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

• Autor(a) principal: ALMEIDA, Sintia Silva de
• Data de Publicação: 2017
• Outros Autores: DORNELES, Elaine Maria Seles, DINIZ, Carlos Augusto Almeida, ABREU, Vinicius Augusto Carvalho de, SOUSA, Cassiana Severiano de, ALVES, Jorianne Thyeska Castro, CARNEIRO, Adriana Ribeiro, VILAS BOAS, Priscilla Carolinne Bagano, SPIER, Sharon, BARH, Debmalya, LAGE, Andrey Pereira, FIGUEIREDO, Henrique Cesar Pereira, AZEVEDO, Vasco Ariston de Carvalho, https://orcid.org/0000-0003-0270-9059, https://orcid.org/0000-0002-2753-1296, https://orcid.org, https://orcid.org/0000-0003-1522-9871, https://orcid.org/0000-0002-4775-2280
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UFPA
• Texto Completo: https://repositorio.ufpa.br/jspui/handle/2011/15726
• topic: Caseous lymphadenitis; Diagnosis; Nitrate reductase; Horse; Sheep; Goats

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.