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

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.

Keywords: chloramphenicol-resistant; genetically modified microorganisms; propidium monoazide treatment; quantitative PCR; viable cells detection.

Figure 1

DNA standard curves for the antibiotic-resistant gene CmR and taxon-specific endogenous genes dxs and dnaA of E. coli and C. glutamicum, respectively, using single-plex or dual-plex quantitative real-time PCR (qPCR) analysis. E—qPCR efficiency; R²—linear correlation coefficient. (A) CmR standard curve for E. coli; (B) dxs standard curve for E. coli; (C) CmR standard curve for C. glutamicum; (D) dnaA standard curve for C. glutamicum.

Figure 2

Quantitative PCR assays targeting CmR/dxs and CmR/dnaA genes using cell-direct dual-plex and propidium monoazide (PMA)-treated qPCR methods. (A,B) CmR and dxs qPCR assays were used to detect genetically modified E. coli cells in serially diluted cell and PMA-treated cell suspensions, respectively; (C,D) CmR and dnaA qPCR assays were used to detect genetically modified C. glutamicum cells in serially diluted cell and PMA-treated cell suspensions, respectively. Black dots indicate the average Cq values of diluted cell points; red dots indicate the average Cq values of PMA-treated diluted cell points.