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BMC veterinary research2021; 17(1); 279; doi: 10.1186/s12917-021-02986-8

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.

Abstract: 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.
© 2021. The Author(s).
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Publication Date: 2021-08-19 PubMed ID: 34412635PubMed Central: PMC8375077DOI: 10.1186/s12917-021-02986-8Google Scholar: Lookup
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  • Journal Article

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 article investigates the reliability of real-time and endpoint colorimetric isothermal assays in diagnosing infections of C. psittaci and Equine herpesvirus-1 in horses, two significant agents of horse abortion. The research indicates that these assays can effectively be used as a quick and practical point of care diagnostic tool in the equine industry.

Research Methods and Goals

  • The researchers aimed to validate the use of Loop Mediated Isothermal Amplification (LAMP) assays to detect two equine abortigenic pathogens: C. psittaci and EHV-1. The LAMP assay is a low-cost, rapid diagnostic test that uses DNA amplification in a constant temperature, making it simple and rapid, and therefore practical for point-of-care (POC) diagnostics.
  • Three methods of LAMP assays were compared: real-time fluorometric LAMP (rtLAMP), colorimetric LAMP using commercially available mix (cLAMP), and SYBR Green DNA binding dye method (sgLAMP). The assays’ performance was tested against established diagnostic qPCR assays to gauge their sensitivity, specificity, and congruence.
  • In addition, the study tested whether these methods could be effectively used at point-of-care, directly in a horse hospital, by personnel who do not have significant previous molecular experience.

Findings of the Study

  • The study found that the validated C. psittaci and EHV-1 rtLAMP assays showed high sensitivity and specificity. The C. psittaci rtLAMP assay displayed a sensitivity of 100% and a specificity of 97.5%. The EHV-1 rtLAMP, meanwhile, showed a sensitivity of 86.96% and an impressive specificity of 100%.
  • When conducted on clinical samples from horses, all three assay methods for C. psittaci (rt-, c-, and sg-LAMP) showed high congruence or agreement, with Kappa values above 0.8, indicating the assays’ robust reliability.
  • The EHV-1 testing also showed high agreement between the three assays, though slightly lower than that of C. psittaci. The cLAMP method showed a Kappa value of 0.784, and the sgLAMP method registered 0.638.
  • Both pathogens’ LAMP assays revealed high agreement (between 94.12% to 100% for C. psittaci and 88.24% to 94.12% for EHV-1) with the reference diagnostic qPCR assays, demonstrating their comparability to laboratory-standard tests.
  • The C. psittaci rt- and c-LAMP assays were also successfully carried out by technicians with no prior molecular experience in the equine hospital, showing that these tests can be effectively performed in a point-of-care setting.

Conclusions of the Research

  • The study concluded that point-of-care options for detecting C. psittaci and EHV-1 in horses are reliable and can be easily used in real-world equine clinical settings.
  • The findings support the capability and applicability of isothermal diagnostics for rapid disease screening in the equine industry, thus potentially limiting economic losses and improving equine health.

Cite This Article

APA
Jelocnik M, Nyari S, Anstey S, Playford N, Fraser TA, Mitchell K, Blishen A, Pollak NM, Carrick J, Chicken C, Jenkins C. (2021). 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 Vet Res, 17(1), 279. https://doi.org/10.1186/s12917-021-02986-8

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 17
Issue: 1
Pages: 279
PII: 279

Researcher Affiliations

Jelocnik, Martina
  • Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Qld, 4557, Australia. mjelocni@usc.edu.au.
Nyari, Sharon
  • Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Qld, 4557, Australia.
Anstey, Susan
  • Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Qld, 4557, Australia.
Playford, Nicole
  • Australia & New Zealand IDEXX Laboratories Pty Ltd, East Brisbane, Qld, 4169, Australia.
Fraser, Tamieka A
  • Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Qld, 4557, Australia.
Mitchell, Keith
  • Scone Equine Group, Scone, NSW, 2337, Australia.
Blishen, Anna
  • Scone Equine Group, Scone, NSW, 2337, Australia.
Pollak, Nina M
  • Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Qld, 4557, Australia.
Carrick, Joan
  • Equine Specialist Consulting, Scone, NSW, 2337, Australia.
Chicken, Catherine
  • Scone Equine Group, Scone, NSW, 2337, Australia.
Jenkins, Cheryl
  • NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW, 2568, Australia.

MeSH Terms

  • Animals
  • Chlamydophila psittaci / isolation & purification
  • Female
  • Fluorometry / methods
  • Fluorometry / veterinary
  • Herpesviridae Infections / diagnosis
  • Herpesviridae Infections / veterinary
  • Herpesvirus 1, Equid / isolation & purification
  • Horse Diseases / diagnosis
  • Horses
  • Molecular Diagnostic Techniques / methods
  • Molecular Diagnostic Techniques / veterinary
  • Nucleic Acid Amplification Techniques / methods
  • Nucleic Acid Amplification Techniques / veterinary
  • Point-of-Care Systems
  • Psittacosis / diagnosis
  • Psittacosis / veterinary
  • Sensitivity and Specificity

Grant Funding

  • PRJ-011174 / Agrifutures Australia
  • DE190100238 / Australian Research Council

Conflict of Interest Statement

The authors declare that they have no competing interests.

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Citations

This article has been cited 6 times.
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