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


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Article Info

DATE

2020-03-05

AUTHORS

Rong Lei, Xinyi Wang, Di Zhang, Yize Liu, Qijun Chen, Ning Jiang

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 R2 value was 0.983 for the concentration range 0.2 ng (4.1 × 107 DNA copies) to 2.0 fg (4.1 × 101 DNA copies). For the pUC57-ema gene DNA, the R2 value was 0.993 for the concentration range 0.2 ng (5.26 × 107 DNA copies) to 2.0 fg (5.26 × 102 DNA copies). For the pUC57-Bc18S gene DNA the R2 value was 0.976 for the concentration range 2.0 ng (4.21 × 108 DNA copies) to 2.0 fg (4.21 × 102 DNA copies). For the pUC57-Te18S gene DNA, the R2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 108 DNA copies) to 2.0 fg (4.16 × 102 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. More... »

PAGES

4096

References to SciGraph publications

  • 2016-08-31. Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum in PARASITES & VECTORS
  • 2013-04-17. Prevalence of Theileria equi, Babesia caballi, and Anaplasma phagocytophilum in horses from the north of Portugal in PARASITOLOGY RESEARCH
  • 2019-07-24. Utilization of recombinase polymerase amplification combined with a lateral flow strip for detection of Perkinsus beihaiensis in the oyster Crassostrea hongkongensis in PARASITES & VECTORS
  • 2013-03-09. Molecular epidemiology of Theileria equi in horses and their association with possible tick vectors in the state of Rio de Janeiro, Brazil in PARASITOLOGY RESEARCH
  • 2018-10-06. Detection of Babesia gibsoni in dogs by combining recombinase polymerase amplification (RPA) with lateral flow (LF) dipstick in PARASITOLOGY RESEARCH
  • 2018-03-02. Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis in PARASITES & VECTORS
  • 1998-04-06. Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998 in PARASITOLOGY RESEARCH
  • 2015-09-04. Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection in PARASITES & VECTORS
  • 2007-09-09. Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil in PARASITOLOGY RESEARCH
  • 2014-03-15. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis in MALARIA JOURNAL
  • 2017-02-07. Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus in VIROLOGY JOURNAL
  • 2007-01-11. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis in PARASITOLOGY RESEARCH
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    http://scigraph.springernature.com/pub.10.1038/s41598-020-60997-1

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    http://dx.doi.org/10.1038/s41598-020-60997-1

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/32139744


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    29 schema:description 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 R2 value was 0.983 for the concentration range 0.2 ng (4.1 × 107 DNA copies) to 2.0 fg (4.1 × 101 DNA copies). For the pUC57-ema gene DNA, the R2 value was 0.993 for the concentration range 0.2 ng (5.26 × 107 DNA copies) to 2.0 fg (5.26 × 102 DNA copies). For the pUC57-Bc18S gene DNA the R2 value was 0.976 for the concentration range 2.0 ng (4.21 × 108 DNA copies) to 2.0 fg (4.21 × 102 DNA copies). For the pUC57-Te18S gene DNA, the R2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 108 DNA copies) to 2.0 fg (4.16 × 102 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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    38 Babesia species
    39 DNA
    40 EMA-1 gene
    41 QRPA
    42 QRPA methods
    43 R2 values
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    45 Theileria equi
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    81 piroplasmosis
    82 plasmid DNA
    83 polymerase amplification
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    90 rRNA gene
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    93 real-time recombinase polymerase amplification
    94 recombinase polymerase amplification
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