A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

Kragh, Martin Laage; Thykier, Mikala; Hansen, Lisbeth Truelstrup

doi:10.1016/j.fm.2019.103310

Cited by 19 publications

(10 citation statements)

References 43 publications

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“…In general, PMA concentrations of 50 µM have been reported to be the optimal concentration for the efficient differentiation between live and dead cells without affecting the viability of L. monocytogenes cells (Kragh et al, 2020). However, when complex matrixes, such as PWW, are used, the concentration of PMAxx needs to be optimized to avoid any impact on cell viability.…”

Section: Viability Of L Monocytogenes Cells Using V-qpcr Techniques mentioning

confidence: 99%

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

et al. 2020

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The significance of viable but non-culturable (VBNC) cells in the food industry is not well known, mainly because of the lack of suitable detection methodologies to distinguish them from dead cells. The study aimed at the selection of the method to differentiate dead and VBNC cells of Listeria monocytogenes in process wash water (PWW) from the fruit and vegetable industry. Different methodologies were examined including (i) flow cytometry, (ii) viability quantitative polymerase chain reaction (v-qPCR) using an improved version of the propidium monoazide (PMAxx) dye as DNA amplificatory inhibitor, and (iii) v-qPCR combining ethidium monoazide (EMA) and PMAxx. The results showed that the flow cytometry, although previously recommended, was not a suitable methodology to differentiate between dead and VBNC cells in PWW, probably because of the complex composition of the water, causing interferences and leading to an overestimation of the dead cells. Based on results obtained, the v-qPCR combined with EMA and PMAxx was the most suitable technique for the detection and quantification of VBNC cells in PWW. Concentrations of 10 µM EMA and 75 µM PMAxx incubated at 40 • C for 40 min followed by a 15-min light exposure inhibited most of the qPCR amplification from dead cells. For the first time, this methodology was validated in an industrial processing line for shredded lettuce washed with chlorine (10 mg/L). The analysis of PWW samples allowed the differentiation of dead and VBNC cells. Therefore, this method can be considered as a rapid and reliable one recommended for the detection of VBNC cells in complex water matrixes such as those of the food industry. However, the complete discrimination of dead and VBNC cells was not achieved, which led to a slight overestimation of the percentage of VBNC cells in PWW, mostly, due to the complex composition of this type of water. More studies are needed to determine the significance of VBNC cells in case of potential cross-contamination of fresh produce during washing.

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Section: Viability Of L Monocytogenes Cells Using V-qpcr Techniques mentioning

confidence: 99%

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

et al. 2020

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“…Combining this with qPCR can quickly and accurately determine the number of viable bacteria in the sample. At present, this method has been applied in the detection of several species, such as Listeria monocytogenes and Lacticaseibacillus paracasei ( Scariot et al, 2018 ; Kragh et al, 2020 ). However, current studies have been limited to the determination of the viable number of a single bacterial species in samples by PMA-qPCR.…”

Section: Introductionmentioning

confidence: 99%

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk

Shi

Fan

et al. 2022

Front. Microbiol.

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The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 1011 and 106 CFU/mL, with R2 > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

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“…For limit of detection (LOD) determination, Probit regression model was performed with a 95% confidence interval, setting the limit at the lowest concentration of CFU mL −1 at which 95% of positive samples were detected (Kralik & Ricchi, 2017). Limit of quantification (LOQ) was determined as the lowest concentration within the linear range of the standard curve (Kragh et al., 2020).…”

Section: Methodsmentioning

confidence: 99%

High‐pressure processing treatment of beef burgers: Effect on Escherichia coli O157 inactivation evaluated by plate count and PMA‐qPCR

Rey

Racca

Ribeiro

et al. 2022

Journal of Food Science

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Propidium monoazide coupled to real time PCR (PMA‐qPCR) is a novel methodology proposed for the quantification of viable bacteria in food after microbial inactivation treatments. The aim of this work was to assess the effectiveness of different pressure levels on the lethality of a pool of Escherichia coli O157 strains in beef burgers by plate count and PMA‐qPCR using uidA as target gene. Also, the effect on native microbiota counts, E. coli O157 counts, and physiochemical parameters of beef burgers during storage in refrigeration and frozen conditions were assessed. The treatment at 600 MPa for 5 min was the most lethal and was selected for the evaluation of bacteria behavior under storage conditions. Native microbiota and E. coli O157 were not recovered during refrigerated and frozen storage (4°C for 7 days and −18°C for 35 days). Cooking weight loss, pH, chromatic parameters, and texture were affected by HPP. Practical Application Practical Application: PMA‐qPCR can be used as an alternative to assess microbial inactivation by different high pressure processing (HPP) conditions (pressure level, holding time and temperature) more rapidly than conventional plate counts. In addition, it has the benefit of being able to quantify viable but nonculturable bacteria from contaminated beef burgers after HPP. Moreover, this novel technique generates less pathogenic residues, which minimizes workers' exposure to human biohazards.

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A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

Cited by 19 publications

References 43 publications

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk

High‐pressure processing treatment of beef burgers: Effect on Escherichia coli O157 inactivation evaluated by plate count and PMA‐qPCR

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