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Updated: Jul 7, 2025
Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR
Published on: February 1, 2014
Propidium Monoazide-Treated, Cell-Direct, Quantitative PCR for Detecting Viable Chloramphenicol-Resistant Escherichia
Yang Qin1, Bo Qu1, Bumkyu Lee1
1Department of Environment Science & Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea.
Abstract:
With the rapid development and commercialization of industrial genetically modified microorganisms (GMMs), public concerns regarding their potential effects are on the rise. It is imperative to promptly monitor the unintended release of viable GMMs into wastewater, the air, and the surrounding ecosystems to prevent the risk of horizontal gene transfer to native microorganisms. In this study, we have developed a method that combines propidium monoazide (PMA) with a dual-plex quantitative PCR (qPCR) approach based on TaqMan probes. This method targets the chloramphenicol-resistant gene (CmR) along with the endogenous genes D-1-deoxyxylulose 5-phosphate synthase (dxs) and chromosomal replication initiator protein (dnaA). It allows for the direct quantitative detection of viable genetically modified Escherichia coli and Corynebacterium glutamicum cells, eliminating the requirement for DNA isolation. The dual-plex qPCR targeting CmR/dxs and CmR/dnaA demonstrated excellent performance across various templates, including DNA, cultured cells, and PMA-treated cells. Repeatability and precision, defined as RSDr% and bias%, respectively, were calculated and found to fall within the acceptable limits specified by the European Network of GMO Laboratories (ENGL). Through PMA-qPCR assays, we determined the detection limits for viable chloramphenicol-resistant E. coli and C. glutamicum strains to be 20 and 51 cells, respectively, at a 95% confidence level. Notably, this method demonstrated superior sensitivity compared to Enzyme-Linked Immunosorbent Assay (ELISA), which has a detection limit exceeding 1000 viable cells for both GM bacterial strains. This approach offers the potential to accurately and efficiently detect viable cells of GMMs, providing a time-saving and cost-effective solution.
Insights
A new propidium monoazide quantitative PCR (PMA-qPCR) method accurately detects viable genetically modified microorganisms (GMMs) in environmental samples. This cost-effective approach offers enhanced sensitivity for monitoring GMM release and preventing horizontal gene transfer.
Area of Science:
- Environmental microbiology
- Molecular biology
- Biotechnology safety
Background:
- Rising public concern over genetically modified microorganisms (GMMs) necessitates robust monitoring methods.
- Unintended release of GMMs poses risks, including horizontal gene transfer to native microbial populations.
- Existing detection methods may lack the sensitivity or efficiency required for timely GMM monitoring.
Purpose of the Study:
- To develop and validate a sensitive method for direct quantitative detection of viable GMMs.
- To assess the risk of GMM release into environmental compartments like wastewater.
- To provide a reliable tool for monitoring GMMs, specifically *Escherichia coli* and *Corynebacterium glutamicum*.
Main Methods:
- Propidium monoazide (PMA) treatment to exclude non-viable cells.
- Dual-plex quantitative PCR (qPCR) using TaqMan probes targeting the *CmR* gene and endogenous genes (*dxs*, *dnaA*).
- Validation across various templates (DNA, cultured cells, PMA-treated cells) and assessment of repeatability and precision.
Main Results:
- The PMA-qPCR method accurately quantifies viable genetically modified *E. coli* and *C. glutamicum* without DNA isolation.
- Detection limits were as low as 20 viable cells for *E. coli* and 51 for *C. glutamicum*.
- The method demonstrated significantly higher sensitivity compared to ELISA, with detection limits exceeding 1000 cells for ELISA.
Conclusions:
- PMA-qPCR offers a sensitive, accurate, and efficient method for detecting viable GMMs.
- This approach provides a time-saving and cost-effective solution for environmental monitoring of GMMs.
- The validated method supports regulatory oversight and public safety concerning GMM commercialization.
