Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood

Miotto, Marília; Barretta, Clarissa; Ossai, Sylvia; Silva, Helen Silvestre da; Kist, Airton; Vieira, Cleide Rosana Werneck; Parveen, Salina

doi:10.1016/j.ijfoodmicro.2019.108467

Cited by 17 publications

(12 citation statements)

References 25 publications

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“…Although the manufacturer recommended a PMAxx concentration of 25 μM, some studies have reported that a higher concentration of PMA more strongly inhibits the qPCR signals from dead cells ( Lv et al, 2019 ; Miotto et al, 2020 ). To determine the optimal concentration of PMA, 200 μl each of the viable cell and dead cell suspensions (concentration: 5 × 10 7 CFU/ml for both) was treated with PMA Enhancer and PMAxx at various concentrations (0, 25, 50, and 100 μM) before DNA extraction and qPCR.…”

Section: Resultsmentioning

confidence: 99%

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR

et al. 2022

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Campylobacter spp. are known as important foodborne gastroenteric pathogens worldwide. Campylobacter spp. can exist in a viable but non-culturable (VBNC) state under unsuitable environmental conditions, which is undetectable by conventional culture methods. Quantitative polymerase chain reaction (qPCR) can be used to detect VBNC Campylobacter spp.; however, both viable and dead bacteria are detected during qPCR and are indistinguishable. Propidium monoazide (PMA), which can only enter dead bacterial cells through a damaged cell wall/cell membrane, binds to DNA and inhibits qPCR. PMA treatment has been performed along with qPCR (PMA-qPCR) to detect viable bacteria. However, the efficacy of detection inhibition differed among studies, and PMA can potentially enter living cells after changes in cell membrane permeability. In this study, we optimized the PMA treatment method by conducting it before qPCR. Two-round PMA treatment completely inhibited the qPCR signals from dead cells, whereas single-round PMA treatment failed to facilitate this. An optimized PMA-qPCR method was developed using commercial chicken meat, and VBNC Campylobacter spp., which are undetectable using conventional culture-based methods, were successfully detected. In conclusion, this study presents a novel, efficient PMA treatment method for the detection of viable Campylobacter spp., including VBNC Campylobacter spp., in chicken meat. We believe that this method will aid the reliable risk assessment of commercial chicken meat.

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Section: Resultsmentioning

confidence: 99%

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR

et al. 2022

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“…At all sampling points analyzed, except for time 0, the quantification obtained by PMAxx-qPCR was closer to the results of colony counting, while non-dyed qPCR overestimated the population in all cases. As we have previously noted, this may be due to the ability of PMAxx to bind the DNA from dead cells, impairing its amplification, so that qPCR will mainly amplify and detect the DNA from viable cells [ 38 , 45 , 50 ]. Thus, at the sampling points where no species were detected by colony counting, PMAxx-qPCR results will be more reliable than qPCR ones since they are more accurate for viable cell counting.…”

Section: Discussionmentioning

confidence: 99%

“…However, the most important aspect revealed by the use of this viable dye was the existence of S. cerevisiae and L. thermotolerans in a VBNC state during fermentation. This behavior has been widely described by several authors in different bacteria and is considered as a survival strategy that permits resilience to unfavorable environmental conditions [ 50 , 69 , 70 ]. These cells retain an intact membrane, undamaged DNA and metabolic activity, but they are not culturable using laboratory media [ 71 ].…”

Section: Discussionmentioning

confidence: 99%

“…This means that only DNA from viable cells (with membrane integrity) will be susceptible to being amplified by qPCR [ 38 , 46 , 47 , 48 , 49 ]. Several studies performed in bacteria and parasites showed that PMA is an excellent choice to determine the viability of cells due to its high specificity and sensitivity in different food matrices, such as oysters, meat and wastewater [ 50 , 51 , 52 ]. Indeed, PMA seemed to be more selective for dead cells than EMA [ 53 , 54 ].…”

Section: Introductionmentioning

confidence: 99%

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Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts

Navarro

Torija

Mas

et al. 2020

Foods

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The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.

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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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Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood

Cited by 17 publications

References 25 publications

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR

Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts

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

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