Table 2.
Initial validation of preformulated lyophilized RPA assay on crude plant extracts and purified Phytophthora ramorum or Pythium splendens DNA.
| Sample | Platform | Average Time Onset of Amplification without Plant Extract a | Average Time Onset of Amplification with Plant Extract | ||
|---|---|---|---|---|---|
|
Phytophthora Genus (FAM) b |
Plant Internal Control (ROX) c |
Phytophthora Genus (FAM) |
Plant Internal Control (ROX) |
||
|
P. ramorum (500 pg/µL) (Positive control) |
Axxin T16-ISO | 7.66 | NR d | - e | - |
| Bio-Rad CFX96 | 12.38 | NR | - | - | |
| ViiA7 RT-PCR | 13.01 | NR | - | - | |
| QuantStudio 6 | 6.69 | NR | - | - | |
| Phytophthora-infected citrus | Axxin T16-ISO | - | - | 7.24 | 8.21 |
| Bio-Rad CFX96 | - | - | 9.08 | 10.46 | |
| ViiA7 RT-PCR | - | - | 12.68 | 14.35 | |
| QuantStudio 6 | - | - | 5.55 | 5.34 | |
|
Pythium splendens (500 pg/µL) (Negative Control) |
Axxin T16-ISO | NR | NR | - | - |
| Bio-Rad CFX96 | NR | NR | - | - | |
| ViiA7 RT-PCR | NR | NR | - | - | |
| QuantStudio 6 | NR | NR | - | - | |
| P. ramorum (0.33 ng/µL) | Axxin T16-ISO | 10.54 | 12.67 | 12.11 | 10.04 |
| Bio-Rad CFX96 | 14.97 | NR | 17.36 | 29.81 | |
| ViiA7 RT-PCR | 12.6 | NR | 13.99 | 8.95 | |
| QuantStudio 6 | 7.53 | NR | 8.11 | 23.21 | |
| P. ramorum (33 pg/µL) | Axxin T16-ISO | 18.90 | NR | 19.71 | 14.22 |
| Bio-Rad CFX96 | 22.66 | NR | NR | 20.45 | |
| ViiA7 RT-PCR | 16.51 | NR | 16.98 | 9.68 | |
| QuantStudio 6 | 9.31 | NR | 11.83 | 22.77 | |
| P. ramorum (3.3 pg/µL) | Axxin T16-ISO | 27.32 | NR | 31.12 | 13.77 |
| Bio-Rad CFX96 | NR | NR | NR | 10.57 | |
| ViiA7 RT-PCR | NR | NR | NR | 9.57 | |
| QuantStudio 6 | 12.99 | NR | 16.01 | 23.09 | |
| P. ramorum (0.33 pg/uL) | Axxin T16-ISO | NR | NR | NR | 10.06 |
| Bio-Rad CFX96 | NR | NR | NR | 15.46 | |
| ViiA7 RT-PCR | NR | NR | NR | 9.28 | |
| QuantStudio 6 | NR | NR | NR | 22.97 | |
a Values are expressed as the average (n = 2) onset of amplification in minutes. b Phytophthora genus-specific probe. c Plant internal control probe. d (NR) refers to reactions that were run but did not have any observed amplification. e (-) refers to reactions that could not be run with or without a plant extract, respectively.