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.
Publication Date: 2016-11-28 PubMed ID: 27929456PubMed Central: PMC5226323DOI: 10.3791/54781Google Scholar: Lookup
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- Journal Article
- Video-Audio Media
- Animal Health
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Disease Management
- Disease Treatment
- Epidemiology
- Equine Health
- Genomics
- Horses
- In Vitro Research
- In Vivo
- Infectious Disease
- Laboratory Methods
- Pathogens
- Polymerase Chain Reaction
- Respiratory Disease
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The researchers developed a process that uses nanoscale real-time PCR, a genetic testing technique, to identify common disease-causing agents in dogs and horses affected with acute respiratory problems. This process allows for high-throughput testing, improving efficiency and accuracy of diagnostic testing.
Understanding the Research
- The study looks into the use of nanoliter scale real-time PCR (Polymerase Chain Reaction) in the detection of respiratory pathogens in animal specimens.
- The key feature of this PCR technique is its ability to run many individual tests simultaneously on a single plate, a process termed as spatial multiplexing. This avoids the problems associated with combining reactions in a single test.
- The researchers designed a panel based on this method, specifically for identifying disease agents present in dogs and horses suffering from acute respiratory illnesses.
Nanoscale Diagnostic PCR Workflow
- The researchers set out a workflow for the nanoscale diagnostic PCR which focuses on procedures that differ from those in microliter scale reactions.
- The workflow includes sample preparation, amplification, and analysis of sequences of target pathogens.
- In the sample respiratory panel used in this study, each of the 18 tests was set up in triplicate, accommodating up to 48 samples per plate. This indicates the high capacity of the process for multiple samples.
Universal Extraction and Pre-Amplification
- In order to facilitate multiple tests, the researchers also optimized a universal extraction and pre-amplification process.
- This standardized process aids the high-throughput preparation of samples, accommodating a variety of matrices and pathogens, either DNA or RNA based.
- The researchers provided representative data for one RNA target (Influenza A Matrix) and one DNA target (Equine Herpesvirus 1) which demonstrates the process’s ability to handle different types of pathogens.
Implications
- This research indicates potential improvements in efficiency and ergonomics of diagnostic testing for respiratory pathogens in animals.
- In particular, the ability to quickly and accurately test for a complete group of pathogens related to specific syndromes would greatly benefit diagnosis and management of acute respiratory diseases.
Cite This Article
APA
Goodman LB, Anderson RR, Slater M, Ortenberg E, Renshaw RW, Chilson BD, Laverack MA, Beeby JS, Dubovi EJ, Glaser AL.
(2016).
High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR.
J Vis Exp(117), 54781.
https://doi.org/10.3791/54781 Publication
Researcher Affiliations
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center; laura.goodman@cornell.edu.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
- Thermo Fisher Scientific Inc.
- Thermo Fisher Scientific Inc.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
- Population Medicine and Diagnostic Sciences, Cornell University Animal Health Diagnostic Center.
MeSH Terms
- Animals
- Dog Diseases / diagnosis
- Dog Diseases / virology
- Dogs
- Herpes Simplex / diagnosis
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Influenza A virus / genetics
- Influenza A virus / isolation & purification
- Orthomyxoviridae Infections / diagnosis
- Real-Time Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Simplexvirus / genetics
- Simplexvirus / isolation & purification
- Specimen Handling
Grant Funding
- U18 FD005144 / FDA HHS
References
This article includes 14 references
- Kalinina O, Lebedeva I, Brown J, Silver J. Nanoliter scale PCR with TaqMan detection.. Nucleic Acids Res 1997 May 15;25(10):1999-2004.
- Morrison T, Hurley J, Garcia J, Yoder K, Katz A, Roberts D, Cho J, Kanigan T, Ilyin SE, Horowitz D, Dixon JM, Brenan CJ. Nanoliter high throughput quantitative PCR.. Nucleic Acids Res 2006;34(18):e123.
- Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.. J Mol Endocrinol 2002 Aug;29(1):23-39.
- McCall MN, Baras AS, Crits-Christoph A, Ingersoll R, McAlexander MA, Witwer KW, Halushka MK. A benchmark for microRNA quantification algorithms using the OpenArray platform.. BMC Bioinformatics 2016 Mar 22;17:138.
- Pozzi A, Previtali C, Cenadelli S, Gandini L, Galli A, Bongioni G. Genetic traceability of cattle using an OpenArray genotyping platform.. Anim Genet 2016 Feb;47(1):133-4.
