فهرست نشریات علمی دسترسی آزاد ایران

Background: Brucellosis is a zoonosis disease which is widespread across the world.Objectives: The aim of the present study is the evaluation of culture-negative blood samples.Materials and Methods: A total of 100 patients with suspected brucellosis were included in this experimental study and givenpositive serological tests. Diagnosis was performed on patients with clinical symptoms of the disease, followed by the detectionof a titer that was equal to or more than 1:160 (in endemic areas) by the standard tube agglutination method. Blood samples werecultured by a BACTEC 9050 system, and subsequently by Brucella agar. At thesame time,DNAfrom all blood samples was extracted byQiagen Kit Company (Qia Amp Mini Kit). A molecular assay of blood samples was carried out by detection of eryD transcriptase andbcsp 31 genes in specific double PCR reactions. The specificity of the primers was evaluated by DNA from pure and approved Brucellacolonies found in the blood samples, by DNA from other bacteria, and by ordinary PCR. DNA extraction from the pure colonies wascarried out by both Qiagen Kit and Chelex 100 methods; the two were compared.Results: 39 cases (39%) had positive resultswhentested by the BACTEC system, and 61 cases (61%) became negative. 23 culture-positiveblood 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 to100%. DNAextraction 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-negativeblood samples. The Chelex 100 method is simpler and safer than the use of Qiagen Kit for DNA extraction.