Table 1.
Predictions of the number of isolates of each pospiviroid species that will be detected using universal and species-specific assays of the Luminex MagPlex-TAG pospiviroid array.
Figure 1.
Flow diagram depicting the five steps of the Luminex MagPlex-TAG pospiviroid array method.
Figure 2.
Specificity of the Luminex MagPlex-TAG pospiviroid array when screened against sequence-characterized pospiviroid isolates.
Performance of the 11-plex bead-based array when screened against a large panel of single and mixed infections of sequence-characterized pospiviroid isolates, obtained from natural infections of a variety of host plants (with mixed infections simulated). Data for healthy tomato (uninfected control) and blank (no template control) samples are included for reference. Asterisks denote mean natural log (ln) median fluorescence intensity (MFI) values that exceed the threshold for that assay within the multiplexed bead-based array.
Table 2.
The sensitivity of the multiplexed Luminex MagPlex-TAG pospiviroid array when tested using a ten-fold serial dilutions of a total RNA extract from a Solanum lycopersicum sample infected with Potato spindle tuber viroid (PSTVd, isolate #N).
Figure 3.
Reproducibility of the Luminex MagPlex-TAG pospiviroid array over ten independent tests.
One sample of Potato spindle tuber viroid (isolate #3077695) was tested in ten independent reactions over several days. Mean natural log (ln) median fluorescence intensity (MFI) values are plotted; error bars show plus or minus (±) one standard deviation. The horizontal bars plotted on each of the bars shows the detection threshold obtained from our kernel density estimation method, for each assay of the array. The small standard deviation of the ln(MFI) values for positively reacting assays in the array (PSTVd, PospUni and PlantIC) demonstrate the high level of precision of the multiplexed bead-based array.
Table 3.
Results of the blind testing of single and mixed infections of pospiviroids in plant samples, showing a comparison of three separate methods for setting thresholds for positivity.
Table 4.
Percentages of false positive and false negative results for Luminex MagPlex-TAG pospiviroid array data when analyzed using three different methods for setting thresholds for positivity.
Table 5.
Details of pospiviroid isolates used for validation of the Luminex MagPlex-TAG pospiviroid array.
Table 6.
Characteristics of the oligonucleotide primers used in this study.