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Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 15.41 MB
Published VersionCC BY V4.0, Open Access
Journal article
Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care
BMC Veterinary Research, Vol.17(1), pp.1-15
2021
Abstract
Background: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for “naked eye” end-point detection when testing ‘real-world’ clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. Results: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91–100%. Conclusions: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing ‘real-world’ samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.
Details
- Title
- Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care
- Authors
- Martina Jelocnik (Corresponding Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacySharon Nyari (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacySusan Anstey (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacyNicole Playford (Author) - Australia & New Zealand IDEXX Laboratories Pty LtdTamieka A. Fraser (Author) - University of the Sunshine Coast, Queensland, School of Science and Engineering - LegacyKeith Mitchell (Author) - Scone Equine GroupAnna Blishen (Author) - Scone Equine GroupNina M. Pollak (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacyJoan Carrick (Author) - Equine Specialist ConsultingCatherine Chicken (Author) - Scone Equine GroupCheryl Jenkins (Author) - New South Wales Department of Primary Industries
- Publication details
- BMC Veterinary Research, Vol.17(1), pp.1-15
- Publisher
- BioMed Central Ltd.
- Date published
- 2021
- DOI
- 10.1186/s12917-021-02986-8
- ISSN
- 1746-6148
- Copyright note
- © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.
- Grant note
- This project is supported by funding from AgriFutures Australia as part of the AgriFutures Thoroughbred Horses Program (project number PRJ-011174) awarded to CJ, MJ and JC.
- Organisation Unit
- Faculty of Science, Health, Education and Engineering; School of Science and Engineering - Legacy; Faculty of Arts, Business and Law; School of Science, Technology and Engineering; Centre for Bioinnovation
- Language
- English
- Record Identifier
- 99578906302621
- Output Type
- Journal article
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