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Background    Corynebacterium pseudotuberculos is 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 T , 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.    Electronic supplementary material  The online version of this article (10.1186/s12917-017-1210-5) contains supplementary material, which is available to authorized users.

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