Rapid Monitoring of Viable Genetically Modified Escherichia coli Using a Cell-Direct Quantitative PCR Method Combined with Propidium Monoazide Treatment

Abstract

The commercialization of industrial genetically modified microorganisms (GMMs) has highlighted their impact on public health and the environment. Rapid and effective monitoring methods detecting live GMMs are essential to enhance current safety management protocols. This study aims to develop a novel cell-direct quantitative polymerase chain reaction (qPCR) method targeting two antibiotic-resistant genes, KmR and nptII, conferring resistance against kanamycin and neomycin, along with propidium monoazide, to precisely detect viable Escherichia coli. The E. coli single-copy taxon-specific gene of D-1-deoxyxylulose 5-phosphate synthase (dxs) was used as the internal control. The qPCR assays demonstrated good performance, with dual-plex primer/probe combinations exhibiting specificity, absence of matrix effects, linear dynamic ranges with acceptable amplification efficiencies, and repeatability for DNA, cells, and PMA-treated cells targeting KmR/dxs and nptII/dxs. Following the PMA-qPCR assays, the viable cell counts for KmR-resistant and nptII-resistant E. coli strains exhibited a bias% of 24.09% and 0.49%, respectively, which were within the acceptable limit of ±25%, as specified by the European Network of GMO Laboratories. This method successfully established detection limits of 69 and 67 viable genetically modified E. coli cells targeting KmR and nptII, respectively. This provides a feasible monitoring approach as an alternative to DNA processing techniques to detect viable GMMs.

Propidium Monoazide-Treated, Cell-Direct, Quantitative PCR for Detecting Viable Chloramphenicol-Resistant Escherichia coli and Corynebacterium glutamicum Cells

“…Sung and Hawkins [33] conducted research on a TaqMan-based real-time PCR assay for cell culture medium, eliminating the need for DNA extraction, and successfully detected fewer than 10 CFU of mycoplasma contaminants in mammalian cell cultures. Furthermore, prior studies investigating cell-direct qPCR analysis of Kanamycin-resistant GM E. coli reported comparable results [32]. In contrast to DNA-based techniques, the utilization of TaqMan-based cell-direct dual-plex qPCR in this study demonstrated exceptional effectiveness in accurately detecting and quantifying both chloramphenicol-resistant E. coli and C. glutamicum cells.…”

“…To ensure uniformity, we selected 18 h cell cultures that exceeded the 0.70 threshold for subsequent CFU and qPCR analyses. Moreover, previous research has confirmed that utilizing matrix substances such as LB broth as dilution substrates does not exhibit any noticeable adverse effects on the efficacy of the cell-direct dual-plex qPCR approach when performed on KmR/dxs and nptII/dxs genes [32] S2).…”

“…To tackle this issue, the cell culture was diluted to a range of 10-fold (10 −1 ) to 100,000-fold (10 −5 ), and then each suspension was treated with 20 mM of PMA solution. Moreover, in the qPCR assays, we employed half the concentration of primers and probes to mitigate PCR inhibitions, a strategy that had demonstrated notable efficacy in a prior study [32]. As a result, the performance of qPCR on treated cells at each dilution point can be verified (Figure S2C,D).…”

Legal and Technical Challenges of Developing Robust Traceability Systems for Genetically Modified Organisms

“…4 The existing literature on GMO traceability primarily focuses on regulatory aspects, technical challenges, and the socio-economic implications. Key studies have analyzed the effectiveness of the EU's regulatory framework (Lu et al, 2020;Jimenez et al, 2021), the technical aspects of detection and monitoring (Fraiture et al, 2015;Ovesná, Demnerová, & Pouchová, 2008), and the public perception and acceptance (Qin et al, 2023;Kunyanga Nkirote Catherine et al, 2024). However, there is a notable gap in research that integrates these perspectives to provide a comprehensive analysis of both legal and technical challenges in traceability.…”