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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 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]