Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids

Population-based sampling has improved pathogen monitoring in the US swine industry by increasing sensitivity while reducing costs. Postmortem tongue fluids (TF) have emerged as a practical option for monitoring porcine reproductive and respiratory syndrome virus (PRRSV) in breeding herds, but limit...

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Main Authors: Isadora F. Machado, Onyekachukwu H. Osemeke, Kent Doolittle, Cesar A. A. Moura, Lucina Galina Pantoja, Giovani Trevisan, Phillip Gauger, Daniel C. L. Linhares
Format: Article
Language:English
Published: MDPI AG 2025-01-01
Series:Veterinary Sciences
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Online Access:https://www.mdpi.com/2306-7381/12/1/59
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author Isadora F. Machado
Onyekachukwu H. Osemeke
Kent Doolittle
Cesar A. A. Moura
Lucina Galina Pantoja
Giovani Trevisan
Phillip Gauger
Daniel C. L. Linhares
author_facet Isadora F. Machado
Onyekachukwu H. Osemeke
Kent Doolittle
Cesar A. A. Moura
Lucina Galina Pantoja
Giovani Trevisan
Phillip Gauger
Daniel C. L. Linhares
author_sort Isadora F. Machado
collection DOAJ
description Population-based sampling has improved pathogen monitoring in the US swine industry by increasing sensitivity while reducing costs. Postmortem tongue fluids (TF) have emerged as a practical option for monitoring porcine reproductive and respiratory syndrome virus (PRRSV) in breeding herds, but limited data exist on optimal storage conditions. This study evaluated PRRSV RNA detection via RT-qPCR in TF samples under various storage times, temperatures, and viral loads. Additionally, the porcine endogenous reference gene (internal sample control, ISC) was assessed for monitoring sample quality. Three studies were conducted: Study 1 compared fresh TF samples to freeze–thaw TF samples, with significantly lower cycle threshold (Ct) values in fresh samples. Studies 2 and 3 evaluated PRRSV RNA stability across six time points (0 to 336 h) and four temperatures (−20 °C, 4 °C, 22 °C, and 34 °C). At 4 °C, Ct value variation was minimal (≤1.1) over 14 days (336 h), with no significant changes within 48 h. In contrast, storage at 22 °C and 34 °C resulted in increased Ct values. The ISC RNA showed similar stability patterns, maintaining quality at 4 °C but degrading at higher temperatures. In conclusion, storing TF at ≤4 °C for up to 14 days for PRRSV and ISC RNA detection is the best scenario, ensuring optimal diagnostic quality.
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spelling doaj-art-ae907d531add4f2da2ddee3fc250c17d2025-01-24T13:52:08ZengMDPI AGVeterinary Sciences2306-73812025-01-011215910.3390/vetsci12010059Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue FluidsIsadora F. Machado0Onyekachukwu H. Osemeke1Kent Doolittle2Cesar A. A. Moura3Lucina Galina Pantoja4Giovani Trevisan5Phillip Gauger6Daniel C. L. Linhares7Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USADepartment of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USAIDEXX Laboratories Inc., Westbrook, ME 04092, USAIowa Select Farms, Iowa Falls, IA 50126, USAGenus plc PIC, Hendersonville, TN 37075, USADepartment of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USADepartment of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USADepartment of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USAPopulation-based sampling has improved pathogen monitoring in the US swine industry by increasing sensitivity while reducing costs. Postmortem tongue fluids (TF) have emerged as a practical option for monitoring porcine reproductive and respiratory syndrome virus (PRRSV) in breeding herds, but limited data exist on optimal storage conditions. This study evaluated PRRSV RNA detection via RT-qPCR in TF samples under various storage times, temperatures, and viral loads. Additionally, the porcine endogenous reference gene (internal sample control, ISC) was assessed for monitoring sample quality. Three studies were conducted: Study 1 compared fresh TF samples to freeze–thaw TF samples, with significantly lower cycle threshold (Ct) values in fresh samples. Studies 2 and 3 evaluated PRRSV RNA stability across six time points (0 to 336 h) and four temperatures (−20 °C, 4 °C, 22 °C, and 34 °C). At 4 °C, Ct value variation was minimal (≤1.1) over 14 days (336 h), with no significant changes within 48 h. In contrast, storage at 22 °C and 34 °C resulted in increased Ct values. The ISC RNA showed similar stability patterns, maintaining quality at 4 °C but degrading at higher temperatures. In conclusion, storing TF at ≤4 °C for up to 14 days for PRRSV and ISC RNA detection is the best scenario, ensuring optimal diagnostic quality.https://www.mdpi.com/2306-7381/12/1/59PRRSVRT-qPCRinternal sample controlswinetongue fluidsdisease monitoring
spellingShingle Isadora F. Machado
Onyekachukwu H. Osemeke
Kent Doolittle
Cesar A. A. Moura
Lucina Galina Pantoja
Giovani Trevisan
Phillip Gauger
Daniel C. L. Linhares
Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids
Veterinary Sciences
PRRSV
RT-qPCR
internal sample control
swine
tongue fluids
disease monitoring
title Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids
title_full Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids
title_fullStr Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids
title_full_unstemmed Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids
title_short Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids
title_sort effect of time and temperature on the detection of prrsv rna and endogenous internal sample control in porcine tongue fluids
topic PRRSV
RT-qPCR
internal sample control
swine
tongue fluids
disease monitoring
url https://www.mdpi.com/2306-7381/12/1/59
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