Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR

Liu, Yarui; Mustapha, Azlin

doi:10.1016/j.ijfoodmicro.2013.10.026

Cited by 61 publications

(65 citation statements)

References 24 publications

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“…According to Spearman's rank correlation, the viable ratios measured through PMA-qPCR significantly correlated with those measured based on the culturable ratios (r D 1; p < 0.01) but not with those measured by qPCR (r D 0.6; p D 0.29). These observations show that qPCR-based viable ratios are higher than those measured by PMA-qPCR or culture assays (Liu and Mustapha 2014). The gradual decline in the viable ratio observed in conjunction with the decreasing controlled viable samples, as measured using qPCR, could be related to membrane protein degradation caused by heating, which in turn, could lead to DNA damage (Masters et al 1998).…”

Section: Comparison Of Culturable or Viable Ratios Asmentioning

confidence: 88%

“…The optimal light exposure time for the PMAqPCR assay varied from those reported in previously published studies. In general, 2-10 min of light exposure has typically been used to detect viable bacteria or viruses (Nocker et al 2006(Nocker et al , 2007Kobayashi et al 2009b;Parshionikar et al 2010;Liu and Mustapha 2014). Therefore, the optimal light exposure time for DNA amplification may vary and depend on the light source and bacterial species used (Liu and Mustapha 2014).…”

Section: Effect Of Light Exposure Times On Pma-qpcrmentioning

confidence: 99%

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Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

Tseng

Hsiao

Chang

et al. 2014

Aerosol Science and Technology

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Airborne Staphylococcus aureus causes a significant proportion of nosocomial infections. The purpose of this study was to combine real-time quantitative polymerase chain reaction with the DNA-binding agent propidium monoazide (PMA-qPCR) to assess exposures to airborne S. aureus. In this work, we generated a S. aureus aerosol and used our assay to detect viable, airborne S. aureus in a study chamber. The biological collection efficiencies of three samplers (the AGI-30 impinger, BioSampler, and Nuclepore filter sampler) were evaluated using the S. aureus aerosols. The effects of storage in collection fluid on S. aureus sampled by the AGI-30 impinger and BioSampler were evaluated. Furthermore, air samples from an intensive care unit (ICU) and a gymnasium (GYM) were subsequently used to test the performance of a BioSampler combined with our PMA-qPCR technique. The BioSampler was more effective than the AGI-30 and Nuclepore filter samplers for preserving the culturability and viability of S. aureus aerosol samples. After sampling by impingement, the loss of viable S. aureus was minimized by treating the cells with PMA prior to storage at ¡20 C and analyzing the samples by qPCR within 3 weeks. In field applications, we noted that traditional culture assays tended to underestimate the viable concentrations of S. aureus by approximately one order of magnitude. Overall, combining qPCR with and without PMA staining may be useful for assessing exposure to airborne S. aureus. However, a complex set of parameters that may affect the efficiency of PMA-qPCR must be taken into account before applying PMA-qPCR to bioaerosol detection.

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Section: Comparison Of Culturable or Viable Ratios Asmentioning

confidence: 88%

Section: Effect Of Light Exposure Times On Pma-qpcrmentioning

confidence: 99%

Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

Tseng

Hsiao

Chang

et al. 2014

Aerosol Science and Technology

View full text Add to dashboard Cite

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“…Thus, the concentration and/or pre-enrichment steps are necessary for most food and water samples to reach the detection limit because food and water usually contain lower level of pathogens; and iv) PCR assays cannot be employed to differentiate between viable and dead bacteria. Due to amplification of DNA from both viable and dead cells within samples, PCR and qPCR assays may lead to overestimation of target cells [53] or may get false-positive results, resulting in unnecessary product recalls and economic losses [54]. Figure 1 shows the presumptive procedure of PCR/qPCR used for detection and identification of Salmonella in food and water [29].…”

Section: Pcr and Quantitative Real-time Pcr (Qpcr)mentioning

confidence: 99%

“…The overestimation may lead to unnecessary product recalls and economic losses [54]. Several methods have been developed for detection of viable foodborne pathogens, including reversetranscriptase PCR (RT-PCR) [94], qPCR assays with biological dyes [33] and commercially available dead/live bacteria viability test kits (Live/Dead ® BacLight ™ Bacterial Viability Kit, Molecular Probes Inc., Eugene, OR).…”

Section: Detection Of Viable Bacteria In Food and Watermentioning

confidence: 99%

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

Hu¹,

Li²

2017

Adv Tech Biol Med

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Salmonella is a leading cause of foodborne diseases. Gastroenteritis caused by nontyphoid Salmonella is still a major infectious disease in the world. About 95% of Salmonella infections are caused by ingestion of contaminated food and water. Rapid, sensitive, and efficient detection and identification of Salmonella from foods and water are critical for minimizing the spread of outbreaks caused by this pathogen. These methods can be applied to track the food source of contamination or by early diagnosis of the infections in a clinical setting. Culture-based methods for detection of Salmonella are laborious and time-consuming, typically taking 5-7 days to obtain a pure culture and serovar identification. In the past few decades, molecular-based technologies have greatly shortened the time for detection and identification of bacterial pathogens from food and water, and drastically increased the specificity and sensitivity of the assays. In this review, we report an update on the development of rapid and efficient methods for the detection and identification of Salmonella in food and water, focusing on the approaches for concentration of Salmonella cells and viable cell detection, as well as the advantages and drawbacks of these methods.

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“…Propidium monoazide (PMA)-real-time PCR based on DNA has been proposed to be a good way to avoid amplification of DNA isolated from dead cells (20). Unfortunately, PMA becomes increasingly toxic at higher concentrations and is very expensive; PMA-real-time PCR methods that include higher PMA concentrations are considered to be counterproductive and cost prohibitive (21).…”

mentioning

confidence: 99%

Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

Liao

Yong

Wang

2017

Appl Environ Microbiol

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This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus. Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log 10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log 10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (Ϫ0.245, Ϫ0.152, and Ϫ0.121 log 10 CFU/day, respectively) were higher than those estimated with the MMs (Ϫ0.134, Ϫ0.0887, and Ϫ0.0732 log 10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs.IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during lowtemperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

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Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR

Cited by 61 publications

References 24 publications

Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures

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