Thermophilic helicase-dependent DNA amplification using the IsoAmp™ SE experimental kit for rapid detection of Streptococcus equi subspecies equi in clinical samples.
Abstract: A simple and portable assay for detection of Streptococcus equi subspecies equi has been developed based on amplification of S. equi-specific sequence using a thermophilic helicase-dependent reaction followed by visual detection of the amplicon in a disposable lateral flow cassette. An experimental kit (IsoAmp™ SE) was evaluated. Analytical sensitivity was 50 copies of S. equi genomic DNA per reaction. The IsoAmp SE assay had 100% specificity when applied to nasal swabs and washes. The assay was more sensitive than culture but less sensitive than nested polymerase chain reaction (PCR). The test requires neither expensive equipment nor extensive training of personnel, provides a practical alternative to culture or PCR assays for detection of S. equi in clinical samples, and expedites identification of atypical colonies of S. equi and Streptococcus zooepidemicus in the laboratory.
Publication Date: 2011-09-13 PubMed ID: 21908346DOI: 10.1177/1040638711416968Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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The researchers proposed an easy and portable testing method for Streptococcus equi subspecies equi, a bacteria that infects horses. The test is based on DNA amplification and was found to be a reliable alternative to traditionally used culture or PCR assays, in terms of specificity, sensitivity, cost, and convenience.
Objective of the Study
- The research aims to develop an efficient, cost-effective, and convenient method for detecting Streptococcus equi subspecies equi, which is a key infectious agent in horses. The proposed method uses thermophilic helicase-dependent DNA amplification in conjunction with visual detection.
Methodology Approach
- The study used the IsoAmp™ SE experimental kit to evaluate the proposed detection assay.
- A thermophilic helicase-dependent reaction was employed for the amplification of S. equi-specific DNA sequence.
- The amplified DNA product, called an amplicon, was then visually detected in a disposable lateral flow cassette.
Results and Findings
- The new assay showed a sensitivity of 50 copies of S. equi genomic DNA per reaction, suggesting it can detect a relatively low amount of the bacterial DNA.
- When applied to nasal swabs and washes, the IsoAmp SE assay had a specificity of 100%, indicating that it didn’t falsely detect other species as S. equi.
- The assay was more sensitive than culture methods but less sensitive than nested polymerase chain reaction (PCR), a common procedure in molecular biology used for amplifying a specific DNA sequence.
Conclusion
- The novel test offers a practical alternative to culture or PCR assays due to its cost-effectiveness and convenience. It does not need expensive equipment or extensive training of personnel to perform.
- In addition to detecting S. equi in clinical samples, it also fast-tracks the identification of atypical colonies of S. equi and Streptococcus zooepidemicus in the lab.
Cite This Article
APA
Artiushin S, Tong Y, Timoney J, Lemieux B, Schlegel A, Kong H.
(2011).
Thermophilic helicase-dependent DNA amplification using the IsoAmp™ SE experimental kit for rapid detection of Streptococcus equi subspecies equi in clinical samples.
J Vet Diagn Invest, 23(5), 909-914.
https://doi.org/10.1177/1040638711416968 Publication
Researcher Affiliations
- 1Sergey Artiushin, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA. scarti1@email.uky.edu
MeSH Terms
- Animals
- DNA Helicases / metabolism
- DNA, Bacterial / genetics
- DNA, Bacterial / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horses
- Nucleic Acid Amplification Techniques / methods
- Nucleic Acid Amplification Techniques / veterinary
- Polymerase Chain Reaction / veterinary
- Sensitivity and Specificity
- Streptococcus equi / genetics
- Streptococcus equi / isolation & purification
- Time Factors
Citations
This article has been cited 7 times.- Ivanov AV, Safenkova IV, Zherdev AV, Dzantiev BB. The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens. Plants (Basel) 2021 Nov 10;10(11).
- Yasukawa K, Yanagihara I, Fujiwara S. Alteration of enzymes and their application to nucleic acid amplification (Review). Int J Mol Med 2020 Nov;46(5):1633-1643.
- Lau HY, Botella JR. Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection. Front Plant Sci 2017;8:2016.
- Barreda-García S, Miranda-Castro R, de-Los-Santos-Álvarez N, Miranda-Ordieres AJ, Lobo-Castañón MJ. Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection. Anal Bioanal Chem 2018 Jan;410(3):679-693.
- Fujiwara A, Kawato K, Kato S, Yasukawa K, Hidese R, Fujiwara S. Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR. Appl Environ Microbiol 2016 May 15;82(10):3022-3031.
- Buchan BW, Ledeboer NA. Emerging technologies for the clinical microbiology laboratory. Clin Microbiol Rev 2014 Oct;27(4):783-822.
- Pumford EA, Lu J, Spaczai I, Prasetyo ME, Zheng EM, Zhang H, Kamei DT. Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics. Biosens Bioelectron 2020 Dec 15;170:112674.
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