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Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus, and foot-and-mouth disease virus
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  • Amaresh Das,
  • Yin Wang,
  • Shawn Babiuk,
  • Jianfa Bai,
  • Kimberly Dodd,
  • Wei Jia
Amaresh Das
US Department of Homeland Security Plum Island Animal Disease Center
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Yin Wang
Kansas State University College of Veterinary Medicine
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Shawn Babiuk
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Jianfa Bai
Kansas State University
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Kimberly Dodd
US Department of Homeland Security Plum Island Animal Disease Center
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Wei Jia
US Department of Homeland Security Plum Island Animal Disease Center
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Abstract

This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV), and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included -actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV, and FMDV) with no cross-reactivity against other differential viruses. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 (CaPV), 7 (PaPV), and 15 (FMDV) copies per assay. The amplification efficiency (AE) and correlation co-efficient (R2), estimated from the standard curves (Ct vs. log10 template dilution), were 94-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96-116% (AE) and >0.98 (R2), respectively, for CaP/FMD rule-out assays and 91-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 (CaPV), 36 (PaPV) and 39 (FMDV) clinical specimens collected from experimentally (CaPV and FMDV) and naturally (PaPV) infected animals, and all tested positive (DSe 100%) except two FMDV specimens that were tested negative (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV, and FMDV in suspected animals and animals with mixed infections.

Peer review status:Published

14 Sep 2020Submitted to Transboundary and Emerging Diseases
16 Sep 2020Submission Checks Completed
16 Sep 2020Assigned to Editor
19 Sep 2020Reviewer(s) Assigned
21 Oct 2020Review(s) Completed, Editorial Evaluation Pending
24 Oct 2020Editorial Decision: Revise Major
17 Feb 20211st Revision Received
18 Feb 2021Assigned to Editor
18 Feb 2021Submission Checks Completed
27 Feb 2021Reviewer(s) Assigned
26 Mar 2021Review(s) Completed, Editorial Evaluation Pending
26 Mar 2021Editorial Decision: Revise Minor
30 Mar 20212nd Revision Received
30 Mar 2021Assigned to Editor
30 Mar 2021Submission Checks Completed
01 Apr 2021Review(s) Completed, Editorial Evaluation Pending
01 Apr 2021Editorial Decision: Accept
27 Apr 2021Published in Transboundary and Emerging Diseases. 10.1111/tbed.14099