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Scientific reports2020; 10(1); 4096; doi: 10.1038/s41598-020-60997-1

Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis.

Abstract: Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R value was 0.983 for the concentration range 0.2 ng (4.1 × 10 DNA copies) to 2.0 fg (4.1 × 10 DNA copies). For the pUC57-ema gene DNA, the R value was 0.993 for the concentration range 0.2 ng (5.26 × 10 DNA copies) to 2.0 fg (5.26 × 10 DNA copies). For the pUC57-Bc18S gene DNA the R value was 0.976 for the concentration range 2.0 ng (4.21 × 10 DNA copies) to 2.0 fg (4.21 × 10 DNA copies). For the pUC57-Te18S gene DNA, the R value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 10 DNA copies) to 2.0 fg (4.16 × 10 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.
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Publication Date: 2020-03-05 PubMed ID: 32139744PubMed Central: PMC7058082DOI: 10.1038/s41598-020-60997-1Google 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 study presents a new method for rapidly diagnosing equine piroplasmosis—a severe, tick-borne protozoan disease in horses. The researchers have created a quick, real-time version of the recombinase polymerase amplification technique, which allows them to simultaneously detect two causative agents of the disease, T. equi and B. caballi. The improved diagnostic protocol is faster and simpler than traditional methods and shows potential for broad use in the field.

Background

  • Equine piroplasmosis (EP) is a serious disease affecting horses. It’s caused by two types of protozoa, T. equi and B. caballi, transmitted through ticks. Importantly, horses can be carriers of these parasites without showing symptoms, posing a danger to areas where the disease is not normally found.
  • Existing tests for EP often lack specificity; they can produce false positives due to cross-reactivity with other species of Babesia—protozoan parasites some tests mistake for B. caballi. This research discusses an improved, more specific method for diagnosing EP using recombinase polymerase amplification (RPA).

Methodology

  • The method uses real-time quantitative recombinase polymerase amplification (qRPA)—an amplification technique used for detecting small amounts of DNA. This allows the simultaneous detection of both causative agents of EP.
  • DNA sequences known as primers and probes were designed to target specific genes in T. equi and B. caballi. These genes—18S rRNA, ema-1 (in T. equi), and bc48 (in B. caballi)—serve as unique identification markers.
  • The researchers used plasmid DNA (pUC57) containing the target genes to test the sensitivity of the qRPA method.

Results

  • The qRPA method’s sensitivity was evaluated across a range of DNA concentrations for each gene target. The method provided robust results, with high correlation (“R value”) between concentration and detection.
  • More importantly, the scientists developed a “duplex” qRPA analysis. This allows them to test for the two parasites simultaneously in one reaction, a critical improvement for efficient diagnosis.
  • The researchers found the best combination for duplex qRPA was primers and probes targeting the bc48 gene of B. caballi and the 18S rRNA gene of T. equi.
  • Duplex qRPA has good specificity and didn’t amplify several similar, non-target parasites. This means the test shows a negative result when the causative agents of EP aren’t present—reducing the risk of false positives.
  • Comparing it to the traditional qPCR method, the researchers found the sensitivity of qRPA was similar when testing DNA extracted from horse blood samples. It was, however, quicker and simpler—highlighting its potential for practical field usage.

Conclusion

  • Overall, this newly developed duplex qRPA method can rapidly identify T. equi and B. caballi, offering a promising tool for on-site EP screening in horses. This method’s speed, simplicity, and dual detection capability make it a potent tool for preventing and managing EP outbreaks in horse populations worldwide.

Cite This Article

APA
Lei R, Wang X, Zhang D, Liu Y, Chen Q, Jiang N. (2020). Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis. Sci Rep, 10(1), 4096. https://doi.org/10.1038/s41598-020-60997-1

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 10
Issue: 1
Pages: 4096
PII: 4096

Researcher Affiliations

Lei, Rong
  • Chinese Academy of Inspection and Quarantine, Beijing, 100176, China.
Wang, Xinyi
  • Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, Shenyang Agricultural University, Shenyang, 110866, China.
  • College of Land and Environment, Shenyang Agricultural University, Shenyang, 110866, China.
Zhang, Di
  • College of Land and Environment, Shenyang Agricultural University, Shenyang, 110866, China.
Liu, Yize
  • Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, Shenyang Agricultural University, Shenyang, 110866, China.
  • The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, 120 Dongling Road, Shenyang, 110866, China.
Chen, Qijun
  • Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, Shenyang Agricultural University, Shenyang, 110866, China.
  • The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, 120 Dongling Road, Shenyang, 110866, China.
Jiang, Ning
  • Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, Shenyang Agricultural University, Shenyang, 110866, China. jiangning@syau.edu.cn.
  • The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, 120 Dongling Road, Shenyang, 110866, China. jiangning@syau.edu.cn.

MeSH Terms

  • Animals
  • Babesiosis / diagnosis
  • DNA Primers
  • Fluorescent Dyes
  • Horse Diseases / diagnosis
  • Horse Diseases / parasitology
  • Horses
  • RNA, Ribosomal, 18S / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Theileriasis / diagnosis

Conflict of Interest Statement

The authors declare no competing interests.

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Citations

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