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Microorganisms2024; 12(4); 777; doi: 10.3390/microorganisms12040777

Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi.

Abstract: subspecies () is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the gene, for the rapid detection of nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the gene, enhancing the overall specificity and sensitivity of the detection. After efficiency optimization, this real-time RAA method can detect 10 or more copies of nucleic acid within 20 min. The method demonstrates high specificity for and does not cross-react with other clinically relevant pathogens. Real-time RAA diagnostic performance was evaluated using 98 nasal swab samples collected from horses and compared with the real-time PCR detection method. Results revealed that 64 and 65 samples tested positive for using real-time RAA and real-time PCR, respectively. The overall agreement between the two assays was 96.94% (95/98), with a kappa value of 0.931 ( < 0.001). Further linear regression analysis indicated a significant correlation in the detection results between the two methods (R = 0.9012, < 0.0001), suggesting that the real-time RAA assay exhibits a detection performance comparable to that of real-time PCR. In conclusion, the real-time RAA assay developed here serves as a highly specific and reliable diagnostic tool for the detection of in equine samples, offering a potential alternative to real-time PCR methods. In conclusion, the real-time RAA nucleic acid diagnostic method, based on the gene, offers rapid and accurate diagnosis of , with the added advantage of minimal equipment requirements, thus contributing to the efficient detection of strangles in horses.
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Publication Date: 2024-04-11 PubMed ID: 38674721PubMed Central: PMC11052427DOI: 10.3390/microorganisms12040777Google 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.

The research article outlines the development of a quick and effective real-time RAA method (a novel nucleic acid diagnostic approach) for detecting Streptococcus equi subsp. equi, the bacterium responsible for causing strangles in equine animals. This method exhibited similar detection performance to the real-time PCR technique, while requiring less equipment.

Overview of Research

  • The research centres around Streptococcus equi subsp. equi (S. equi), a subtype of the Streptococcus equi bacteria, which causes strangles, a highly infectious disease in horses and other equine species.
  • Due to the global spread of strangles, there is a need for efficient disease control routines; therefore, the researchers developed a novel nucleic acid diagnostic tool named the real-time Recombinase-Aided Amplification (RAA) assay to quickly detect S. equi.

Process and Evaluations of the Real-time RAA Method

  • The real-time RAA technique uses specific probes and primers that target the seqA gene, a gene unique to S. equi, increasing the assay’s sensitivity and specificity.
  • After optimizing its efficiency, it was found that the real-time RAA method could detect 10 or more copies of its target nucleic acid within 20 minutes, demonstrating high specificity for S. equi with no cross-reaction to other clinically relevant pathogens.
  • In terms of performance, the real-time RAA was compared with the real-time Polymerase Chain Reaction (PCR) method using 98 nasal swab samples collected from horses.
  • Of the samples, 64 tested positive for S. equi using the real-time RAA method, while 65 tested positive using the real-time PCR method.
  • There was a high level of agreement (96.94%) between the results, with a kappa value of 0.931, indicating strong agreement between the two tests.
  • A linear regression analysis maintained a significant correlation between the detection results of the two methods (R = 0.9012, P < 0.0001).

Conclusion

  • As a conclusion, the real-time RAA method proved to be a reliable diagnostic method for detecting S. equi in equine samples, with performance levels comparable to those of the real-time PCR method.
  • Notably, the real-time RAA technique requires minimal equipment, thus providing a feasible and cost-effective alternative to real-time PCR.
  • Finally, the study suggests that this new diagnostic tool can contribute to the efficient detection and ensuing control of strangles in horses.

Cite This Article

APA
Zu H, Sun R, Li J, Guo X, Wang M, Guo W, Wang X. (2024). Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi. Microorganisms, 12(4), 777. https://doi.org/10.3390/microorganisms12040777

Publication

Microorganisms
ISSN: 2076-2607
NlmUniqueID: 101625893
Country: Switzerland
Language: English
Volume: 12
Issue: 4
PII: 777

Researcher Affiliations

Zu, Haoyu
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Sun, Rongkuan
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
  • College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Li, Jiaxin
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Guo, Xing
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Wang, Min
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Guo, Wei
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
  • Institute of Western Agriculture, The Chinese Academy of Agricultural Sciences, Changji 831100, China.
Wang, Xiaojun
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
  • Institute of Western Agriculture, The Chinese Academy of Agricultural Sciences, Changji 831100, China.

