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Pathogens
Volume 15
Issue 8
10.3390/pathogens15080791
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Article

Analytical Validation and Preliminary Diagnostic Performance Evaluation of GenoPATHX™ Multiplex qPCR for Quantitative Detection of Key Salmonella Serovars in Poultry Matrices

by
Rejoice Nyarku
,
Emmanuel Kuufire
,
Viona Osei
,
Kingsley E. Bentum
,
Asmaa Elrefaey
,
Emmanuel Piiru
,
Tyric James
,
Yilkal Woube
,
Temesgen Samuel
and
Woubit Abebe
*
Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA
*
Author to whom correspondence should be addressed.
Pathogens 2026, 15(8), 791; https://doi.org/10.3390/pathogens15080791
Submission received: 30 May 2026 / Revised: 22 July 2026 / Accepted: 23 July 2026 / Published: 25 July 2026
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Abstract

Rapid detection and quantification of epidemiologically important Salmonella enterica serovars are critical for poultry surveillance, food safety monitoring, and risk-based intervention strategies. This study performed comprehensive analytical validation together with a preliminary field-based diagnostic performance evaluation of GenoPATHX™, a multiplex probe-based qPCR platform designed for the simultaneous detection and quantification of priority Salmonella serovars in poultry-associated matrices. The platform consists of two multiplex panels, designated the Chicken Key Performance Indicator (CKPI) and Turkey Key Performance Indicator (TKPI), each designed to detect priority poultry-associated Salmonella serovars together with a genus-level S. enterica marker. Analytical performance was evaluated for amplification efficiency, linearity, limit of detection (LoD95), limit of quantification (LoQ), repeatability, intermediate precision, analytical specificity (inclusivity/exclusivity), robustness, matrix effects, and performance in artificially inoculated matrices. Diagnostic performance was further assessed using naturally contaminated poultry environmental samples. The assay demonstrated robust amplification performance in both singleplex and multiplex formats, with high linearity (R2 = 0.987–0.999) and LoD95 values ranging from 60 to 545 genome equivalents per reaction. Complete analytical inclusivity and high exclusivity were achieved for the evaluated isolate panel. In field samples, the direct GenoPATHX™ workflow demonstrated 81.0% sensitivity, 91.3% specificity, and substantial agreement with the USDA-FSIS reference culture method (κ = 0.73). Overall, GenoPATHX™ exhibited robust analytical performance and enabled rapid, same-day quantitative detection of priority Salmonella serovars in poultry-associated matrices, supporting its application for poultry surveillance and food safety monitoring.
Keywords: Salmonella enterica; multiplex qPCR; quantitative detection; poultry matrices; diagnostic performance evaluation Salmonella enterica; multiplex qPCR; quantitative detection; poultry matrices; diagnostic performance evaluation

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MDPI and ACS Style

Nyarku, R.; Kuufire, E.; Osei, V.; Bentum, K.E.; Elrefaey, A.; Piiru, E.; James, T.; Woube, Y.; Samuel, T.; Abebe, W. Analytical Validation and Preliminary Diagnostic Performance Evaluation of GenoPATHX™ Multiplex qPCR for Quantitative Detection of Key Salmonella Serovars in Poultry Matrices. Pathogens 2026, 15, 791. https://doi.org/10.3390/pathogens15080791

AMA Style

Nyarku R, Kuufire E, Osei V, Bentum KE, Elrefaey A, Piiru E, James T, Woube Y, Samuel T, Abebe W. Analytical Validation and Preliminary Diagnostic Performance Evaluation of GenoPATHX™ Multiplex qPCR for Quantitative Detection of Key Salmonella Serovars in Poultry Matrices. Pathogens. 2026; 15(8):791. https://doi.org/10.3390/pathogens15080791

Chicago/Turabian Style

Nyarku, Rejoice, Emmanuel Kuufire, Viona Osei, Kingsley E. Bentum, Asmaa Elrefaey, Emmanuel Piiru, Tyric James, Yilkal Woube, Temesgen Samuel, and Woubit Abebe. 2026. "Analytical Validation and Preliminary Diagnostic Performance Evaluation of GenoPATHX™ Multiplex qPCR for Quantitative Detection of Key Salmonella Serovars in Poultry Matrices" Pathogens 15, no. 8: 791. https://doi.org/10.3390/pathogens15080791

APA Style

Nyarku, R., Kuufire, E., Osei, V., Bentum, K. E., Elrefaey, A., Piiru, E., James, T., Woube, Y., Samuel, T., & Abebe, W. (2026). Analytical Validation and Preliminary Diagnostic Performance Evaluation of GenoPATHX™ Multiplex qPCR for Quantitative Detection of Key Salmonella Serovars in Poultry Matrices. Pathogens, 15(8), 791. https://doi.org/10.3390/pathogens15080791

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