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Microorganisms2023; 11(11); 2633; doi: 10.3390/microorganisms11112633

Development of a Real-Time Quantitative PCR Based on a TaqMan-MGB Probe for the Rapid Detection of Theileria haneyi.

Abstract: Equine piroplasmosis (EP) is a parasitic disease caused by (), () and (). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of . In this study, we developed a new qPCR method to detect based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in or A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of infections in horses.
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Publication Date: 2023-10-26 PubMed ID: 38004645PubMed Central: PMC10673206DOI: 10.3390/microorganisms11112633Google 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.

Overview

  • This study developed a new real-time quantitative PCR (qPCR) assay using a TaqMan-MGB probe for the rapid and specific detection of Theileria haneyi, a parasite causing equine piroplasmosis (EP).
  • The newly developed qPCR assay showed high specificity and sensitivity and was validated against a nested PCR (nPCR) method using clinical samples, demonstrating its potential for improving diagnosis of T. haneyi infections in horses.

Introduction to the Problem

  • Equine piroplasmosis (EP) is a significant parasitic disease affecting horses, caused by protozoans including Theileria equi, Theileria haneyi, and Babesia caballi.
  • The disease is reportable to the World Organization for Animal Health (WOAH), reflecting its importance in animal health and trade regulations.
  • Existing diagnostic methods include traditional PCR techniques, but real-time quantitative PCR (qPCR) had not been widely applied for detection of T. haneyi.
  • Rapid, sensitive, and specific diagnostic tools are essential for timely treatment and for epidemiological surveillance.

Development of the New qPCR Assay

  • The target gene selected for detection was chr1sco (chromosome 1 single-copy open reading frame gene) unique to T. haneyi, with no orthologs in T. equi or B. caballi, ensuring assay specificity.
  • A TaqMan minor groove binder (MGB) probe was designed for the qPCR assay; this probe type enhances specificity and sensitivity by stabilizing the DNA-probe hybrid.
  • To create a quantitative assay, a plasmid containing the chr1sco gene fragment was constructed to serve as a standard for generating standard curves, enabling quantification of parasite DNA in samples.

Evaluation of Assay Performance

  • Specificity Testing:
    • The qPCR assay was tested against DNA from other common equine infectious pathogens.
    • It showed no cross-reactivity, confirming its high specificity for T. haneyi.
  • Sensitivity Testing:
    • Standard plasmid dilutions were used to evaluate the assay’s detection limit.
    • The assay was able to detect very low levels of chr1sco DNA, indicating high sensitivity.

Validation with Clinical Samples

  • A total of 96 clinical samples from horses were analyzed by both the new qPCR assay and an optimized nested PCR (nPCR) assay previously used for T. haneyi detection.
  • The new qPCR showed 85.42% agreement with the nPCR results, indicating strong concordance between methods.
  • This validation supports the utility of the qPCR as a reliable diagnostic tool for clinical and epidemiological applications.

Significance and Implications

  • The novel qPCR assay provides:
    • Rapid turnaround time and quantitative data useful for clinical decision-making.
    • Increased sensitivity and specificity for T. haneyi compared to some traditional PCR methods.
    • A standardized method that can be used in epidemiological surveys to better understand the prevalence and distribution of equine piroplasmosis.
  • Improved diagnosis of T. haneyi infections will facilitate better disease management and control in the equine industry.

Cite This Article

APA
Zhou B, Yang G, Hu Z, Chen K, Guo W, Wang X, Du C. (2023). Development of a Real-Time Quantitative PCR Based on a TaqMan-MGB Probe for the Rapid Detection of Theileria haneyi. Microorganisms, 11(11), 2633. https://doi.org/10.3390/microorganisms11112633

Publication

Microorganisms
ISSN: 2076-2607
NlmUniqueID: 101625893
Country: Switzerland
Language: English
Volume: 11
Issue: 11
PII: 2633

Researcher Affiliations

Zhou, Bingqian
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Yang, Guangpu
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Hu, Zhe
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Chen, Kewei
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Guo, Wei
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Wang, Xiaojun
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Du, Cheng
  • State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.

Grant Funding

  • 2022YFD1800200 / the National Key Research and Development Program of China
  • TD2022C006 / the Natural Science Foundation of Heilongjiang Province (Team Program)

Conflict of Interest Statement

The authors declare no conflict of interest.

