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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.

SUBMITTER: Qin Y

PROVIDER: S-EPMC10743000 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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

Qin Yang Y Qu Bo B Lee Bumkyu B

Genes 20231127 12

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 (qPC ...[more]

PMID: 38136957

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Project description:IntroductionToxigenic Vibrio cholerae serogroup O1 and O139 are the pathogens responsible for the global cholera epidemic. V. cholerae can settle in the water and spread via the fecal-oral route. Rapid and accurate monitoring of live V. cholerae in environmental water has become an important strategy to prevent and control cholera transmission. Conventional plate counting is widely used to detect viable bacteria but requires time and effort.MethodsThis study aims to develop a new assay that combines triplex droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment for quantitatively detecting live V. cholerae O1/O139 and cholera enterotoxin. Specific primers and probes were designed according to the conserved regions of gene rfb O1, rfb O139, and ctxA. The amplification procedures and PMA treatment conditions were optimized. The specificity, sensitivity, and ability of PMA-ddPCR to detect viable bacteria-derived DNA were evaluated in simulated seawater samples.Results and discussionThe results revealed that the optimal primer concentrations of rfb O1, rfb O139, and ctxA were 1 μM, while the concentrations of the three probes were 0.25, 0.25, and 0.4 μM, respectively. The best annealing temperature was 58°C to obtain the most accurate results. The optimal strategy for distinguishing dead and live bacteria from PMA treatment was incubation at the concentration of 20 μM for 15 min, followed by exposure to a 650-W halogen lamp for 20 min. In pure culture solutions, the limit of detection (LODs) of V. cholerae O1 and O139, and ctxA were 127.91, 120.23 CFU/mL, and 1.5 copies/reaction in PMA-triplex ddPCR, respectively, while the LODs of the three targets were 150.66, 147.57 CFU/mL, and 2 copies/reaction in seawater samples. The PMA-ddPCR sensitivity was about 10 times higher than that of PMA-qPCR. When detecting spiked seawater samples with live bacterial concentrations of 1.53 × 102 and 1.53 × 105 CFU/mL, the assay presented a higher sensitivity (100%, 16/16) than qPCR (50.00%, 8/16) and a perfect specificity (100%, 9/9). These results indicate that the developed PMA-triplex ddPCR is superior to the qPCR regarding sensitivity and specificity and can be used to rapidly detect viable toxigenic V. cholerae O1 and O139 in suspicious seawater samples.

Dataset Information

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

Publications

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

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