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PeerJ2024; 12; e17955; doi: 10.7717/peerj.17955

Enhancement of loop-mediated isothermal amplification (LAMP) with guanidine hydrochloride for the detection of Streptococcus equi subspecies equi (Strangles).

Abstract: subspecies , commonly referred to as "strangles", poses a significant biosecurity challenge across equine farms worldwide. The continuous prevalence and highly transmissibility of strangles necessitates a rapid and accurate diagnostic procedure. However, current "gold-standard" techniques, such as cultures and quantitative polymerase chain reaction (qPCR), are unreliable or inaccessible, and require lengthy periods between sample collection and results. Moreover, the lack of a standardized detection protocol can lead to variations in results. This study aimed to develop a reproducible and field-deployable diagnostic assay to detect strangles in real-time. Utilising the rapid technique loop-mediated isothermal amplification (LAMP), we developed an assay targeting a conserved region of the specific M gene (SeM). Additionally, we optimised our assay with guanidine hydrochloride (GuHCl) to enhance the assay's performance and detection capabilities. The Str-LAMP was able to detect within 13 minutes and 20 seconds for both synthetic DNA and clinical isolates, with a limit of detection (LOD) of  53 copies/µl. Our assay demonstrated high repeatability with the inter-coefficient of variation ranging from 0.17% to 3.93%. Furthermore, the clinical sensitivity and specificity was calculated at 91.3% and 93.3%, respectively, with a correct classification rate of 91.8%. The implementation of this newly developed strangles assay can be employed as an efficient aid for in-field surveillance programs. The assay's reproducibility can allow for equine managers to undertake routine self-surveillance on their properties, without the requirement of specialised training. The Str-LAMP assay has the potential to be a valuable tool to help mitigate potential strangles outbreaks.
©2024 Knox and Beddoe.
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Publication Date: 2024-10-08 PubMed ID: 39421427PubMed Central: PMC11484460DOI: 10.7717/peerj.17955Google 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.

Overview

  • This research focused on developing a rapid, reliable, and field-deployable diagnostic test for detecting Streptococcus equi subspecies equi, the bacterium responsible for strangles in horses.
  • The study improved a DNA amplification method called loop-mediated isothermal amplification (LAMP) by adding guanidine hydrochloride, enhancing speed and accuracy of detection.

Background and Problem Statement

  • Streptococcus equi subspecies equi causes strangles, a highly contagious respiratory disease in horses that poses biosecurity risks on horse farms worldwide.
  • Strangles is prevalent and easily transmitted, making rapid and accurate detection critical for controlling outbreaks.
  • Current diagnostic methods (like bacterial cultures and qPCR) are often slow, inaccessible, or inconsistent due to lack of standardization.
  • There is a need for a standardized, quick, and field-friendly diagnostic tool that can be easily used by equine managers without specialized laboratory training.

Objective

  • To develop and optimize a reproducible LAMP assay that can detect S. equi subspecies equi rapidly and accurately in both synthetic DNA and clinical samples.
  • To enhance the LAMP assay’s performance by incorporating guanidine hydrochloride (GuHCl), known for improving nucleic acid amplification sensitivity.

Methods and Assay Development

  • The assay targeted a conserved region of the highly specific M gene (SeM) unique to S. equi subspecies equi.
  • The research team optimized LAMP conditions with guanidine hydrochloride to accelerate reaction times and increase detection sensitivity.
  • They tested the assay using both synthetic DNA templates and clinical isolates (actual samples from infected horses) to assess performance.

Key Findings

  • The enhanced LAMP assay (Str-LAMP) detected the target bacterium within 13 minutes and 20 seconds on average.
  • The limit of detection (LOD) was 53 copies per microliter, indicating high sensitivity.
  • Repeatability was strong, with inter-coefficient of variation between 0.17% and 3.93%, demonstrating reliable reproducibility between tests.
  • Clinical testing showed sensitivity (true positive rate) of 91.3% and specificity (true negative rate) of 93.3%, yielding an overall correct classification rate of 91.8%.

Implications and Applications

  • The rapid, reliable Str-LAMP assay can be used directly in the field, facilitating early diagnosis and control of strangles outbreaks.
  • Its ease of use and consistent performance allow equine farm managers to perform routine self-surveillance without requiring specialized training or expensive equipment.
  • This assay could standardize strangles detection protocols, reducing variability and improving biosecurity measures on equine properties.
  • Overall, the Str-LAMP assay represents a promising tool to help mitigate transmission risks and manage strangles outbreaks globally.

Conclusion

  • The study successfully demonstrated that adding guanidine hydrochloride to LAMP significantly improves detection speed and sensitivity for S. equi subspecies equi.
  • The developed Str-LAMP assay is a valuable, field-deployable diagnostic method that supports rapid and accurate strangles diagnosis and enhanced biosecurity management.

Cite This Article

APA
Knox A, Beddoe T. (2024). Enhancement of loop-mediated isothermal amplification (LAMP) with guanidine hydrochloride for the detection of Streptococcus equi subspecies equi (Strangles). PeerJ, 12, e17955. https://doi.org/10.7717/peerj.17955

Publication

PeerJ
ISSN: 2167-8359
NlmUniqueID: 101603425
Country: United States
Language: English
Volume: 12
Pages: e17955
PII: e17955

Researcher Affiliations

Knox, Alexandra
  • Department of Animal, Plant and Soil Science, La Trobe University, Bundoora, Victoria, Australia.
Beddoe, Travis
  • Department of Animal, Plant and Soil Science, La Trobe University, Bundoora, Victoria, Australia.

MeSH Terms

  • Nucleic Acid Amplification Techniques / methods
  • Nucleic Acid Amplification Techniques / veterinary
  • Horses
  • Animals
  • Horse Diseases / diagnosis
  • Horse Diseases / microbiology
  • Molecular Diagnostic Techniques / methods
  • Streptococcal Infections / veterinary
  • Streptococcal Infections / diagnosis
  • Streptococcal Infections / microbiology
  • Guanidine
  • Sensitivity and Specificity
  • Streptococcus equi / genetics
  • Streptococcus equi / isolation & purification
  • Reproducibility of Results
  • DNA, Bacterial / genetics
  • Limit of Detection
  • Streptococcus

Conflict of Interest Statement

Travis Beddoe is an Academic Editor for PeerJ.

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Citations

This article has been cited 1 times.
  1. Wong YN, Hau PT, Chau EC, Ng LC, Murillo M, Fung J, Po WW, Yu RC, Kam MK, Tam EW, Tsang CC, Chow FW. Enhanced Specificity in Colorimetric LAMP Assay for Sarocladium kiliense Detection Using a Combination of Two Additives. J Fungi (Basel) 2024 Dec 11;10(12).
    doi: 10.3390/jof10120857pubmed: 39728353google scholar: lookup
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