Detection of Streptococcus equi subsp. equi in guttural pouch lavage samples using a loop-mediated isothermal nucleic acid amplification microfluidic device.
Abstract: Rapid point-of-care (POC) detection of Streptococcus equi subsp. equi (S. equi) would theoretically reduce the spread of strangles by identifying index and carrier horses. Objective: That the eqbE isothermal amplification (LAMP) assay, and the same eqbE LAMP assay tested in a microfluidic device format, are comparable to a triplex real-time quantitative polymerase chain reaction (qPCR) assay that is commonly used in diagnostic labs. Methods: Sixty-eight guttural pouch lavage (GPL) specimens from horses recovering from strangles. Methods: Guttural pouch lavage specimens were tested for S. equi retrospectively using the benchtop eqbE LAMP, the eqbE LAMP microfluidic device, and compared to the triplex qPCR, that detects 2 S. equi-specific genes, eqbE and SEQ2190, as the reference standard using the receiver operating characteristic area under the curve (ROC). Results: The 27/68 specimens were positive by benchtop eqbE LAMP, 31/64 by eqbE LAMP microfluidic device, and 12/67 by triplex qPCR. Using the triplex PCR as the reference, the benchtop eqbE LAMP showed excellent discrimination (ROC Area = 0.813, 95% confidence interval [CI] = 0.711-0.915) as did the LAMP microfluidic device (ROC Area = 0.811, 95% CI = 0.529-0.782). There was no significant difference between the benchtop LAMP and LAMP microfluidic device (ROC Area 0.813 ± 0.055 vs 0.811 ± 0.034, P = .97). Conclusions: The eqbE LAMP microfluidic device detected S. equi in GPL specimens from convalescent horses. This assay shows potential for development as a POC device for rapid, sensitive, accurate, and cost-efficient detection of S. equi.
© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.
Publication Date: 2021-03-17 PubMed ID: 33728675PubMed Central: PMC8163136DOI: 10.1111/jvim.16105Google Scholar: Lookup
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- Journal Article
Summary
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The study investigates and affirms a rapid diagnostic method for detecting a responsible bacterium for strangles in horses, Streptococcus equi, using a isothermal amplification assay in a microfluidic device format. The previous common diagnostic test method, a real-time quantitative polymerase chain reaction, is compared and deemed less efficient.
Objective
The main goal of this research was to compare methods of detection for Streptococcus equi subsp. equi (S. equi), responsible for strangles in horses. More specifically, the study aimed to evaluate the effectiveness of the eqbE Loop-mediated isothermal amplification(LAMP) assay in benchtop and microfluidic device formats, comparing these to the popularly used triplex real-time quantitative polymerase chain reaction (qPCR) assay.
Methodology
- The research utilized 68 guttural pouch lavage (GPL) specimens taken from horses that were recovering from strangles.
- Three types of tests were done on the specimens – the benchtop eqbE LAMP, the eqbE LAMP microfluidic device, and the triplex qPCR, with the latter serving as the reference standard
- Detection of S. equi in GPL specimens was then compared to the triplex qPCR, which detects two separate S. equi-specific genes(eqbE and SEQ2190).
Results
- Out of 68 samples, 27 were found to be positive by the benchtop eqbE LAMP method, 31 by the eqbE LAMP microfluidic device, and only 12 were detected by the standard triplex qPCR method.
- The performance of the two LAMP methods — benchtop and microfluidic device — were found to be comparable with excellent discrimination as per the Receiver Operating Characteristic (ROC) Area, leaving no significant difference between the benchtop LAMP and LAMP microfluidic device.
Conclusion
In conclusion, it was found that the eqbE LAMP microfluidic device was quite effective in detecting S. equi in GPL specimens from convalescent horses. This discovery suggests that the eqbE LAMP assay, particularly in the microfluidic device format, could potentially be developed as a rapid, sensitive, accurate, and cost-efficient point-of-care (POC) device for detecting S. equi in horses. This would contribute greatly to controlling the spread of strangles by embracing faster identification of carrier and index horses with the disease.
Cite This Article
APA
Boyle AG, Rankin SC, O'Shea K, Stefanovski D, Peng J, Song J, Bau HH.
(2021).
Detection of Streptococcus equi subsp. equi in guttural pouch lavage samples using a loop-mediated isothermal nucleic acid amplification microfluidic device.
J Vet Intern Med, 35(3), 1597-1603.
https://doi.org/10.1111/jvim.16105 Publication
Researcher Affiliations
- Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA.
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
- Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA.
- Department of Mechanical Engineering and Applied Mechanics, School of Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
- Department of Mechanical Engineering and Applied Mechanics, School of Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
- Department of Mechanical Engineering and Applied Mechanics, School of Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
MeSH Terms
- AAA Domain
- Animals
- Horse Diseases / diagnosis
- Horses
- Lab-On-A-Chip Devices
- Molecular Diagnostic Techniques
- Nucleic Acid Amplification Techniques
- Nucleic Acids
- Retrospective Studies
- Streptococcal Infections / diagnosis
- Streptococcal Infections / veterinary
- Streptococcus
- Streptococcus equi / genetics
- Therapeutic Irrigation / veterinary
Grant Funding
- R21 AI128059 / NIAID NIH HHS
- Grayson-Jockey Club Research Foundation
- Boehringer Ingelheim
Conflict of Interest Statement
The University of Pennsylvania has applied for patent protection for the microfluidic device technology, with H. H. Bau and J. Song named as coinventors. No other authors have a conflict of interest.
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Citations
This article has been cited 4 times.- Knox A, Zerna G, Beddoe T. Current and Future Advances in the Detection and Surveillance of Biosecurity-Relevant Equine Bacterial Diseases Using Loop-Mediated Isothermal Amplification (LAMP). Animals (Basel) 2023 Aug 18;13(16).
- Garner C, Stephen C, Pant SD, Ghorashi SA. Comparison of PCR-HRM, colorimetric LAMP and culture based diagnostic assays in the detection of endometritis caused by Streptococcus equi subsp. zooepidemicus in mares. Vet Res Commun 2023 Jun;47(2):495-509.
- Zhang E, Ou H, Jia L, Zhang W, Wang Y, Wang X. Comparative analysis of loop-mediated isothermal amplification combined with microfluidic chip technology and q-PCR in the detection of clinical infectious pathogens. J Clin Lab Anal 2022 Aug;36(8):e24565.
- Knox A, Beddoe T. Enhancement of loop-mediated isothermal amplification (LAMP) with guanidine hydrochloride for the detection of Streptococcus equi subspecies equi (Strangles). PeerJ 2024;12:e17955.
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