title: The Optimization of Molecular Detection of Clinical Isolates of Brucella in Blood Cultures by eryD Transcriptase Gene for Confirmation of Culture-Negative Samples.
abstract: BACKGROUND
  Brucellosis is a zoonosis disease which is widespread across the world.
  
  
  OBJECTIVE
  The aim of the present study is the evaluation of culture-negative blood samples.
  
  
  METHODS
  A total of 100 patients with suspected brucellosis were included in this experimental study and given positive serological tests. Diagnosis was performed on patients with clinical symptoms of the disease, followed by the detection of a titer that was equal to or more than 1:160 (in endemic areas) by the standard tube agglutination method. Blood samples were cultured by a BACTEC 9050 system, and subsequently by Brucella agar. At the same time, DNA from all blood samples was extracted by Qiagen Kit Company (Qia Amp Mini Kit). A molecular assay of blood samples was carried out by detection of eryD transcriptase and bcsp 31 genes in specific double PCR reactions. The specificity of the primers was evaluated by DNA from pure and approved Brucella colonies found in the blood samples, by DNA from other bacteria, and by ordinary PCR. DNA extraction from the pure colonies was carried out by both Qiagen Kit and Chelex 100 methods; the two were compared.
  
  
  RESULTS
  39 cases (39%) had positive results when tested by the BACTEC system, and 61 cases (61%) became negative. 23 culture-positive blood samples were randomly selected for PCR reactions; all showed 491 bp for the eryD gene and 223 bp for the bcsp 31 gene. Interestingly, out of 14 culture-negative blood samples, 13 cases showed positive bonds in PCR. The specificity of the PCR method was equal to 100%. DNA extraction from pure cultures was done by both Chelex 100 and Qiagen Kit; these showed the same results for all samples.
  
  
  CONCLUSIONS
  The results prove that the presented double PCR method could be used to detect positive cases from culture-negative blood samples. The Chelex 100 method is simpler and safer than the use of Qiagen Kit for DNA extraction.
authors.affiliation: Department of Immunology and Microbiology, Arak University of Medical Sciences, Arak, IR Iran.
authors.forename: Mahsa
authors.initials: M
authors.lastname: Tabibnejad
authors.affiliation: Brucellosis Research Center, Hamadan University of Medical Sciences, Hamadan, IR Iran.
authors.forename: Mohammad Yousef
authors.initials: MY
authors.lastname: Alikhani
authors.affiliation: Tuberculosis and Pediatric Infectious Research Center and Department of Microbiology, Arak University of Medical Sciences, Arak, IR Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Arjomandzadegan
authors.affiliation: Brucellosis Research Center, Hamadan University of Medical Sciences, Hamadan, IR Iran.
authors.forename: Seyed Hamid
authors.initials: SH
authors.lastname: Hashemi
authors.affiliation: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion, Hamadan, IR Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Naseri
keywords: Brucella; Chelex 100; Detection; eryD Gene