• 농업과학연구
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• 제49권4호
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• pp.795-807
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• 2022

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^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) 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.

• 한국식품과학회지
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• 제36권5호
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• pp.828-832
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• 2004

본 연구에서는 유전자 재조합 되지 않은(non-GM) 대두와 유전자 재조합된(GM) 대두가 혼입되어 제조된 두부에서 효소 면역 측정법을 이용하여 non-GM 대두의 혼입량을 추정하고자 하였다. 두부의 SDS-PAGE 실행 결과 non-GM 두부에서만 나타나는 특이 단백질 non-GM 113kDa 밴드와 non-GM과 GM 두부에서 모두 나타나는 non-GM 24kDa 밴드를 선별하고 이들을 토끼에 면역하여 항체생성 여부를 ELISA한 결과 non-GM 113kDa과 non-GM 24kDa 단백질 모두 항체가 형성됨을 확인하였고 $10^{-1}-10^{-6}$의 단백질 희석배수에서 두부를 이들 항체에 대하여 ELISA함으로써 원료대두의 GM여부를 확인할 수 있었다. 이들 중, 보다 감도가 높았던 non-GM 113kDa 단백질을 $10^{-7}-10^{-6}$의 배수로 희석하여 ELISA 흡광도와 non-GM 단백질의 관계를 나타내는 표준곡선을 작성하였고, 임의로 non-GM 대두와 GM 대두를 혼합하여 제조한 두부의 ELISA 흡광도를 이 표준곡선과 비교하여 non-GM 원료와 GM 원료 작물의 혼입율을 측정한 결과, 높은 정확도를 보였다.

• Asian-Australasian Journal of Animal Sciences
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• 제19권12호
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• pp.1761-1768
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• 2006

In this study, the comparative effects of transgenic corn (Mon 810 and Event 176) and isogenic corn (DK729) were investigated for their influence on in vitro rumen fermentation. This study consisted of three treatments with 0.25 g rice straw, 0.25 g of corn (Mon810/Event176/DK 729) mixed with 30 ml rumen fluid-basal medium in a serum bottle. They were prepared in oxygen free conditions and incubated at $39^{\circ}C$ in a shaking incubator. The influence of transgenic corn on the number of bacterial population, F. succinogenes (cellulolytic) and S. bovis (amylolytic), was quantified using RT-PCR. Fermentative parameters were measured at 0, 2, 4, 8, 12 and 24 h and substrate digestibility was measured at 12 and 24 h. No significant differences were observed in digestibility of dry matter, NDF, ADF at 12 and 24 h for both transgenic and isogenic form of corns (p>0.05) as well as in fermentative parameters. Fluid pH remained unaffected by hybrid trait and decreased with VFA accumulation as incubation time progressed. No influence of corn trait itself was seen on concentration of total VFA, acetic, propionic, butyric and valeric acids. There were no significant differences (p<0.05) in total gas production, composition of gas (methane and hydrogen) at all times of sampling, as well as in NH3-N production. Bacterial quantification using RT-PCR showed that the population number was not affected by transgenic corn. From this study it is concluded that transgenic corn (Mon810 and Event 176) had no adverse effects on rumen fermentation and digestibility compared to isogenic corn. However, regular monitoring of these transgenic feeds is needed by present day researchers to enable consumers with the option to select their preferred food source for animal or human consumption.