Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR
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. c...
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doaj-2617b0b122ba445db0d7fb71401d8efa2021-07-02T15:32:59ZengFarnameIranian Journal of Medical Microbiology1735-86122345-43422019-02-01126390398Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCRMohammad Khezri0Masoud Rezaei1Ashraf Mohabbati Mobarez2mehdi zolfaghari3 Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran Department of Seafood Science, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran Department of Medical Bacteriology, Faculty of Medical, Tarbiat Modares University, Tehran, Iran Department of Seafood Processing, Faculty of Fisheries and Environmental Sciences, Agricultur and Natural Resource University of Gorgan, Gorgan, Iran 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.http://ijmm.ir/article-1-889-en.htmlEscherichia coli O157:H7Bacteria viabilityReverse Transcriptase PCR |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mohammad Khezri Masoud Rezaei Ashraf Mohabbati Mobarez mehdi zolfaghari |
spellingShingle |
Mohammad Khezri Masoud Rezaei Ashraf Mohabbati Mobarez mehdi zolfaghari Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR Iranian Journal of Medical Microbiology Escherichia coli O157:H7 Bacteria viability Reverse Transcriptase PCR |
author_facet |
Mohammad Khezri Masoud Rezaei Ashraf Mohabbati Mobarez mehdi zolfaghari |
author_sort |
Mohammad Khezri |
title |
Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR |
title_short |
Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR |
title_full |
Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR |
title_fullStr |
Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR |
title_full_unstemmed |
Detection of Viable But Non-Culturable State of Escherichia coli O157:H7 Using Reverse Transcription PCR |
title_sort |
detection of viable but non-culturable state of escherichia coli o157:h7 using reverse transcription pcr |
publisher |
Farname |
series |
Iranian Journal of Medical Microbiology |
issn |
1735-8612 2345-4342 |
publishDate |
2019-02-01 |
description |
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. |
topic |
Escherichia coli O157:H7 Bacteria viability Reverse Transcriptase PCR |
url |
http://ijmm.ir/article-1-889-en.html |
work_keys_str_mv |
AT mohammadkhezri detectionofviablebutnonculturablestateofescherichiacolio157h7usingreversetranscriptionpcr AT masoudrezaei detectionofviablebutnonculturablestateofescherichiacolio157h7usingreversetranscriptionpcr AT ashrafmohabbatimobarez detectionofviablebutnonculturablestateofescherichiacolio157h7usingreversetranscriptionpcr AT mehdizolfaghari detectionofviablebutnonculturablestateofescherichiacolio157h7usingreversetranscriptionpcr |
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