References

This article includes 30 references
  1. Wise L.N., Kappmeyer L.S., Mealey R.H., Knowles D.P. Review of equine piroplasmosis. J. Vet. Intern. Med. 2013;27:1334–1346. doi: 10.1111/jvim.12168.
    doi: 10.1111/jvim.12168pubmed: 24033559google scholar: lookup
  2. Wise L.N., Pelzel-McCluskey A.M., Mealey R.H., Knowles D.P. Equine piroplasmosis. Vet. Clin. N. Am. Equine Pract. 2014;30:677–693. doi: 10.1016/j.cveq.2014.08.008.
    doi: 10.1016/j.cveq.2014.08.008pubmed: 25300637google scholar: lookup
  3. Scoles G.A., Ueti M.W. Vector ecology of equine piroplasmosis. Annu. Rev. Entomol. 2015;60:561–580. doi: 10.1146/annurev-ento-010814-021110.
    doi: 10.1146/annurev-ento-010814-021110pubmed: 25564746google scholar: lookup
  4. Giubega S., Ilie M.S., Luca I., Florea T., Dreghiciu C., Oprescu I., Morariu S., Dărăbuș G. Seroprevalence of Anti-Theileria equi Antibodies in Horses from Three Geographically Distinct Areas of Romania. Pathogens. 2022;11:669. doi: 10.3390/pathogens11060669.
    doi: 10.3390/pathogens11060669pmc: PMC9229635pubmed: 35745523google scholar: lookup
  5. Camino E., de la Cruz M.L., Dominguez L., Carvajal K.A., Fores P., de Juan L., Cruz-Lopez F. Epidemiological Situation of the Exposure to Agents Causing Equine Piroplasmosis in Spanish Purebred Horses in Spain: Seroprevalence and Associated Risk Factors. J. Equine Vet. Sci. 2018;67:81–86. doi: 10.1016/j.jevs.2018.03.012.
    doi: 10.1016/j.jevs.2018.03.012google scholar: lookup
  6. Onyiche T.E., Taioe M.O., Molefe N.I., Biu A.A., Luka J., Omeh I.J., Yokoyama N., Thekisoe O. Equine piroplasmosis: An insight into global exposure of equids from 1990 to 2019 by systematic review and meta-analysis. Parasitology. 2020;147:1411–1424. doi: 10.1017/S0031182020001407.
    doi: 10.1017/S0031182020001407pmc: PMC10317785pubmed: 32741382google scholar: lookup
  7. Zobba R., Ardu M., Niccolini S., Chessa B., Manna L., Cocco R., Pinna Parpaglia M.L. Clinical and Laboratory Findings in Equine Piroplasmosis. J. Equine Vet. Sci. 2008;28:301–308. doi: 10.1016/j.jevs.2008.03.005.
    doi: 10.1016/j.jevs.2008.03.005google scholar: lookup
  8. Almazán C., Scimeca R.C., Reichard M.V., Mosqueda J. Babesiosis and Theileriosis in North America. Pathogens. 2022;11:168. doi: 10.3390/pathogens11020168.
    doi: 10.3390/pathogens11020168pmc: PMC8874406pubmed: 35215111google scholar: lookup
  9. Allsopp M.T., Cavalier-Smith T., De Waal D.T., Allsopp B.A. Phylogeny and evolution of the piroplasms. Pt 2Parasitology. 1994;108:147–152. doi: 10.1017/S0031182000068232.
    doi: 10.1017/S0031182000068232pubmed: 8159459google scholar: lookup
  10. Knowles D.P., Kappmeyer L.S., Haney D., Herndon D.R., Fry L.M., Munro J.B., Sears K., Ueti M.W., Wise L.N., Silva M., et al. Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria: Implications for apicomplexan parasite surveillance. Int. J. Parasitol. 2018;48:679–690. doi: 10.1016/j.ijpara.2018.03.010.
    doi: 10.1016/j.ijpara.2018.03.010pubmed: 29885436google scholar: lookup
  11. Cunha C.W., Kappmeyer L.S., McGuire T.C., Dellagostin O.A., Knowles D.P. Conformational dependence and conservation of an immunodominant epitope within the babesia equi erythrocyte-stage surface protein equi merozoite antigen 1. Clin. Diagn. Lab. Immunol. 2002;9:1301–1306. doi: 10.1128/CDLI.9.6.1301-1306.2002.
    doi: 10.1128/CDLI.9.6.1301-1306.2002pmc: PMC130086pubmed: 12414764google scholar: lookup
  12. Ueti M.W., Palmer G.H., Kappmeyer L.S., Scoles G.A., Knowles D.P. Expression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus. J. Clin. Microbiol. 2003;41:5803–5809. doi: 10.1128/JCM.41.12.5803-5809.2003.
    doi: 10.1128/JCM.41.12.5803-5809.2003pmc: PMC308990pubmed: 14662988google scholar: lookup
  13. Bastos R.G., Sears K.P., Dinkel K.D., Kappmeyer L., Ueti M.W., Knowles D.P., Fry L.M. Development of an Indirect ELISA to Detect Equine Antibodies to Theileria haneyi. Pathogens. 2021;10:270. doi: 10.3390/pathogens10030270.
    doi: 10.3390/pathogens10030270pmc: PMC7997436pubmed: 33673478google scholar: lookup
  14. Coultous R.M., McDonald M., Raftery A.G., Shiels B.R., Sutton D.G.M., Weir W. Analysis of Theileria equi diversity in The Gambia using a novel genotyping method. Transbound. Emerg. Dis. 2020;67:1213–1221. doi: 10.1111/tbed.13454.
    doi: 10.1111/tbed.13454pubmed: 31845493google scholar: lookup
  15. Mshelia P.W., Kappmeyer L., Johnson W.C., Kudi C.A., Oluyinka O.O., Balogun E.O., Richard E.E., Onoja E., Sears K.P., Ueti M.W. Molecular detection of Theileria species and Babesia caballi from horses in Nigeria. Parasitol. Res. 2020;119:2955–2963. doi: 10.1007/s00436-020-06797-y.
