Detection of Corynebacterium pseudotuberculosis by SYBR Green Real Time PCR

Abstract

Corynebacterium pseudotuberculosis, a Gram positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic debilitating disease of sheep, goats and other warm blooded animals. Definite diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, the present study was undertaken with the aim of evaluating a SYBR Green real time PCR assay targeting ABC transporter gene for the detection of C. pseudotuberculosis in DNA isolated from culture and pus samples from sheep and goats. The analytical sensitivity and specificity of the PCR primers were evaluated. The test demonstrated tenfold more sensitivity than conventional PCR and detected down to 100 femtogram of genomic DNA of pure C. pseudotuberculosis. The real-time PCR was found to be specific for ABC transporter gene of C. pseudotuberculosis, as no cross reactivity was detected with a variety of known bacterial isolates. The real time PCR was employed for direct detection of C. pseudotuberculosis in pus samples collected from clinically suspected cases of caseous lymphadenitis. It is concluded that, this real time assay may be used as a valuable diagnostic tool for the rapid and specific detection of C. pseudotuberculosis in clinical samples, large scale epidemiological studies and clarification of pathogenesis.