Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

Gerber, Priscilla Freitas; O'Neill, Kevin; Owolodun, Olajide; Wang, Chong; Harmon, Karen; Zhang, Jianqiang; Halbur, Patrick G; Zhou, Lei; Meng, Xiang-Jin; Opriessnig, Tanja

doi:10.1128/JCM.02685-12

Author(s)

Gerber, Priscilla Freitas

O'Neill, Kevin

Owolodun, Olajide

Wang, Chong

Harmon, Karen

Zhang, Jianqiang

Halbur, Patrick G

Zhou, Lei

Meng, Xiang-Jin

Opriessnig, Tanja

Publication Date

2013

Abstract

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) realtime reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days-2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays (VetMax from Applied Biosystems, Foster City, CA [abbreviated AB]; VetAlert from Tetracore, Rockville,MD[TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold,<30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.

Citation

Journal of Clinical Microbiology, 51(2), p. 547-556

ISSN

1098-660X

0095-1137

Link

https://hdl.handle.net/1959.11/21248

Language

en

Publisher

American Society for Microbiology

Title

Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

Type of document

Journal Article

Entity Type

Publication

Author(s)	Gerber, Priscilla Freitas O'Neill, Kevin Owolodun, Olajide Wang, Chong Harmon, Karen Zhang, Jianqiang Halbur, Patrick G Zhou, Lei Meng, Xiang-Jin Opriessnig, Tanja
Publication Date	2013
Abstract	The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) realtime reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days-2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays (VetMax from Applied Biosystems, Foster City, CA [abbreviated AB]; VetAlert from Tetracore, Rockville,MD[TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold,<30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.
Citation	Journal of Clinical Microbiology, 51(2), p. 547-556
ISSN	1098-660X 0095-1137
Link	https://hdl.handle.net/1959.11/21248
Language	en
Publisher	American Society for Microbiology
Title	Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types
Type of document	Journal Article
Entity Type	Publication

Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

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