- Grigorenko E, Fisher C, Patel S, Chancey C, Rios M, Nakhasi HL, Duncan RC. Multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an OpenArray platform.. J Mol Diagn 2014 Jan;16(1):136-44.
- Shu B, Wu KH, Emery S, Villanueva J, Johnson R, Guthrie E, Berman L, Warnes C, Barnes N, Klimov A, Lindstrom S. Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.. J Clin Microbiol 2011 Jul;49(7):2614-9.
- Elia G, Decaro N, Martella V, Campolo M, Desario C, Lorusso E, Cirone F, Buonavoglia C. Detection of equine herpesvirus type 1 by real time PCR.. J Virol Methods 2006 Apr;133(1):70-5.
- Dreier J, Störmer M, Kleesiek K. Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays.. J Clin Microbiol 2005 Sep;43(9):4551-7.
- Pusterla N, Hussey GS. Equine herpesvirus 1 myeloencephalopathy.. Vet Clin North Am Equine Pract 2014 Dec;30(3):489-506.
- Firth C, Lipkin WI. The genomics of emerging pathogens.. Annu Rev Genomics Hum Genet 2013;14:281-300.
- Lecuit M, Eloit M. The potential of whole genome NGS for infectious disease diagnosis.. Expert Rev Mol Diagn 2015;15(12):1517-9.
- Dunne G, Gurfield N. Local veterinary diagnostic laboratory, a model for the one health initiative.. Vet Clin North Am Small Anim Pract 2009 Mar;39(2):373-84.
- Moutailler S, Valiente Moro C, Vaumourin E, Michelet L, Tran FH, Devillers E, Cosson JF, Gasqui P, Van VT, Mavingui P, Vourc'h G, Vayssier-Taussat M. Co-infection of Ticks: The Rule Rather Than the Exception.. PLoS Negl Trop Dis 2016 Mar;10(3):e0004539.
Citations
This article has been cited 7 times.- Mitchell PK, Cronk BD, Voorhees IEH, Rothenheber D, Anderson RR, Chan TH, Wasik BR, Dubovi EJ, Parrish CR, Goodman LB. Method comparison of targeted influenza A virus typing and whole-genome sequencing from respiratory specimens of companion animals. J Vet Diagn Invest 2021 Mar;33(2):191-201.
- Stout AE, Hofmar-Glennon HG, André NM, Goodman LB, Anderson RR, Mitchell PK, Thompson BS, Lejeune M, Whittaker GR, Goodrich EL. Infectious disease surveillance of apparently healthy horses at a multi-day show using a novel nanoscale real-time PCR panel. J Vet Diagn Invest 2021 Jan;33(1):80-86.
- Tufts DM, Goodman LB, Benedict MC, Davis AD, VanAcker MC, Diuk-Wasser M. Association of the invasive Haemaphysalis longicornis tick with vertebrate hosts, other native tick vectors, and tick-borne pathogens in New York City, USA. Int J Parasitol 2021 Feb;51(2-3):149-157.
- Yuan Q, Llanos-Soto SG, Gangloff-Kaufmann JL, Lampman JM, Frye MJ, Benedict MC, Tallmadge RL, Mitchell PK, Anderson RR, Cronk BD, Stanhope BJ, Jarvis AR, Lejeune M, Renshaw RW, Laverack M, Lamb EM, Goodman LB. Active surveillance of pathogens from ticks collected in New York State suburban parks and schoolyards. Zoonoses Public Health 2020 Sep;67(6):684-696.
- Tallmadge RL, Anderson R, Mitchell PK, Forbes ZC, Werner B, Gioia G, Moroni P, Glaser A, Thachil AJ, Goodman LB. Characterization of a novel Mycoplasma cynos real-time PCR assay. J Vet Diagn Invest 2020 Nov;32(6):793-801.
- Cronk BD, Caserta LC, Laverack M, Gerdes RS, Hynes K, Hopf CR, Fadden MA, Nakagun S, Schuler KL, Buckles EL, Lejeune M, Diel DG. Infection and tissue distribution of highly pathogenic avian influenza A type H5N1 (clade 2.3.4.4b) in red fox kits (Vulpes vulpes). Emerg Microbes Infect 2023 Dec;12(2):2249554.
- Molesan A, Goodman L, Ford J, Lovering SJ, Kelly K. The Causes of Canine Myocarditis and Myocardial Fibrosis Are Elusive by Targeted Molecular Testing: Retrospective Analysis and Literature Review. Vet Pathol 2019 Sep;56(5):761-777.
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