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Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection.

ABSTRACT: Brucellosis is a worldwide re-emerging zoonosis. It has an economic impact due to abortion and loss of fertility in livestock. In this study, Real-time recombinase polymerase amplification (RT-RPA-BP26) targeting Brucella spp. bp26 gene and Lateral flow dipstick (LFD-RPA-IS711) combined with SYBR- Green recombinase polymerase amplification (RPA) targeting insertion sequence IS711 region of Brucella spp. bp26 gene, was developed to detect Brucella spp. from different sample types in domestic animals. The sensitivity and specificity of the two developed RPAs were compared with real-time PCR, PCR, and Rose Bengal Plate Test (RBPT). The analytical sensitivity and detection limit of Real-time RPA and LFD RPA were four and six copies per reaction respectively. The detection of six colony forming units (CFU) of the bacteria-bearing construct with the target sequence was within 20?min at 40?°C for Real-time RPA and 37?°C for LFD RPA. The LFD RPA could work at temperatures between 30 and 35?°C and could be completed within 10-30?min. No significant differences were observed when comparing the results from Real-time RPA and LFD RPA to Real-time PCR and PCR. Both methods showed no cross reactivity with Chlamydia abortus, Toxoplasma gondii, Salmonella typhimurium, and Escherichia coli. In conclusion, RPA is a useful and convenient field and point of care test for brucellosis.

SUBMITTER: Gumaa MM

PROVIDER: S-EPMC7127669 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection.

Gumaa M M MM Cao Xiaoan X Li Zhaocai Z Lou Zhongzi Z Zhang Nianzhang N Zhang Zhijun Z Zhou Jizhang J Fu Baoquan B

Molecular and cellular probes 20190808

Brucellosis is a worldwide re-emerging zoonosis. It has an economic impact due to abortion and loss of fertility in livestock. In this study, Real-time recombinase polymerase amplification (RT-RPA-BP26) targeting Brucella spp. bp26 gene and Lateral flow dipstick (LFD-RPA-IS711) combined with SYBR- Green recombinase polymerase amplification (RPA) targeting insertion sequence IS711 region of Brucella spp. bp26 gene, was developed to detect Brucella spp. from different sample types in domestic anim ...[more]

PMID: 31401295

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Project 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.

Dataset Information

Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection.

Publications

Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection.

Similar Datasets

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