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
  • 2015-09-04. Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection 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
  • 2014-03-15. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis in MALARIA JOURNAL
  • 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
  • 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
  • 2007-09-09. Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of 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
  • 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
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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

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    PUBMED

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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 R<sup>2</sup> value was 0.983 for the concentration range 0.2 ng (4.1 × 10<sup>7</sup> DNA copies) to 2.0 fg (4.1 × 10<sup>1</sup> DNA copies). For the pUC57-ema gene DNA, the R<sup>2</sup> value was 0.993 for the concentration range 0.2 ng (5.26 × 10<sup>7</sup> DNA copies) to 2.0 fg (5.26 × 10<sup>2</sup> DNA copies). For the pUC57-Bc18S gene DNA the R<sup>2</sup> value was 0.976 for the concentration range 2.0 ng (4.21 × 10<sup>8</sup> DNA copies) to 2.0 fg (4.21 × 10<sup>2</sup> DNA copies). For the pUC57-Te18S gene DNA, the R<sup>2</sup> value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 10<sup>8</sup> DNA copies) to 2.0 fg (4.16 × 10<sup>2</sup> 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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    36 schema:keywords Babesia caballi
    37 Babesia species
    38 DNA
    39 EMA-1 gene
    40 EP screening
    41 QRPA
    42 QRPA methods
    43 Rapid isothermal duplex real-time recombinase polymerase amplification
    44 Theileria equi
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    46 amplification method
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    51 bc48 gene
    52 best combination
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    54 caballi
    55 carriers
    56 combination
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    58 concentration
    59 concentration range
    60 detection
    61 diagnosis
    62 disease
    63 duplex qRPA analysis
    64 duplex qRPA assay
    65 duplex real-time recombinase polymerase amplification
    66 duplex strategy
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    68 equine babesiosis
    69 equine piroplasmosis
    70 fast identification
    71 fast simultaneous detection
    72 gene DNA
    73 genes
    74 good specificity
    75 great potential
    76 horse blood specimens
    77 horses
    78 identification
    79 infectious carriers
    80 isothermal duplex real-time recombinase polymerase amplification
    81 lack specificity
    82 method
    83 negative amplification
    84 non-enzootic areas
    85 operating process
    86 pUC57 plasmid DNA
    87 pUC57-Bc18S gene DNA
    88 pUC57-bc48 gene DNA
    89 pUC57-ema gene DNA
    90 parasites
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    92 plasmid DNA
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    95 positive amplification
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    102 qRPA analysis
    103 qRPA assay
    104 quantitative recombinase polymerase amplification (qRPA) method
    105 rRNA gene
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    108 reaction
    109 real horse blood specimens
    110 real-time quantitative recombinase polymerase amplification (qRPA) method
    111 real-time recombinase polymerase amplification
    112 recombinase polymerase amplification
    113 recombinase polymerase amplification method
    114 regulatory tests
    115 results
    116 risk
    117 screening
    118 sensitivity
    119 sensitivity of qRPA
    120 sero-epidemiological methods
    121 severe disease
    122 similar parasites
    123 simultaneous detection
    124 site EP screening
    125 species
    126 specificity
    127 specimens
    128 strategies
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    130 target genes
    131 test
    132 tick-borne protozoa Theileria equi
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