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

Sintia Almeida, Elaine M.S. Dorneles, Carlos Diniz, Vinícius Abreu, Cassiana Sousa, Jorianne Alves, Adriana Carneiro, Priscilla Bagano, Sharon Spier, Debmalya Barh, Andrey P. Lage, Henrique Figueiredo, Vasco Azevedo

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background: 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 19410T, 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.

Original languageEnglish (US)
Article number290
JournalBMC Veterinary Research
Volume13
Issue number1
DOIs
StatePublished - Sep 25 2017
Externally publishedYes

Fingerprint

Corynebacterium pseudotuberculosis
Ovis
Equus
Sheep
Polymerase Chain Reaction
Nitrates
assays
Nitrate Reductase
Multiplex Polymerase Chain Reaction
nitrates
nitrate reductase
lymphangitis
Lymphangitis
caseous lymphadenitis
Genes
New World Camelids
Lymphadenitis
llamas
genes
nitrate reduction

Keywords

  • Caseous lymphadenitis
  • Diagnosis
  • Goats
  • Horse
  • Nitrate reductase
  • Sheep

ASJC Scopus subject areas

  • veterinary(all)

Cite this

Almeida, S., Dorneles, E. M. S., Diniz, C., Abreu, V., Sousa, C., Alves, J., ... Azevedo, V. (2017). Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi. BMC Veterinary Research, 13(1), [290]. https://doi.org/10.1186/s12917-017-1210-5

Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi. / Almeida, Sintia; Dorneles, Elaine M.S.; Diniz, Carlos; Abreu, Vinícius; Sousa, Cassiana; Alves, Jorianne; Carneiro, Adriana; Bagano, Priscilla; Spier, Sharon; Barh, Debmalya; Lage, Andrey P.; Figueiredo, Henrique; Azevedo, Vasco.

In: BMC Veterinary Research, Vol. 13, No. 1, 290, 25.09.2017.

Research output: Contribution to journalArticle

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 Veterinary Research, vol. 13, no. 1, 290. https://doi.org/10.1186/s12917-017-1210-5
Almeida, Sintia ; Dorneles, Elaine M.S. ; Diniz, Carlos ; Abreu, Vinícius ; Sousa, Cassiana ; Alves, Jorianne ; Carneiro, Adriana ; Bagano, Priscilla ; Spier, Sharon ; Barh, Debmalya ; Lage, Andrey P. ; Figueiredo, Henrique ; Azevedo, Vasco. / Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi. In: BMC Veterinary Research. 2017 ; Vol. 13, No. 1.
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abstract = "Background: 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 19410T, 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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AU - Almeida, Sintia

AU - Dorneles, Elaine M.S.

AU - Diniz, Carlos

AU - Abreu, Vinícius

AU - Sousa, Cassiana

AU - Alves, Jorianne

AU - Carneiro, Adriana

AU - Bagano, Priscilla

AU - Spier, Sharon

AU - Barh, Debmalya

AU - Lage, Andrey P.

AU - Figueiredo, Henrique

AU - Azevedo, Vasco

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N2 - Background: 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 19410T, 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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KW - Caseous lymphadenitis

KW - Diagnosis

KW - Goats

KW - Horse

KW - Nitrate reductase

KW - Sheep

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