FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Journal of veterinary internal medicine2021; 35(3); 1597-1603; doi: 10.1111/jvim.16105

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.
Get Full Text
Publication Date: 2021-03-17 PubMed ID: 33728675PubMed Central: PMC8163136DOI: 10.1111/jvim.16105Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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 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

Journal of veterinary internal medicine
ISSN: 1939-1676
NlmUniqueID: 8708660
Country: United States
Language: English
Volume: 35
Issue: 3
Pages: 1597-1603

Researcher Affiliations

Boyle, Ashley G
  • Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA.
Rankin, Shelley C
  • Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
O'Shea, Kathleen
  • Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Stefanovski, Darko
  • Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA.
Peng, Jing
  • Department of Mechanical Engineering and Applied Mechanics, School of Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Song, Jinzhao
  • Department of Mechanical Engineering and Applied Mechanics, School of Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Bau, Haim H
  • 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.

References

This article includes 21 references
  1. Newton JR, Verheyen K, Talbot NC, Timoney JF, Wood JL, Lakhani KH, Chanter N. Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi.. Equine Vet J 2000 Nov;32(6):515-26.
    pubmed: 11093626doi: 10.2746/042516400777584721google scholar: lookup
  2. Timoney JF, Artiushin SC. Detection of Streptococcus equi in equine nasal swabs and washes by DNA amplification.. Vet Rec 1997 Oct 25;141(17):446-7.
    pubmed: 9369003doi: 10.1136/vr.141.17.446google scholar: lookup
  3. Meehan M, Lewis MJ, Byrne C, O'Hare D, Woof JM, Owen P. Localization of the equine IgG-binding domain in the fibrinogen-binding protein (FgBP) of Streptococcus equi subsp. equi.. Microbiology (Reading) 2009 Aug;155(Pt 8):2583-2592.
    pubmed: 19423628doi: 10.1099/mic.0.028845-0google scholar: lookup
  4. Båverud V, Johansson SK, Aspan A. Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus.. Vet Microbiol 2007 Oct 6;124(3-4):219-29.
    pubmed: 17531409doi: 10.1016/j.vetmic.2007.04.020google scholar: lookup
  5. Heather Z, Holden MT, Steward KF, Parkhill J, Song L, Challis GL, Robinson C, Davis-Poynter N, Waller AS. A novel streptococcal integrative conjugative element involved in iron acquisition.. Mol Microbiol 2008 Dec;70(5):1274-92.
    pmc: PMC3672683pubmed: 18990191doi: 10.1111/j.1365-2958.2008.06481.xgoogle scholar: lookup
  6. Webb K, Barker C, Harrison T, Heather Z, Steward KF, Robinson C, Newton JR, Waller AS. Detection of Streptococcus equi subspecies equi using a triplex qPCR assay.. Vet J 2013 Mar;195(3):300-4.
    pmc: PMC3611602pubmed: 22884566doi: 10.1016/j.tvjl.2012.07.007google scholar: lookup
  7. Parida M, Sannarangaiah S, Dash PK, Rao PV, Morita K. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases.. Rev Med Virol 2008 Nov-Dec;18(6):407-21.
    pmc: PMC7169140pubmed: 18716992doi: 10.1002/rmv.593google scholar: lookup
  8. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA.. Nucleic Acids Res 2000 Jun 15;28(12):E63.
    pmc: PMC102748pubmed: 10871386doi: 10.1093/nar/28.12.e63google scholar: lookup
  9. Mauk MG, Song J, Liu C, Bau HH. Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests.. Biosensors (Basel) 2018 Feb 26;8(1).
    pmc: PMC5872065pubmed: 29495424doi: 10.3390/bios8010017google scholar: lookup
  10. Liao SC, Peng J, Mauk MG, Awasthi S, Song J, Friedman H, Bau HH, Liu C. Smart Cup: A Minimally-Instrumented, Smartphone-Based Point-of-Care Molecular Diagnostic Device.. Sens Actuators B Chem 2016 Jun 28;229:232-238.
