Search

Background and Aims: Many bacteria including Escherichia coli may enter into a viable but non-culturable (VBNC) state under unfavorable stresses, which are unable to be detected by culture-based methods. In this study, the use of Reverse Transcription PCR (RT-PCR) for detection of VBNC state of E. coli O157:H7 was investigated. Materials and Methods: Escherichia coli O157:H7 was inoculated in distilled water and 30 percent salt water at room temperature. The cultivability of bacteria was determined using routine culture and colony counting on MacConkey agar. The RT-PCR of 16S rRNA gene involved direct extraction and purification of RNA, DNase I treatment for removing DNA contamination, cDNA synthesis and electrophoresis of PCR products of cDNA was used to detect viable E. coli O157:H7 under studied treatments and was compared with the results of RT-PCR of 16S rRNA gene of heat- killed bacteria. Results: The cultivability of bacteria was maintained during the study period in distilled water; however, the use of 30% NaCl caused the bacteria to be non-cultivable on day 4. The RTPCR of 16S rRNA showed the positive expression of this gene in cultivable and non-cultivable bacteria during the study period, whereas heat-killed bacteria were negative for this gene, which indicated the efficacy of RT-PCR of 16S rRNA in differentiation of alive from dead bacteria. Conclusions: Escherichia coli O157:H7 entered into the VBNC under 30 percent NaCl which can be associated with serious human health problems. RT-PCR can be used to detect bacteria in the VBNC state. [ABSTRACT FROM AUTHOR]

Background: DNA-based computing is an emerging research aspect that enables the in-vivo computation and decision making with significant correctness. Recent papers show that the expression level of miRNAs are related to the progress status of some diseases such as cancers and DNA computing is introduced as a low cost and concise technique for detection of these biomarkers. In this paper, DNAbased logic gates are implemented in the laboratory to detect the level of miR-21 as the biomarker of cancer. Materials and Methods: At the first, required strands for designing DNA gates are synthesized. Then, double stranded gate is generated in laboratory using a temperature gradient that followed by electrophoresis process. This double strand is the computation engine for detecting the miR-21 biomarker. miR-21 is as input in designed gate. At the end, the expression level of miR-21 is identified by measuring the generated fluorescent. Results: at the first stage, the proposed DNA-based logic gate is evaluated by using the synthesized input strands and then it is experimented on a tumor tissue. Experimental results on synthesized strands show that its detection quality/correctness is 2.5x better than conventional methods. Conclusion: Experimental results on the tumor tissues are successful and are matched with those are extracted from real time PCR results. Also, the results show that this method is significantly more suitable than real time PCR in view of time and cost. [ABSTRACT FROM AUTHOR]

Background and Aims: Brucellosis is an infectious zoonotic disease responsible for reproductive disorders among animals and significant economic losses to the meat and milk supplychain. The purpose of this study was therefore to evaluate and diagnose the species and strains of Brucella isolates among cattles via multiplex polymerase chain reaction. Materials and Methods: In this study, blood samples were taken from 22 cows diagnosed as having brucellosis by PCR test. Blood samples were cultured in BACTEC vials and incubated at 37 degrees Celsius for 5 days following which each sample was cultured on Brucella agar and kept for 3 days under Co2 condition.The 16SrRNA gene was used as the target for the detection of Brucella genus and the bcsp31 gene was used for the detection of Brucella biovars. The PCR products were electrophoresed on %1 agarose and compared with standard strains. Results: The PCR results showed a total of 22 positive samples for Brucella among cattles. Out of the 22 cattle positive samples, 19 were identified as B.abortus (biovars 1, 2, 3, 4, 5) and 3 as B. melitensis (biovars 1, 2, 3). Conclusions: Brucella abortus biovars 1 and 3 and B .melitensis biovar 1 are the most prevalent biovars among cattles in Lorestan province, Iran. Moreover, multiplex PCR method could be used to improve the qualitative level and the Brucella diagnosis speed in laboratories. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a protozoan parasitic disease and a global health problem. The aim of this study is to diagnose the parasitic infection in humans for epidemiological identification and providing control programs using proprietary co-designed primers in three species of Leishmania. Materials and Methods: 30 common Leishmania isolates were gathered from different centers in Iran. Having been cultured in RPMI-1640 Medium, DNA was extracted and the gene ITS2-rRNA was amplified by PCR. The amplicons were examined by electrophoresis on agarose gel 2%. Also, in FLASH PCR method, a specific probe and florence colour were used to investigae the amplicon existence on sample. Results: The results of the investigations by PCR and FLASH PCR methods show that these methods are sensitive and specific for diagnosis of Leishmania Conclusion: In this study, identification of Leishmania parasite using specific primer pairs was successful and TaqMan could be one of the most sensitive diagnostic methods to identify parasite load for the ITS2 region of Leishmania [ABSTRACT FROM AUTHOR]

Background and purpose: The purpose of this study was to design a recombinant vector pEFGP- N1 containing the full length of HIV-1Vpr gene. To the best of our knowledge, the cloning of Vpr gene in pEGFP_N1 is not previously done. Materials and methods: As a source of Vpr gene the pUC19-Vpr recombinant vector was confirmed by digestion with restriction enzymes BglII and NotI in order to separate the Vpr gene. The specific primers for Vpr gene including the restriction sites of XhoI and KpnI were designed according to the multiple cloning site of pEGFP-N1 and PCR reaction was performed using the pUC 19-Vpr vector as a template. The PCR product was undergone electrophoresis and gel extraction. Digestion reaction was done on both the extracted PCR product and the pEGFP-N1vector. The recombinant pEGFP-N1-Vpr vector was achieved by the ligation reaction using the T4 DNA ligase and it was transformed into the E-coli (DH5α) and propagated. Finally, confirmation was done through the restriction enzyme digestion and PCR amplification. Results: The recombinant vector pUC19-Vpr was confirmed using the restriction enzymes digestion, and then Vpr gene was successfully amplified using its specific primers including restriction sites for XhoI and KpnI. The PCR product was confirmed by electrophoresis. Finally, a recombinant vector pEGFP-N1 containing the full length of human immunodeficiency virus-1 Vpr gene (pEGFP-N1-Vpr) was successfully constructed. Conclusion: HIV-1 Vpr gene in its full length size could be ligated into the pEGFP-N1. [ABSTRACT FROM AUTHOR]