    doi: 10.1007/s00436-020-06797-ypmc: PMC7431391pubmed: 32647992google scholar: lookup
  16. Bhoora R.V., Collins N.E., Schnittger L., Troskie C., Marumo R., Labuschagne K., Smith R.M., Dalton D.L., Mbizeni S. Molecular genotyping and epidemiology of equine piroplasmids in South Africa. Ticks Tick-Borne Dis. 2020;11:101358. doi: 10.1016/j.ttbdis.2019.101358.
    doi: 10.1016/j.ttbdis.2019.101358pubmed: 31870636google scholar: lookup
  17. Navarro E., Serrano-Heras G., Castaño M.J., Solera J. Real-time PCR detection chemistry. Clin. Chim. Acta. 2015;439:231–250. doi: 10.1016/j.cca.2014.10.017.
    doi: 10.1016/j.cca.2014.10.017pubmed: 25451956google scholar: lookup
  18. Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M.W., Shipley G.L., et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009;55:611–622. doi: 10.1373/clinchem.2008.112797.
    doi: 10.1373/clinchem.2008.112797pubmed: 19246619google scholar: lookup
  19. Kralik P., Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front. Microbiol. 2017;8:108. doi: 10.3389/fmicb.2017.00108.
    doi: 10.3389/fmicb.2017.00108pmc: PMC5288344pubmed: 28210243google scholar: lookup
  20. Chen K., Hu Z., Li J., Wang J., Liu D., Qi T., Guo W., Du C., Wang X. Prevalence and molecular epidemiology of equine piroplasmosis in China: A neglected tick-borne disease. Sci. China Life Sci. 2021;65:445–447. doi: 10.1007/s11427-021-2021-3.
    doi: 10.1007/s11427-021-2021-3pubmed: 34939161google scholar: lookup
  21. Whelan J.A., Russell N.B., Whelan M.A. A method for the absolute quantification of cDNA using real-time PCR. J. Immunol. Methods. 2003;278:261–269. doi: 10.1016/S0022-1759(03)00223-0.
    doi: 10.1016/S0022-1759(03)00223-0pubmed: 12957413google scholar: lookup
  22. Bishop R.P., Kappmeyer L.S., Onzere C.K., Odongo D.O., Githaka N., Sears K.P., Knowles D.P., Fry L.M. Equid infective Theileria cluster in distinct 18S rRNA gene clades comprising multiple taxa with unusually broad mammalian host ranges. Parasit. Vectors. 2020;13:261. doi: 10.1186/s13071-020-04131-0.
    doi: 10.1186/s13071-020-04131-0pmc: PMC7236219pubmed: 32430015google scholar: lookup
  23. Sears K., Knowles D., Dinkel K., Mshelia P.W., Onzere C., Silva M., Fry L. Imidocarb Dipropionate Lacks Efficacy against Theileria haneyi and Fails to Consistently Clear Theileria equi in Horses Co-Infected with T. haneyi. Pathogens. 2020;9:1035. doi: 10.3390/pathogens9121035.
    doi: 10.3390/pathogens9121035pmc: PMC7764667pubmed: 33321715google scholar: lookup
  24. Schrader C., Schielke A., Ellerbroek L., Johne R. PCR inhibitors—Occurrence, properties and removal. J. Appl. Microbiol. 2012;113:1014–1026. doi: 10.1111/j.1365-2672.2012.05384.x.
    doi: 10.1111/j.1365-2672.2012.05384.xpubmed: 22747964google scholar: lookup
  25. Criado-Fornelio A., Martinez-Marcos A., Buling-Sarana A., Barba-Carretero J.C. Molecular studies on Babesia, Theileria and Hepatozoon in Southern Europe. Part I. Epizootiological aspects. Vet. Parasitol. 2003;113:189–201. doi: 10.1016/S0304-4017(03)00078-5.
    doi: 10.1016/S0304-4017(03)00078-5pubmed: 12719133google scholar: lookup
  26. Collins M.S., Bashiruddin J.B., Alexander D.J. Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity. Arch. Virol. 1993;128:363–370. doi: 10.1007/BF01309446.
    doi: 10.1007/BF01309446pubmed: 8435046google scholar: lookup
  27. Valera M.J., Torija M.J., Mas A., Mateo E. Acetobacter malorum and Acetobacter cerevisiae identification and quantification by Real-Time PCR with TaqMan-MGB probes. Food Microbiol. 2013;36:30–39. doi: 10.1016/j.fm.2013.03.008.
    doi: 10.1016/j.fm.2013.03.008pubmed: 23764217google scholar: lookup
  28. Watzinger F., Ebner K., Lion T. Detection and monitoring of virus infections by real-time PCR. Mol. Asp. Med. 2006;27:254–298. doi: 10.1016/j.mam.2005.12.001.
    doi: 10.1016/j.mam.2005.12.001pmc: PMC7112306pubmed: 16481036google scholar: lookup
  29. Kutyavin I.V., Afonina I.A., Mills A., Gorn V.V., Lukhtanov E.A., Belousov E.S., Singer M.J., Walburger D.K., Lokhov S.G., Gall A.A., et al. 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 2000;28:655–661. doi: 10.1093/nar/28.2.655.
    doi: 10.1093/nar/28.2.655pmc: PMC102528pubmed: 10606668google scholar: lookup
  30. Jiang Y., Zhang S., Qin H., Meng S., Deng X., Lin H., Xin X., Liang Y., Chen B., Cui Y., et al. Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus. Eur. J. Med. Res. 2021;26:147. doi: 10.1186/s40001-021-00608-5.
    doi: 10.1186/s40001-021-00608-5pmc: PMC8677905pubmed: 34920757google scholar: lookup

