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Veterinary research communications2022; 47(2); 495-509; doi: 10.1007/s11259-022-10047-0

Comparison of PCR-HRM, colorimetric LAMP and culture based diagnostic assays in the detection of endometritis caused by Streptococcus equi subsp. zooepidemicus in mares.

Abstract: Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the causative agents of equine endometritis. In this study, a panel of different bacterial species, and colonies derived from bacteriological cultures of 38 clinical samples, were subjected to Loop-Mediated Isothermal Amplification (LAMP) assay and PCR, followed by high-resolution melt (HRM) curve analysis. All clinical samples were genotyped into three distinct groups based on HRM curve analysis. Differences in melting curve profiles were a reflection of DNA variation in sorD gene which was confirmed by DNA sequencing. A mathematical model based on Genetic Confidence Percentage (GCP) was used in HRM curve analysis and a cut-off point value was established which differentiated S. zooepidemicus isolates without requiring visual interpretation of curve profiles. The accuracy of PCR-HRM and bacterial culture in detection of S. zooepidemicus were identical with 100% sensitivity and specificity, while LAMP assay had similar specificity but a lower sensitivity (89.5%). PCR-HRM and LAMP assay provided an effective detection method with a turn-around time of six hours for PCR-HRM and 120 min for LAMP assay, compared to a minimum three days that was required when routine bacteriological culture method was used. In summary, results indicate that LAMP had the quickest turnaround, and HRM curve analysis could potentially be used for genotyping without DNA sequencing. Any mare suspected of endometritis will benefit from developed rapid diagnostic tests for detection of S. zooepidemicus and proper treatment prior to being bred and will mitigate unnecessary treatment and antibiotic resistance.
© 2022. The Author(s), under exclusive licence to Springer Nature B.V.
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Publication Date: 2022-12-20 PubMed ID: 36538151PubMed Central: PMC9765344DOI: 10.1007/s11259-022-10047-0Google Scholar: Lookup
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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.

This research investigated the efficacy of different testing methods – PCR-HRM, colorimetric LAMP, and traditional culture– in detecting endometritis caused by Streptococcus equi subsp. zooepidemicus in mares. The study found that both PCR-HRM and culture-based methods were 100% accurate, but the LAMP method was slightly less sensitive. Importantly, the LAMP method was the fastest, and HRM curve analysis could be used for genotyping without the need for DNA sequencing.

PCR-HRM, colorimetric LAMP, and culture-based diagnostic assays

  • The research explored three different diagnostic methods for detecting strains of streptococcus equi subsp. zooepidemicus, one of the bacteria responsible for equine endometritis which is an inflammation of the innermost lining of the uterus.
  • The methods were PCR-HRM (Polymerase Chain Reaction – High-Resolution Melt), colorimetric LAMP (Loop-Mediated Isothermal Amplification), and traditional culture-based diagnostic assays. PCR-HRM is a method of genetic testing that can identify variations in DNA sequences while colorimetric LAMP is a rapid, easy and cost-effective diagnostic method that does not require expensive equipment. Traditional culture-based diagnostics grow bacteria in the lab to confirm its presence.

Comparative Analysis of the three methods

  • In the study, all three diagnostic methods were used on clinical samples taken from mares, and the effectiveness, accuracy and turnaround time of each were measured.
  • The results showed that PCR-HRM and traditional bacterial cultures were both 100% reliable for detecting the S. zooepidemicus, while the LAMP assay was slightly less sensitive at 89.5%.
  • However, the LAMP assay proved significantly quicker than both PCR-HRM and traditional culture methods, with a detection speed of 120 minutes compared to six hours for PCR-HRM and a minimum of three days for traditional cultures.
  • Further, the study discovered that HRM curve analysis could be a useful tool for genotyping, as it can detect variations in DNA without needing DNA sequencing, effectively saving time and resources.

Impact on clinical practice

  • The outcomes of the study suggest potential for improvements in diagnosing endometritis in mares, which can lead to faster and more accurate treatment. Faster diagnostic methods like the LAMP assay can ensure that horses receive necessary treatment quickly, potentially improving their recovery rates.
  • Moreover, the ability to genotype using HRM curve analysis without resorting to DNA sequencing can potentially reduce costs and speed up diagnostics further.

Cite This Article

APA
Garner C, Stephen C, Pant SD, Ghorashi SA. (2022). 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, 47(2), 495-509. https://doi.org/10.1007/s11259-022-10047-0

Publication

Veterinary research communications
ISSN: 1573-7446
NlmUniqueID: 8100520
Country: Switzerland
Language: English
Volume: 47
Issue: 2
Pages: 495-509

Researcher Affiliations

Garner, Charlotte
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia.
Stephen, Cyril
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia.
  • Graham Centre for Agricultural Innovation, Wagga Wagga, Australia.
Pant, Sameer Dinkar
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia.
  • Graham Centre for Agricultural Innovation, Wagga Wagga, Australia.
Ghorashi, Seyed Ali
  • School of Agricultural, Environmental and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia. aghorashi@csu.edu.au.
  • Graham Centre for Agricultural Innovation, Wagga Wagga, Australia. aghorashi@csu.edu.au.

MeSH Terms

  • Horses
  • Animals
  • Female
  • Endometritis / diagnosis
  • Endometritis / veterinary
  • Streptococcus equi / genetics
  • Streptococcal Infections / diagnosis
  • Streptococcal Infections / veterinary
  • Streptococcal Infections / microbiology
  • Colorimetry / veterinary
  • Polymerase Chain Reaction / veterinary
  • Horse Diseases / diagnosis

Grant Funding

  • 40702 / Charles Sturt University
  • 40825 / Graham Centre for Agricultural Innovation

Conflict of Interest Statement

The authors declare that they have no competing interests.

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