Fig. 5: Testing efficiency of three extraction kits using clinical samples stored in the ZY preservative and spiked with BCoV.
Stool samples were collected in the Zymo DNA/RNA shield buffer (ZY) preservative from 20 COVID-19 outpatients enrolled in a clinical trial of Peginterferon Lambda-1a. All samples were spiked with 10 μL of undiluted BCoV vaccine. In parallel, the same set of samples were processed without any spike-in. RNA from these samples were extracted using the MagMAX Viral/Pathogen Kit (MM; green), QIAamp Viral RNA Mini Kit (QA; orange), or Zymo Quick-RNA Viral Kit (ZY; purple). U stands for undetermined and indicates samples without no detectable RNA above the LoB. a RNA from samples with BCoV spiked in were assayed for SARS-CoV-2 RNA using ddPCR targeting the N1 gene. Anonymized sample identities are listed on the x-axis and absolute concentration is listed on the y-axis. Two-sided paired T tests were performed on n = 11 independent extractions (9 samples for which no RNA was detected across any kit were excluded). b Scatter plot of the absolute concentration of SARS-CoV-2 RNA derived from samples without any spike-in (x-axis) versus those with 10 μL of undiluted BCoV spiked in (y-axis), measured using ddPCR targeting the N1 gene. Thirty samples that yielded no detectable RNA above LoB in both sets of samples were left out of the plot. A linear regression is plotted in black and 95% confidence interval is shaded in gray, centered around the line of best fit. Samples with undetermined concentrations in either one of the cases are not included in the linear regression analysis. The associated regression equation generated from the n = 29 remaining extractions is y = 0.450 + 0.950x, R2adj = 0.870, R = 0.940, and p = 3.3E−14. c RNA extracted from samples with BCoV spiked were assayed for BCoV RNA using ddPCR targeting the M gene. A cumulative box plot shows the absolute concentrations of BCoV RNA across the clinical samples (n = 20 independent biological samples). The lower bound of the box marks the first quartile, the higher bound of the box marks the third quartile, the horizontal line marks the median, and the whiskers mark the outlier-removed minima and maxima. Source data are provided as a Source data file.
