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 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 thesame time, DNAfrom 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 resultswhentested 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%. 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-negative blood samples. The Chelex 100 method is simpler and safer than the use of Qiagen Kit for DNA extraction.

Introduction: Extracting of genomic DNA with the proper quality and quantity is critical in the molecular identification of insects, whose morphological identification is problematic and affected by specific stage of life, size and sex. Also, storing insects under inappropriate conditions can have damaging effects on the quantity and quality of extracted DNA. Thus, choosing an appropriate protocol to provide pure DNA from these insects is essential. Materials and Methods: In the present study to achieve an efficient method for DNA extraction from insect, we have applied and compared three common methods including (CTAB, Chelex and using commercial kit (animal tissue DNA isolation kit)). The quantity and quality of the extracted DNA were measured in two thermal conditions when DNA was stored at both-20 ° C and-80 ° C. The concentration of DNA extracted was measured on the Thermo Scientific NanoDrop 2000c. Results: The greatest and lowest average nucleic acid concentration of parasitoids were recorded for CTAB and Chelex. The mean DNA yield of parasitoids (Scelionidae) from different collection preservatives indicated that the greatest average DNA yield was recorded for 95% and 99. 8% ethanol respectively, and 75% ethanol had the lowest value of average DNA yield. Conclusion: CTAB method as a suitable method. and storing insects in-80° C at 99. 8% ethanol as suitable methods with high performance to DNA extraction of parasitoids was suggested.

Background and Objective: Formalin-fixed paraffin-embedded tissues are a valuable source of DNA for molecular studies. We designed and optimized an efficient procedure for DNA extraction from formalin-fixed paraffin embedded tissues.Materials and Methods: Seventy three blocks of cervical paraffin-embedded tissues were investigated. DNA was extracted using 45 minutes boiling in alkaline solution together with 10 beads of Chelex-20, followed by phenol-chloroform extraction and alcohol precipitation.Results: This method produced DNA suitable for amplification using primers specific for human SMN and b globin genes in 98.63% and 82.2% of samples respectively. We also detected human papillomavirus DNA in 58.33% of appropriate samples.Conclusion: This procedure provides simple and efficient method for recovery of amplifiable genomic and viral DNA from paraffin wax embedded tissues.