year 12, Issue 6 (January - February 2019)
Iran J Med Microbiol 2019, 12(6): 390-398 |
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1- Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran
2- Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran , rezai_ma@modares.ac.ir
3- Department of Medical Bacteriology, Faculty of Medical, Tarbiat Modares University, Tehran, Iran
4- Department of Seafood Processing, Faculty of Fisheries and Environmental Sciences, Agricultur and Natural Resource University of Gorgan, Gorgan, Iran
2- Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran , rezai_ma@modares.ac.ir
3- Department of Medical Bacteriology, Faculty of Medical, Tarbiat Modares University, Tehran, Iran
4- Department of Seafood Processing, Faculty of Fisheries and Environmental Sciences, Agricultur and Natural Resource University of Gorgan, Gorgan, Iran
Abstract: (8660 Views)
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 30percent NaCl caused the bacteria to be non-cultivable on day 4. The RT-PCR 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.
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 30percent NaCl caused the bacteria to be non-cultivable on day 4. The RT-PCR 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.
Type of Study: Original Research Article |
Subject:
Food Microbiology
Received: 2018/11/15 | Accepted: 2019/01/22 | ePublished: 2019/03/29
Received: 2018/11/15 | Accepted: 2019/01/22 | ePublished: 2019/03/29
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