title: High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR.
abstract: Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center; laura.goodman@cornell.edu.
authors.forename: Laura B
authors.initials: LB
authors.lastname: Goodman
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: Renee R
authors.initials: RR
authors.lastname: Anderson
authors.affiliation: Thermo Fisher Scientific Inc.
authors.forename: Marcia
authors.initials: M
authors.lastname: Slater
authors.affiliation: Thermo Fisher Scientific Inc.
authors.forename: Elen
authors.initials: E
authors.lastname: Ortenberg
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: Randall W
authors.initials: RW
authors.lastname: Renshaw
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: Brittany D
authors.initials: BD
authors.lastname: Chilson
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Laverack
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: John S
authors.initials: JS
authors.lastname: Beeby
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Dubovi
authors.affiliation: Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Glaser
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000175:diagnosis / Q000821:virology; D004285:Dogs; D006561:Herpes Simplex / Q000175:diagnosis; D006734:Horse Diseases / Q000175:diagnosis / Q000821:virology; D006736:Horses; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018139:Simplexvirus / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling