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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 저널정보
- 충남대학교 농업과학연구소 Korean Journal of Agricultural Science Korean Journal of Agricultural Science Vol.49 No.4
- 발행연도
- 2022.12
- 수록면
- 847 - 859 (13page)
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초록· 키워드
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The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10<SUP>-4</SUP>) diluted cell suspension, with a limit of detection at 95% confidence (LOD<SUB>95%</SUB>) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.
목차
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
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