Conflict of Interest Statement

The authors declare no conflicts of interest.

References

This article includes 36 references
  1. Newton JR, Wood JLN, Dunn KA, DeBrauwere MN, Chanter N. Naturally occurring persistent and asymptomatic infection of the guttural pouches of horses with Streptococcus equi. Vet. Rec. 1997;140:84–90.
    doi: 10.1136/vr.140.4.84pubmed: 9032908google scholar: lookup
  2. Neamat-Allah AN, Damaty HM. Strangles in Arabian horses in Egypt: Clinical, epidemiological, hematological, and biochemical aspects. Vet. World 2016;9:820–826.
    doi: 10.14202/vetworld.2016.820-826pmc: PMC5021829pubmed: 27651668google scholar: lookup
  3. Waller A. Streptococcus equi: Breaking its strangles-hold. Vet. Rec. 2018;182:316–318.
    doi: 10.1136/vr.k1231pubmed: 29545493google scholar: lookup
  4. Ikhuoso OA, Monroy JC, Rivas-Caceres RR, Cipriano-Salazar M, Barbabosa Pliego A. Streptococcus equi in Equine: Diagnostic and Healthy Performance Impacts. J. Equine Vet. Sci. 2020;85:102870.
    doi: 10.1016/j.jevs.2019.102870pubmed: 31952639google scholar: lookup
  5. Rendle D, de Brauwere N, Hallowell G, Ivens P, McGlennon A, Newton R, White J, Waller A. Streptococcus equi infections: Current best practice in the diagnosis and management of ‘strangles’. UK-Vet Equine 2021;5:S3–S15.
    doi: 10.12968/ukve.2021.5.2.S.3google scholar: lookup
  6. Mallicote M. Update on Streptococcus equi subsp equi infections. Vet. Clin. N. Am. Equine Pract. 2015;31:27–41.
    doi: 10.1016/j.cveq.2014.11.003pubmed: 25600455google scholar: lookup
  7. Seyum F, Mahendra P. Clinical and microbiological observations on strangles in donkeys. Haryana Vet. 2015;54:64–66.
  8. Tonpitak W, Sornklien C, Wutthiwithayaphong S. Characterization of a Streptococcus equi ssp. equi Isolate from a Strangles Outbreak in Thailand. J. Equine Vet. Sci. 2016;38:30–32.
    doi: 10.1016/j.jevs.2016.01.001google scholar: lookup
  9. Torpiano P, Nestorova N, Vella C. Streptococcus equi subsp. equi meningitis, septicemia and subdural empyema in a child. IDCases 2020;21:e00808.
    doi: 10.1016/j.idcr.2020.e00808pmc: PMC7256365pubmed: 32489871google scholar: lookup
  10. Bohlman T, Waddell H, Schumaker B. A case of bacteremia and pneumonia caused by Streptococcus equi subspecies equi infection in a 70-year-old female following horse exposure in rural Wyoming. Ann. Clin. Microbiol. Antimicrob. 2023;22:65.
    doi: 10.1186/s12941-023-00602-1pmc: PMC10399059pubmed: 37533031google scholar: lookup
  11. Judy CE, Chaffin MK, Cohen ND. Empyema of the guttural pouch (auditory tube diverticulum) in horses: 91 cases (1977–1997). J. Am. Vet. Med. Assoc. 1999;215:1666–1670.
    doi: 10.2460/javma.1999.215.11.1666pubmed: 14567432google scholar: lookup
  12. Boyle AG, Timoney JF, Newton JR, Hines MT, Waller AS, Buchanan BR. Streptococcus equi Infections in Horses: Guidelines for Treatment, Control, and Prevention of Strangles-Revised Consensus Statement. J. Vet. Intern. Med. 2018;32:633–647.
    doi: 10.1111/jvim.15043pmc: PMC5867011pubmed: 29424487google scholar: lookup
  13. Liu Y. Identification of a Novel Genotype of Streptococcus equi Subspecies equi in a Donkey Suffering from Strangles. Pak. Vet. J. 2019;39:609–611.
