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BMC veterinary research2017; 13(1); 290; doi: 10.1186/s12917-017-1210-5

Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi.

Abstract: Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis. Methods: In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level. Results: A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410, were compared to the results of nitrate reductase identification by biochemical test. The McNemar's Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16-6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90-0.98)]. Conclusions: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.
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Publication Date: 2017-09-25 PubMed ID: 28946887PubMed Central: PMC5613524DOI: 10.1186/s12917-017-1210-5Google 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 research aimed to develop a highly efficient test, known as a quadruplex PCR assay, which can identify the Corynebacterium pseudotuberculosis bacterium and differentiate between its two varieties which cause serious health conditions in different animals. The newly-developed test demonstrated great success in identifying 97.13% of the bacterial strains tested.

Objectives

  • The research sought to improve the identification of the Corynebacterium pseudotuberculosis bacterium which is classified into two biovars, nitrate-negative biovar Ovis and nitrate-positive biovar Equi.
  • The main objective was to develop a quadruplex PCR assay. This assay would enable simultaneous detection and distinction between both biovars of the bacterium.

Methods

  • The researchers looked into the genomes of C. pseudotuberculosis to identify genes involved in the nitrate reduction pathway. This was done with the goal of improving a species identification three-primer multiplex PCR assay.
  • They included the nitrate reductase gene (narG) into the PCR assay along with the 16S, rpoB, and pld genes. This was expected to bolster the diagnosis accuracy of the PCR assay at the biovar level.

Results

  • The research team managed to create a novel quadruplex PCR assay for identifying C. pseudotuberculosis species and differentiating their biovar.
  • The quadruplex PCR results of 348 strains were measured and compared to those of the nitrate reductase identification done by a biochemical test. These strains came from various hosts, including goats, sheep, horses, cattle, buffalo, llamas, and humans, as well as a vaccine strain and a type strain.
  • No significant difference was observed when comparing the results from the newly-developed quadruplex PCR and the biochemical test using McNemar’s Chi-squared test. A concordance of 97.13% (338 out of 348 strains) was noted.

Conclusions

  • The quadruplex assay’s capability to differentiate between the two biovars of C. pseudotuberculosis adds value to its application in clinical microbiology laboratory testing.

Cite This Article

APA
Almeida S, Dorneles EMS, Diniz C, Abreu V, Sousa C, Alves J, Carneiro A, Bagano P, Spier S, Barh D, Lage AP, Figueiredo H, Azevedo V. (2017). Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi. BMC Vet Res, 13(1), 290. https://doi.org/10.1186/s12917-017-1210-5

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 13
Issue: 1
Pages: 290
PII: 290

Researcher Affiliations

Almeida, Sintia
  • Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. sintiaalmeida@gmail.com.
Dorneles, Elaine M S
  • Escola de Veterinária, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Diniz, Carlos
  • Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
  • Departamento de Medicina Veterinária, Federal University of Lavras, Lavras, MG, Brazil.
Abreu, Vinícius
  • Centro de Energia Nuclear na Agricultura, University of Sao Paulo, Piracicaba, SP, Brazil.
Sousa, Cassiana
  • Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Alves, Jorianne
  • Instituto de Ciências Biológicas, Federal University of Para, Belém, PA, Brazil.
Carneiro, Adriana
  • Instituto de Ciências Biológicas, Federal University of Para, Belém, PA, Brazil.
Bagano, Priscilla
  • Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Spier, Sharon
  • Department of Medicine and Epidemiology, UC Davis School of Veterinary Medicine, Davis, CA, USA.
Barh, Debmalya
  • Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
  • Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, WB, India.
Lage, Andrey P
  • Escola de Veterinária, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Figueiredo, Henrique
  • Aquacen - National Reference Laboratory for Aquatic Animal Diseases, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Azevedo, Vasco
  • Instituto de Ciências Biológicas, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. vasco@icb.ufmg.br.

MeSH Terms

  • Corynebacterium pseudotuberculosis / classification
  • Corynebacterium pseudotuberculosis / genetics
  • Corynebacterium pseudotuberculosis / isolation & purification
  • DNA, Bacterial / genetics
  • Genome, Bacterial
  • Polymerase Chain Reaction / methods
  • Species Specificity

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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