Citations

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  1. Tao Y, Duan S, Yu K, Cheng X, Li X, Zhang W, Zhang Y, Wei F. Development of TaqMan Real-Time Fluorescent Quantitative PCR Method for Identification and Quantification of Sinomenium acutum-Originated Herbal Drugs. Molecules 2025 Sep 16;30(18).
    doi: 10.3390/molecules30183763pubmed: 41011655google scholar: lookup
  2. Zhang Y, Zhang M, Su H, Liu J, Zhao F, Zhao Y, Li X, Yang Y, Cao G, Zhang Y. Rapid Identification of the SNP Mutation in the ABCD4 Gene and Its Association with Multi-Vertebrae Phenotypes in Ujimqin Sheep Using TaqMan-MGB Technology. Animals (Basel) 2025 Aug 5;15(15).
    doi: 10.3390/ani15152284pubmed: 40805073google scholar: lookup
  3. Li G, Wang J, Tian L, Ding M, Liu Y, Wang J. Rapid and Ultrasensitive Detection of Staphylococcus aureus by a One-Pot System Integrating Pyrococcus furiosus Argonaute with Loop-Mediated Isothermal Amplification. J Microbiol Biotechnol 2025 Jul 14;35:e2504034.
    doi: 10.4014/jmb.2504.04034pubmed: 40659552google scholar: lookup
  4. Zeng Z, Yang J, Dai J, Zou L, Lin Z, Liu Y, Guan W, Li F, Zheng K, Yuan S, Sun F, He F, Hong Y, Li H, Liu W, Men G, Zhang X, Lan Y, Deng X, Li L, Lin Y, Lai H, Qian P, Fan Q, Jiang M, Li J, Tang G, Mo Q, Deng X, Huang J, Deng X, Yang Z. Application of novel dark-quencher labeled probes in multiplex qRT-PCR assays for rapid detection of SARS-CoV-2 variants. J Thorac Dis 2025 Apr 30;17(4):2159-2173.
    doi: 10.21037/jtd-24-853pubmed: 40400917google scholar: lookup
  5. Ulucesme MC, Ozubek S, Aktas M. Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats. Vet Sci 2024 Oct 1;11(10).
    doi: 10.3390/vetsci11100466pubmed: 39453058google scholar: lookup
  6. Mendoza FJ, Pérez-Écija A, Kappmeyer LS, Suarez CE, Bastos RG. New insights in the diagnosis and treatment of equine piroplasmosis: pitfalls, idiosyncrasies, and myths. Front Vet Sci 2024;11:1459989.
    doi: 10.3389/fvets.2024.1459989pubmed: 39205808google scholar: lookup
  7. Shang P, Xu L, Cheng T. Serological and Molecular Detection of Citrus Tristeza Virus: A Review. Microorganisms 2024 Jul 27;12(8).
    doi: 10.3390/microorganisms12081539pubmed: 39203383google scholar: lookup
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