    doi: 10.29261/pakvetj/2019.087google scholar: lookup
  14. Pusterla N, Bowers J, Barnum S, Hall JA. Molecular detection of Streptococcus equi subspecies equi in face flies (Musca autumnalis) collected during a strangles outbreak on a Thoroughbred farm. Med. Vet. Entomol. 2020;34:120–122.
    doi: 10.1111/mve.12394pubmed: 31280485google scholar: lookup
  15. Robinson C, Waller AS, Frykberg L, Flock M, Zachrisson O, Guss B, Flock JI. Intramuscular vaccination with Strangvac is safe and induces protection against equine strangles caused by Streptococcus equi. Vaccine 2020;38:4861–4868.
    doi: 10.1016/j.vaccine.2020.05.046pubmed: 32507408google scholar: lookup
  16. McGlennon A, Waller A, Verheyen K, Slater J, Grewar J, Aanensen D, Newton R. Surveillance of strangles in UK horses between 2015 and 2019 based on laboratory detection of Streptococcus equi. Vet. Rec. 2021;189:e948.
    doi: 10.1002/vetr.948pubmed: 34570896google scholar: lookup
  17. Jaramillo-Morales C, James K, Barnum S, Vaala W, Chappell DE, Schneider C, Craig B, Bain F, Barnett DC, Gaughan E. Voluntary Biosurveillance of Streptococcus equi Subsp. equi in Nasal Secretions of 9409 Equids with Upper Airway Infection in the USA. Vet. Sci. 2023;10:78.
    doi: 10.3390/vetsci10020078pmc: PMC9962190pubmed: 36851382google scholar: lookup
  18. Rotinsulu DA, Ewers C, Kerner K, Amrozi A, Soejoedono RD, Semmler T, Bauerfeind R. Molecular Features and Antimicrobial Susceptibilities of Streptococcus equi ssp. equi Isolates from Strangles Cases in Indonesia. Vet. Sci. 2023;10:49.
    doi: 10.3390/vetsci10010049pmc: PMC9867300pubmed: 36669050google scholar: lookup
  19. Arafa AA, Hedia RH, Ata NS, Ibrahim ES. Vancomycin resistant Streptococcus equi subsp. equi isolated from equines suffering from respiratory manifestation in Egypt. Vet. World 2021;14:1808–1814.
    doi: 10.14202/vetworld.2021.1808-1814pmc: PMC8404119pubmed: 34475702google scholar: lookup
  20. Bell DSH, Goncalves E. Diabetogenic effects of cardioprotective drugs. Diabetes Obes. Metab. 2021;23:877–885.
    doi: 10.1111/dom.14295pubmed: 33319474google scholar: lookup
  21. Pringle J, Venner M, Tscheschlok L, Bachi L, Riihimaki M. Long term silent carriers of Streptococcus equi ssp. equi following strangles; carrier detection related to sampling site of collection and culture versus qPCR. Vet. J. 2019;246:66–70.
    doi: 10.1016/j.tvjl.2019.02.003pubmed: 30902191google scholar: lookup
  22. Durham AE, Kemp-Symonds J. Failure of serological testing for antigens A and C of Streptococcus equi subspecies equi to identify guttural pouch carriers. Equine Vet. J. 2021;53:38–43.
    doi: 10.1111/evj.13276pubmed: 32374892google scholar: lookup
  23. Duran MC, Goehring LS. Equine strangles: An update on disease control and prevention. Austral J. Vet. Sci. 2021;53:23–31.
    doi: 10.4067/S0719-81322021000100023google scholar: lookup
  24. 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;13:75.
    doi: 10.1186/s12917-017-0989-4pmc: PMC5364677pubmed: 28335829google scholar: lookup
  25. Stöckle SD, Winter JC, Schöpe SS, Gehlen H. Possibilities and limitations of the prevention of strangles outbreaks on horse farms. Pferdeheilkunde 2019;35:258–264.