    pmc: PMC4756427pubmed: 26900258doi: 10.1016/j.snb.2016.01.073google scholar: lookup
  11. Liu C, Mauk MG, Hart R, Qiu X, Bau HH. A self-heating cartridge for molecular diagnostics.. Lab Chip 2011 Aug 21;11(16):2686-92.
    pubmed: 21734986doi: 10.1039/c1lc20345bgoogle scholar: lookup
  12. Chuang TL, Wei SC, Lee SY, Lin CW. A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification.. Biosens Bioelectron 2012 Feb 15;32(1):89-95.
    pmc: PMC7127281pubmed: 22209071doi: 10.1016/j.bios.2011.11.037google scholar: lookup
  13. Roskos K, Hickerson AI, Lu HW, Ferguson TM, Shinde DN, Klaue Y, Niemz A. Simple system for isothermal DNA amplification coupled to lateral flow detection.. PLoS One 2013;8(7):e69355.
    pmc: PMC3724848pubmed: 23922706doi: 10.1371/journal.pone.0069355google scholar: lookup
  14. Song J, Liu C, Mauk MG, Peng J, Schoenfeld T, Bau HH. A Multifunctional Reactor with Dry-Stored Reagents for Enzymatic Amplification of Nucleic Acids.. Anal Chem 2018 Jan 16;90(2):1209-1216.
    pmc: PMC6310013pubmed: 29226671doi: 10.1021/acs.analchem.7b03834google scholar: lookup
  15. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-Free Point-of-Care Molecular Detection of Zika Virus.. Anal Chem 2016 Jul 19;88(14):7289-94.
    pmc: PMC4955015pubmed: 27306491doi: 10.1021/acs.analchem.6b01632google scholar: lookup
  16. Boyle AG, Rankin SC, D￮ L, Boston RC, Wheeler-Aceto H. Streptococcus equi Detection Polymerase Chain Reaction Assay for Equine Nasopharyngeal and Guttural Pouch Wash Samples.. J Vet Intern Med 2016 Jan-Feb;30(1):276-81.
    pmc: PMC4913660pubmed: 26678318doi: 10.1111/jvim.13808google scholar: lookup
  17. Boyle AG, Stefanovski D, Rankin SC. Comparison of nasopharyngeal and guttural pouch specimens to determine the optimal sampling site to detect Streptococcus equi subsp equi carriers by DNA amplification.. BMC Vet Res 2017 Mar 23;13(1):75.
    pmc: PMC5364677pubmed: 28335829doi: 10.1186/s12917-017-0989-4google scholar: lookup
  18. Liu C, Geva E, Mauk M, Qiu X, Abrams WR, Malamud D, Curtis K, Owen SM, Bau HH. An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases.. Analyst 2011 May 21;136(10):2069-76.
    pmc: PMC4360993pubmed: 21455542doi: 10.1039/c1an00007agoogle scholar: lookup
  19. Song J, Pandian V, Mauk MG, Bau HH, Cherry S, Tisi LC, Liu C. Smartphone-Based Mobile Detection Platform for Molecular Diagnostics and Spatiotemporal Disease Mapping.. Anal Chem 2018 Apr 3;90(7):4823-4831.
    pmc: PMC5928517pubmed: 29542319doi: 10.1021/acs.analchem.8b00283google scholar: lookup
  20. Harris SR, Robinson C, Steward KF, Webb KS, Paillot R, Parkhill J, Holden MT, Waller AS. Genome specialization and decay of the strangles pathogen, Streptococcus equi, is driven by persistent infection.. Genome Res 2015 Sep;25(9):1360-71.
    pmc: PMC4561494pubmed: 26160165doi: 10.1101/gr.189803.115google scholar: lookup
  21. Slovis NM, Browne N, Bozorgmanesh R. Point-of-Care Diagnostics in Equine Practice.. Vet Clin North Am Equine Pract 2020 Apr;36(1):161-171.
    pubmed: 32145834doi: 10.1016/j.cveq.2019.12.007google scholar: lookup

Citations

This article has been cited 4 times.
  1. 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).
    doi: 10.3390/ani13162663pubmed: 37627456google scholar: lookup
  2. 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.
    doi: 10.1007/s11259-022-10047-0pubmed: 36538151google scholar: lookup
  3. 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.
    doi: 10.1002/jcla.24565pubmed: 35754145google scholar: lookup
  4. 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.
    doi: 10.7717/peerj.17955pubmed: 39421427google scholar: lookup
Subscribe to our newsletter
Join 99,658 horse owners like you who receive our email updates on horse health and nutrition.