Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

摘要

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ mu l of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P multocida. The real-time PCR was found to be specific for KMT1 gene of P multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.