obtained from CSF samples, whereas the internal control (CIC) amplified less efficiently, as expected, because of competition (gray line).
Fig 3.
GenMark eSensor RVP signal strength for HRV in samples stratified by sequence results.
Un-masking of the RVP results for all 62 samples revealed that the 49 positive PCR amplicons identified were derived from those samples that were also positive for HRV on the GenMark panel. Sequencing of these amplicons revealed that 33 were EV-D68 and 16 were HRV. The mean signal strength in nA of the RVP results for those samples identified as EV-D68 was significantly lower than for the HRV samples (20.3 nA versus 129.7 nA, respectively, p < 0.00001).
Table 3.
Differentiating EV-D68 from HRV by RVP signal of 40Na.
Table 4.
Differentiating EV-D68 from HRV by RVP signal of 50nA.