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Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay

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Abstract

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 101 CFU and 2.28 × 102 CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.

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Acknowledgments

This study was funded by a project of NFBSFARA scheme, ICAR, Government of India. We sincerely thank Indian Veterinary Research Institute (IVRI) for providing necessary facilities to carry out this work. We acknowledge Division of Bacteriology, Division of Biological Products and Division of Veterinary Public Health of our institute for providing Brucella and non-Brucella spp. for this study.

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Authors and Affiliations

  1. Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243122, India

    Bikash R. Prusty, B. P. Mishra & Praveen K. Gupta

  2. Division of Bacteriology, Indian Veterinary Research Institute, Izatnagar, India

    Pallab Chaudhuri

  3. Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, India

    V. K. Chaturvedi

  4. Center for Wildlife, Indian Veterinary Research Institute, Izatnagar, India

    Mohini Saini

Authors
  1. Bikash R. Prusty
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  2. Pallab Chaudhuri
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  3. V. K. Chaturvedi
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  4. Mohini Saini
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  5. B. P. Mishra
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  6. Praveen K. Gupta
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Correspondence to Bikash R. Prusty or Praveen K. Gupta.

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Prusty, B.R., Chaudhuri, P., Chaturvedi, V.K. et al. Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay. Indian J Microbiol 56, 142–147 (2016). https://doi.org/10.1007/s12088-015-0563-3

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  • DOI: https://doi.org/10.1007/s12088-015-0563-3

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