طراحي فرايندي حساس به منظور تشخيص سريع بروسلوز انساني در سرم آلوده بر پايه واكنش زنجيره اي پليمراز

Sensitive Procedure for Rapid Detection of Human Brucellosis, Based on PCR Method in Contaminated Serum Samples

Abstract Background and objective: Brucellosis is a zoonosis transmittable to humans poses a significant public health problem in many developing countries and requires rapid and accurate diagnostic methods. Here, our aim was to develop a diagnostic polymerase chain reaction (PCR) assay in artificially contaminated serum samples as a model for rapid and accurate laboratory confirmation of human brucellosis. Material and methods: In this study, initially the standard Brucella abortus strain (2308) were cultured on Brucella agar medium and then colonies were inactivated by formalin 10 %. Genomic DNA was extracted from inactivated bacterial colonies. Serial dilutions of bacterial-DNA were prepared in fetal bovine serum (FBS) and water and subsequently DNA extraction were repeated on these artificially contaminated samples. The two pairs of primers amplified two different fragments included in: a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR) and a sequence 16S rRNA of B. abortus (primers F4/R2). Results: The two primers assayed showed a difference in sensitivity for detecting Brucella DNA, ranging between 5 pg and 50 pg for artificially contaminated serum samples and 50Fg and 5 pg for contaminated control samples. Therefore, the sensitivity of PCR using F4/R2 primers was greater than the PCR using JPF/JPR primers. Conclusion: Although the sensitivity of PCR using these primers was affected by serum inhibitors, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.