Validation of a quantitative PCR assay for the detection and quantification of the nematode Litylenchus crenatae in American beech leaf tissue
- 1The Ohio State University
Abstract
Beech leaf disease (BLD), caused by the migratory plant pathogenic nematode, Litylenchus crenatae (LC, Anguinidae) is presently one of the most concerning forest diseases in the United States. BLD has spread rapidly across the landscape and is currently found in 15 U.S. states and Ontario, Canadian, making monitoring a top priority. Here, we report on the development and testing of a probe-based qPCR detection assay that integrates previously reported LC-specific primers with a newly designed probe, enabling sensitive and reproducible quantification of nematodes in plant and non-plant matrices. This assay exhibited strong in silico and in planta specificity and high quantitative performance (R2 = 0.988-0.996), with amplification efficiencies of 90.0-96.8%. The assay also performed consistently on LC-spiked spore trap membranes used to monitor the fungal aerobiome, despite matrix-associated shifts in amplification efficiency. These results support the utility of the assay for monitoring and surveillance. The reliability was further evaluated by two independent diagnostic laboratories, demonstrating high inter-laboratory concordance in detection and strong agreement in relative quantification. The analytical sensitivity and reproducible performance of the assay align with key validation principles outlined by the APS Diagnostic Assay Validation Network (DAVN) and support its application as a molecular tool in experimental, diagnostic, and surveillance contexts, with important implications for national and international LC monitoring and disease management.
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BibTeX
@misc{torresbedoya2026validation,
title = {{Validation of a quantitative PCR assay for the detection and quantification of the nematode Litylenchus crenatae in American beech leaf tissue}},
author = {E. Torres-Bedoya and A. Miles and J. Ker and F. Rotondo and K. Snover-Clift and P. Bonello},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.03.749213},
url = {https://www.biorxiv.org/content/10.64898/2026.09.03.749213v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Validation of a quantitative PCR assay for the detection and quantification of the nematode Litylenchus crenatae in American beech leaf tissue
AU - E. Torres-Bedoya
AU - A. Miles
AU - J. Ker
AU - F. Rotondo
AU - K. Snover-Clift
AU - P. Bonello
PY - 2026
DA - 2026/09/03
PB - bioRxiv
DO - 10.64898/2026.09.03.749213
UR - https://www.biorxiv.org/content/10.64898/2026.09.03.749213v1
AB - Beech leaf disease (BLD), caused by the migratory plant pathogenic nematode, Litylenchus crenatae (LC, Anguinidae) is presently one of the most concerning forest diseases in the United States. BLD has spread rapidly across the landscape and is currently found in 15 U.S. states and Ontario, Canadian, making monitoring a top priority. Here, we report on the development and testing of a probe-based qPCR detection assay that integrates previously reported LC-specific primers with a newly designed probe, enabling sensitive and reproducible quantification of nematodes in plant and non-plant matrices. This assay exhibited strong in silico and in planta specificity and high quantitative performance (R2 = 0.988-0.996), with amplification efficiencies of 90.0-96.8%. The assay also performed consistently on LC-spiked spore trap membranes used to monitor the fungal aerobiome, despite matrix-associated shifts in amplification efficiency. These results support the utility of the assay for monitoring and surveillance. The reliability was further evaluated by two independent diagnostic laboratories, demonstrating high inter-laboratory concordance in detection and strong agreement in relative quantification. The analytical sensitivity and reproducible performance of the assay align with key validation principles outlined by the APS Diagnostic Assay Validation Network (DAVN) and support its application as a molecular tool in experimental, diagnostic, and surveillance contexts, with important implications for national and international LC monitoring and disease management.
ER -