ZIKV RNA Isolation and Quantification

Plasma and PBMC were isolated from EDTA-treated whole blood on Ficoll paque at 1860 x rcf for 30 minutes as described in Dudley et. al. [24 (link)]. Serum was isolated from clot-activator tubes without additive. Viral RNA (vRNA) was extracted as previously described with a Maxwell 16 MDx instrument (Promega, Madison, WI) and evaluated using qRT-PCR [24 (link), 34 (link), 50 (link)]. RNA concentration was determined by interpolation onto an internal standard curve of seven ten-fold serial dilutions of a synthetic ZIKV RNA segment based on ZIKV-FP. The limit of detection of this assay is 100 copies vRNA/ml plasma or serum.

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Breitbach M.E., Newman C.M., Dudley D.M., Stewart L.M., Aliota M.T., Koenig M.R., Shepherd P.M., Yamamoto K., Crooks C.M., Young G., Semler M.R., Weiler A.M., Barry G.L., Heimsath H., Mohr E.L., Eichkoff J., Newton W., Peterson E., Schultz-Darken N., Permar S.R., Dean H., Capuano S 3.r.d., Osorio J.E., Friedrich T.C, & O’Connor D.H. (2019). Primary infection with dengue or Zika virus does not affect the severity of heterologous secondary infection in macaques. PLoS Pathogens, 15(8), e1007766.

Publication 2019

Maxwell 16 MDx

Assay Blood Clot Dilutions Edta Ficoll Plasma Promega Serum Viral rna Zikv

Corresponding Organization : University of Wisconsin–Madison

Other organizations : University of Minnesota, Takeda (United States), Duke University

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Variable analysis

independent variables

Plasma and PBMC isolation from EDTA-treated whole blood on Ficoll paque at 1860 x rcf for 30 minutes
Serum isolation from clot-activator tubes without additive
Viral RNA (vRNA) extraction using Maxwell 16 MDx instrument (Promega, Madison, WI)
VRNA evaluation using qRT-PCR

dependent variables

RNA concentration determined by interpolation onto an internal standard curve of seven ten-fold serial dilutions of a synthetic ZIKV RNA segment based on ZIKV-FP

control variables

EDTA-treated whole blood
Clot-activator tubes without additive
Limit of detection of 100 copies vRNA/ml plasma or serum

controls

Positive control: Seven ten-fold serial dilutions of a synthetic ZIKV RNA segment based on ZIKV-FP
Negative control: Not explicitly mentioned

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