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Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

Wylie TN, Wylie KM, Buller RS, Cannella M, Storch GA - J. Clin. Microbiol. (2015)

Bottom Line: This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness.As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO.The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.

No MeSH data available.


Related in: MedlinePlus

Amplification plot showing WashU RT-PCR assay EV-D68 sensitivity. The PCR amplification cycle number is displayed on the x axis while log(ΔRn) is shown on the y axis. Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. ΔRn is Rn minus the baseline and is plotted against PCR cycle number. The light green and light purple lines show detection of the 2014 EV-D68 outbreak strain using the WashU design 1 and design 2 assays, respectively. The brown and dark purple lines show detection of the more distant 1962 Fermon EV-D68 type strain using the WashU design 2 and design 1 assays, respectively. The incorporation of degenerate bases and mixed primers in WashU design 2 shows a significant increase in sensitivity (6.7 cycles earlier detection) for the Fermon type strain (brown line), with minimal decrease in sensitivity to the 2014 outbreak strain (light purple) (<0.5 cycles difference).
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Figure 2: Amplification plot showing WashU RT-PCR assay EV-D68 sensitivity. The PCR amplification cycle number is displayed on the x axis while log(ΔRn) is shown on the y axis. Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. ΔRn is Rn minus the baseline and is plotted against PCR cycle number. The light green and light purple lines show detection of the 2014 EV-D68 outbreak strain using the WashU design 1 and design 2 assays, respectively. The brown and dark purple lines show detection of the more distant 1962 Fermon EV-D68 type strain using the WashU design 2 and design 1 assays, respectively. The incorporation of degenerate bases and mixed primers in WashU design 2 shows a significant increase in sensitivity (6.7 cycles earlier detection) for the Fermon type strain (brown line), with minimal decrease in sensitivity to the 2014 outbreak strain (light purple) (<0.5 cycles difference).

Mentions: We tested our two assays and the two versions of the CDC assay on a set of clinical samples from the 2014 outbreak (Table 2). We also included the Fermon strain of EV-D68 obtained from the Children's Hospital Colorado. The two WashU assays performed similarly on the samples, with <1 cycle difference between the two assays for 12 of the 14 samples. The published CDC assay (FAM reporter) performed less well, failing to detect 6 of the 14 samples. However, the modified CDC assay (i.e., with the substitution of FAM with Cy5) enabled the detection of all 14 samples. However, the CT values were higher for the modified CDC assay than those for the WashU assays. The WashU assays but not the CDC assays detected the Fermon strain. Strikingly, the WashU design 2 assay detected Fermon 6.7 RT-PCR cycles earlier than WashU design 1 assay, and the amplification curve indicated improved amplitude and amplification efficiency (Fig. 2).


Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay.

Wylie TN, Wylie KM, Buller RS, Cannella M, Storch GA - J. Clin. Microbiol. (2015)

Amplification plot showing WashU RT-PCR assay EV-D68 sensitivity. The PCR amplification cycle number is displayed on the x axis while log(ΔRn) is shown on the y axis. Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. ΔRn is Rn minus the baseline and is plotted against PCR cycle number. The light green and light purple lines show detection of the 2014 EV-D68 outbreak strain using the WashU design 1 and design 2 assays, respectively. The brown and dark purple lines show detection of the more distant 1962 Fermon EV-D68 type strain using the WashU design 2 and design 1 assays, respectively. The incorporation of degenerate bases and mixed primers in WashU design 2 shows a significant increase in sensitivity (6.7 cycles earlier detection) for the Fermon type strain (brown line), with minimal decrease in sensitivity to the 2014 outbreak strain (light purple) (<0.5 cycles difference).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508392&req=5

Figure 2: Amplification plot showing WashU RT-PCR assay EV-D68 sensitivity. The PCR amplification cycle number is displayed on the x axis while log(ΔRn) is shown on the y axis. Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. ΔRn is Rn minus the baseline and is plotted against PCR cycle number. The light green and light purple lines show detection of the 2014 EV-D68 outbreak strain using the WashU design 1 and design 2 assays, respectively. The brown and dark purple lines show detection of the more distant 1962 Fermon EV-D68 type strain using the WashU design 2 and design 1 assays, respectively. The incorporation of degenerate bases and mixed primers in WashU design 2 shows a significant increase in sensitivity (6.7 cycles earlier detection) for the Fermon type strain (brown line), with minimal decrease in sensitivity to the 2014 outbreak strain (light purple) (<0.5 cycles difference).
Mentions: We tested our two assays and the two versions of the CDC assay on a set of clinical samples from the 2014 outbreak (Table 2). We also included the Fermon strain of EV-D68 obtained from the Children's Hospital Colorado. The two WashU assays performed similarly on the samples, with <1 cycle difference between the two assays for 12 of the 14 samples. The published CDC assay (FAM reporter) performed less well, failing to detect 6 of the 14 samples. However, the modified CDC assay (i.e., with the substitution of FAM with Cy5) enabled the detection of all 14 samples. However, the CT values were higher for the modified CDC assay than those for the WashU assays. The WashU assays but not the CDC assays detected the Fermon strain. Strikingly, the WashU design 2 assay detected Fermon 6.7 RT-PCR cycles earlier than WashU design 1 assay, and the amplification curve indicated improved amplitude and amplification efficiency (Fig. 2).

Bottom Line: This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness.As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO.The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.

No MeSH data available.


Related in: MedlinePlus