Ethidium and propidium monoazide: comparison of potential toxicity on<i>Vibrio</i>sp. viability

Copin, S.; Mougin, Julia; Raguenet, Virginie; Robert-Pillot, Annick; Midelet, Graziella; Grard, Thierry; Bonnin-Jusserand, Maryse

doi:10.1111/lam.13412

Cited by 3 publications

(3 citation statements)

References 27 publications

(33 reference statements)

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“…Currently, PMA dye combined with fluorescent qPCR (PMA-qPCR) is widely used as a rapid detection method in food, medicine, and the environment. It detects not only bacteria, such as E. coli ( Miotto et al, 2020 ; Deshmukh et al, 2021 ), Salmonella ( Techathuvanan and D'Souza, 2020 ), Lactobacillus ( Yang et al, 2021 ), Vibrio ( Copin et al, 2021 ), and Listeria monocytogenes ( Kragh et al, 2020 ), but also fungi, such as C.albicans ( Asadzadeh et al, 2018 ), and even virus ( Zeng et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

A new method for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants based on Propidium Monoazide combined with real-time PCR

et al. 2022

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Rapid detection of antibacterial and bacteriostatic properties is an important part of the quality and safety supervision of disinfectants. In this study, propidium monoazide (PMA) was used in combination with real-time PCR (PMA-qPCR) to detect the antibacterial and bacteriostatic activity of disinfectants against three commonly used indicator bacteria, Escherichia coli, Staphylococcus aureus, and Candida albicans, utilizing specifically designed primers. The method for preparing membrane-damaged bacteria was optimized to improve the ability of the PMA dye to distinguish between live and dead indicator bacteria. Finally, this method could simultaneously detect viable numbers of the indicator bacteria after the disinfectants were used. The R2 values of the PMA-qPCR standard curves were 0.9986, 0.9980, and 0.9962 for E. coli, S. aureus, and C. albicans, respectively, and the detection range was 103 ~ 106 CFU/ml, showing no significant difference in accuracy compared to that of the plate counting method (p > 0.05). The method established here is the first application of PMA-qPCR to detect the antibacterial and bacteriostatic activity of disinfectants. This technique markedly simplifies the detection steps of antibacterial and bacteriostatic activity, reduces the detection time (3 h compared to 48 ~ 72 h for the plate counting method), improves the quality supervision efficiency of disinfectants, and guarantees healthy and safe lives.

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Section: Introductionmentioning

confidence: 99%

A new method for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants based on Propidium Monoazide combined with real-time PCR

et al. 2022

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“…The traditional pathogenic culture method and immunoassay method are cumbersome, unreliable, and prone to false-negative results, especially in distinguishing homologous strains and VBNC cells. It is necessary to find more accurate detection techniques for V. vulnificus detection (Copin et al, 2021). In recent years, with the development of heat-resistant DNA polymerases (e.g., Taq DNA polymerase) and thermal cycling instruments, molecular biology techniques represented by polymerase chain reaction (PCR) have gradually advanced from the research laboratories.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, this bottleneck could be broken by combining photo-reactive DNA-binding dye, such as propidium monoazide (PMA) and ethidium monoazide (EMA), with nucleic acid molecular diagnostics (Codony et al, 2020). A previous study revealed (Copin et al, 2021) that pre-treatment with different concentration gradients of EMA in seafood and environmental samples had a lethal effect on V. vulnificus strain compared with PMA. PMA could penetrate into the damaged and dead cells and selectively bind to the DNA in these cells, which can effectively inhibit their PCR amplification (Elizaquível et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

Zhang

et al. 2022

Front. Microbiol.

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Vibrio vulnificus (V. vulnificus) is one of the most common pathogenic Vibrio species to humans; therefore, the establishment of timely and credible detection methods has become an urgent requirement for V. vulnificus illness surveillance. In this study, an assay combining droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment was developed for detecting V. vulnificus. The primers/probes targeting the V. vulnificus hemolysin A (vvhA) gene, amplification procedures, and PMA processing conditions involved in the assay were optimized. Then, we analyzed the specificity, sensitivity, and ability to detect live cell DNA while testing the performance of PMA-ddPCR in clinical samples. The optimal concentrations of primers and probes were 1.0 and 0.3 μM, respectively. The annealing temperature achieving the highest accuracy in ddPCR assay was 60°C. With an initial V. vulnificus cell concentration of 108 CFU/mL (colony-forming units per milliliter), the optimal strategy to distinguish live cells from dead cells was to treat samples with 100 μM PMA for 15 min in the dark and expose them to LED light with an output wavelength of 465 nm for 10 min. The specificity of the PMA-ddPCR assay was tested on 27 strains, including seven V. vulnificus strains and 20 other bacterial strains. Only the seven V. vulnificus strains were observed with positive signals in specificity analysis. Comparative experiments on the detection ability of PMA-ddPCR and PMA-qPCR in pure cultures and plasma samples were performed. The limit of detection (LOD) and the limit of quantitation (LOQ) in pure culture solutions of V. vulnificus were 29.33 and 53.64 CFU/mL in PMA-ddPCR, respectively. For artificially clinical sample tests in PMA-ddPCR, V. vulnificus could be detected at concentrations as low as 65.20 CFU/mL. The sensitivity of the PMA-ddPCR assay was 15- to 40-fold more sensitive than the PMA-qPCR in this study. The PMA-ddPCR assay we developed provides a new insight to accurately detect live cells of V. vulnificus in clinical samples, which is of great significance to enhance public health safety and security capability and improve the emergency response level for V. vulnificus infection.

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Microplastics accumulation in mangroves increasing the resistance of its colonization Vibrio and Shewanella

Tan

Xie

et al. 2022

Chemosphere

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Ethidium and propidium monoazide: comparison of potential toxicity onVibriosp. viability

Cited by 3 publications

References 27 publications

A new method for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants based on Propidium Monoazide combined with real-time PCR

A new method for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants based on Propidium Monoazide combined with real-time PCR

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

Microplastics accumulation in mangroves increasing the resistance of its colonization Vibrio and Shewanella

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