    doi: 10.21836/PEM20190308google scholar: lookup
  26. Lindahl S, Båverud V, Egenvall A, Aspán A, Pringle J. Comparison of sampling sites and laboratory diagnostic tests for S. equi subsp. equi in horses from confirmed strangles outbreaks. J. Vet. Intern. Med. 2013;27:542–547.
    doi: 10.1111/jvim.12063pubmed: 23527817google scholar: lookup
  27. Waller AS. New Perspectives for the Diagnosis, Control, Treatment, and Prevention of Strangles in Horses. Vet. Clin. N. Am.-Equine 2014;30:591–607.
    doi: 10.1016/j.cveq.2014.08.007pubmed: 25300634google scholar: lookup
  28. 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;195:300–304.
    doi: 10.1016/j.tvjl.2012.07.007pmc: PMC3611602pubmed: 22884566google scholar: lookup
  29. Harms C, Mapes S, Akana N, Coatti Rocha D, Pusterla N. Detection of modified-live equine intranasal vaccine pathogens in adult horses using quantitative PCR. Vet. Rec. 2014;175:510.
    doi: 10.1136/vr.102592pubmed: 25274853google scholar: lookup
  30. Cordoni G, Williams A, Durham A, Florio D, Zanoni RG, La Ragione RM. Rapid diagnosis of strangles (Streptococcus equi subspecies equi) using PCR. Res. Ve.t Sci. 2015;102:162–166.
    doi: 10.1016/j.rvsc.2015.08.008pubmed: 26412537google scholar: lookup
  31. Pusterla N, Leutenegger CM, Barnum SM, Byrne BA. Use of quantitative real-time PCR to determine viability of Streptococcus equi subspecies equi in respiratory secretions from horses with strangles. Equine Vet. J. 2018;50:697–700.
    doi: 10.1111/evj.12809pubmed: 29341315google scholar: lookup
  32. Riihimaki M, Aspan A, Ljung H, Pringle J. Long term dynamics of a Streptococcus equi ssp equi outbreak, assessed by qPCR and culture and seM sequencing in silent carriers of strangles. Vet. Microbiol. 2018;223:107–112.
    doi: 10.1016/j.vetmic.2018.07.016pubmed: 30173735google scholar: lookup
  33. Zheng YZ, Chen JT, Li J, Wu XJ, Wen JZ, Liu XZ, Lin LY, Liang XY, Huang HY, Zha GC. Reverse Transcription Recombinase-Aided Amplification Assay with Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus. Front. Cell Infect. Microbiol. 2021;11:613304.
    doi: 10.3389/fcimb.2021.613304pmc: PMC7882697pubmed: 33598439google scholar: lookup
  34. Fan G, Zhang R, He X, Tian F, Nie M, Shen X, Ma X. RAP: A Novel Approach to the Rapid and Highly Sensitive Detection of Respiratory Viruses. Front. Bioeng. Biotechnol. 2021;9:766411.
    doi: 10.3389/fbioe.2021.766411pmc: PMC8602363pubmed: 34805120google scholar: lookup
  35. Liu YY, Ren WC, Xue ZT, Miao YD, Wang W, Zhang XX, Yao C, Shang YY, Li SS, Mi FL. Real-time recombinase-aided amplification assay for rapid amplification of the gene of s. Eur. J. Clin. Microbiol. 2023;42:963–972.
    doi: 10.1007/s10096-023-04626-5pubmed: 37256455google scholar: lookup
  36. Cao YH, Fang TS, Shen JL, Zhang GD, Guo DH, Zhao LA, Jiang Y, Zhi S, Zheng L, Lv XF. Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing Escherichia coli. J. AOAC Int. 2023;106:1246–1253.
    doi: 10.1093/jaoacint/qsad063pubmed: 37252814google scholar: lookup

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