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Transboundary and emerging diseases2022; 69(5); e1338-e1349; doi: 10.1111/tbed.14464

Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi.

Abstract: Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.
© 2022 Wiley-VCH GmbH.
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Publication Date: 2022-02-08 PubMed ID: 35089645DOI: 10.1111/tbed.14464Google 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 article presents the development of a new method to simultaneously detect and differentiate two protozoan parasites that cause Equine Piroplasmosis. The new method, a duplex real-time PCR assay, was designed according to conserved gene sequences and used double-quenched probes to reduce false positives. The assay was validated and showed high agreement with existing assays.

Overview of the Study

  • The study focuses on the development of a duplex real-time PCR detection method that allows for simultaneous detection and differentiation of T. equi and B. caballi, two parasites that cause Equine Piroplasmosis (EP), a tick-borne disease in horses.
  • Existing real-time PCR methods for detecting EP based on 18S rRNA analysis were not able to detect all genotypes of EP, particularly a genotype A of T. equi.
  • The newly developed real-time PCR detection method was specifically designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi, including Chinese strains

Technique and Methodology

  • The article specifies that double-quenched probes were used in this new PCR detection method. The use of these probes leads to less background and more signal which significantly decreases the number of false positives that are typical with conventional single-quenched probes.
  • The new PCR assays demonstrated good specificity, sensitivity, repeatability and reproducibility in the tests conducted.

Validation Process

  • The usefulness and reliability of the newly developed real-time PCR method were validated by comparison with a nested PCR assay and another previously developed real-time PCR for EP in the analysis of 506 clinical samples. These samples were collected over the years 2019 and 2020 from eleven provinces and regions of China.
  • Agreements between the new duplex real-time PCR assay and the nPCR assay or the earlier developed real-time PCR assay were found to be 92.5% for T. equi and 99.4% for B. caballi and 87.4% for T. equi and 97.2% for B. caballi. This indicates that the new method was able to match the detection of the parasites in the majority of instances.
  • It was also found that the positivity rate of this new method for detecting T. equi was 43.87%, while for detecting B. caballi it was 5.10%, which points to T.equi being a more prevalent parasite in the tested group.

Significance of the Findings and Future Applications

  • The development of this duplex real-time PCR assay is significant as it can lead to accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.
  • With its capabilities of detecting and differentiating between these two parasites that cause EP, it brings more informative and reliable diagnosis and promotes better disease management and control.

Cite This Article

APA
Chen K, Hu Z, Yang G, Guo W, Qi T, Liu D, Wang Y, Du C, Wang X. (2022). Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi. Transbound Emerg Dis, 69(5), e1338-e1349. https://doi.org/10.1111/tbed.14464

Publication

Transboundary and emerging diseases
ISSN: 1865-1682
NlmUniqueID: 101319538
Country: Germany
Language: English
Volume: 69
Issue: 5
Pages: e1338-e1349

Researcher Affiliations

Chen, Kewei
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Hu, Zhe
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Yang, Guangpu
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Guo, Wei
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Qi, Ting
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Liu, Diqiu
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Yaoxin
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Du, Cheng
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Wang, Xiaojun
  • State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

MeSH Terms

  • Animals
  • Babesia / genetics
  • Babesiosis / diagnosis
  • Babesiosis / epidemiology
  • Babesiosis / parasitology
  • Cattle
  • Cattle Diseases
  • Horse Diseases / diagnosis
  • Horse Diseases / epidemiology
  • Horse Diseases / parasitology
  • Horses
  • RNA, Ribosomal, 18S / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / veterinary
  • Reproducibility of Results
  • Theileria / genetics
  • Theileriasis / diagnosis
  • Theileriasis / epidemiology
  • Theileriasis / parasitology

Grant Funding

  • 2020YFE0203400 / National Key Research and Development Project of China
  • 2017YFD0500400 / National Key Research and Development Project of China
  • 31772720 / National Natural Science Foundation of China
  • C2018071 / Natural Science Foundation of Heilongjiang Province of China

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This article has been cited 8 times.
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