title: Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses.
abstract: Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91-99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28-1.06 and 0.01-0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Amaresh
authors.initials: A
authors.lastname: Das
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Gordon
authors.initials: G
authors.lastname: Ward
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Andre
authors.initials: A
authors.lastname: Lowe
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Lizhe
authors.initials: L
authors.lastname: Xu
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Karen
authors.initials: K
authors.lastname: Moran
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Randall
authors.initials: R
authors.lastname: Renshaw
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Edward
authors.initials: E
authors.lastname: Dubovi
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Monica
authors.initials: M
authors.lastname: Reising
authors.affiliation: Reagents and Vaccines Services Section (Das, Ward, Lowe, Xu, Jia), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Diagnostic Services Section (Moran), Foreign Animal Disease Diagnostic Laboratory, Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, NY.|Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY (Renshaw, Dubovi).|Statistics Section, Center for Veterinary Biologics, Policy Evaluation and Licensing, Ames, IA (Reising).
authors.forename: Wei
authors.initials: W
authors.lastname: Jia
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D009923:Orf virus; D018157:Parapoxvirus / Q000302:isolation & purification*; D011213:Poxviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology
keywords: Conventional and real-time PCR; parapoxviruses; validation; zoonotic disease.