Assessment of Viral Limit of Detection in Spiked, Unassembled High-Throughput Sequencing Datasets
High-throughput sequencing (HTS) technology is applied to plant virus diagnosis using virus discovery pipelines, and direct detection of plant viruses from unassembled HTS is needed. The e-probe diagnostic nucleic acid assay Microbe Finder (EDNA-MiFi) is a pipeline for pathogen detection for unassem...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
The American Phytopathological Society
2025-06-01
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| Series: | PhytoFrontiers |
| Subjects: | |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/PHYTOFR-11-24-0121-FI |
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| Summary: | High-throughput sequencing (HTS) technology is applied to plant virus diagnosis using virus discovery pipelines, and direct detection of plant viruses from unassembled HTS is needed. The e-probe diagnostic nucleic acid assay Microbe Finder (EDNA-MiFi) is a pipeline for pathogen detection for unassembled HTS datasets. The application of EDNA-MiFi as a diagnostic tool was demonstrated in detecting plant pathogens of different taxa. This study demonstrates that the limit of detection of EDNA-MiFi for sensitive virus detection and quantification can be calculated. A synthetic construct mimicking the Arabis mosaic virus (ArMV) RNA2 was synthesized in vitro. E-probes for the ArMV-RNA2 were designed de novo and curated for target specificity. The new and specific e-probes of ArMV RNA2 were 20 and 60 nt long and were queried against 28 HTS unassembled spiked genome libraries obtained by spiking a serially diluted gradient of the synthetic construct carrying the ArMV RNA2 with purified genomic DNA from Vitis vinifera L. The experiment generated 28 HTS spiked datasets simulating a range of virus abundance levels, such as stages of virus infection. The limit of detection of EDNA-MiFi using the synthetic ArMV-RNA2 construct in vitro and e-probes 20 nt long varies depending on the E value, with 0.0000001 ng (14.1 copies) at an E value of 1, which is comparable to the qPCR limit of detection. Other reliable detections were at 0.001 ng at an E value of 1e−1 and 0.01 at an E value of 1e−2. EDNA-MiFi rapidly queries the spiked genome libraries with sets of predetermined plant virus e-probes, allowing for rapid and sensitive detection without requiring genome sequence assembly. [Figure: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. |
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| ISSN: | 2690-5442 |