The Experts below are selected from a list of 1194 Experts worldwide ranked by ideXlab platform
Rosa Aznar - One of the best experts on this subject based on the ideXlab platform.
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application of Propidium Monoazide quantitative pcr for selective detection of live escherichia coli o157 h7 in vegetables after inactivation by essential oils
International Journal of Food Microbiology, 2012Co-Authors: Patricia Elizaquivel, Rosa Aznar, Gloria SánchezAbstract:The use of Propidium Monoazide (PMA) is enjoying increased popularity among researchers in different fields of microbiology. Its use in combination with real-time PCR (qPCR) represents one of the most successful approaches to detect viable cells. PMA-qPCR has successfully been used to evaluate the efficacy of various disinfection technologies in different microorganisms. Initially, in this study the effect of four essential oils (EOs), cumin, clove, oregano and cinnamon, was evaluated on suspensions of the enterohemorrhagic Escherichia coli O157:H7 by PMA-qPCR, LIVE/DEAD BacLight flow cytometry analysis (LIVE/DEAD-FCM), and plate count. E. coli O157:H7 cells treated with EOs at killing concentrations were permeable to PMA which was confirmed by LIVE/DEAD-FCM. However, the PMA-qPCR assay allows specific quantification among the autochthonous microbiota of food products. Therefore, the PMA-qPCR assay was used to evaluate its applicability in artificially contaminated iceberg lettuce and soya sprouts. Amplification signal was negative for the spiking tests performed with any of the EO-killed E. coli cells. It demonstrates that the PMA-qPCR assay is a suitable technique for monitoring E. coli O157:H7 inactivation by essential oils in fresh-cut vegetables.
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Discrimination of Infectious Hepatitis A Viruses by Propidium Monoazide Real-Time RT-PCR
Food and Environmental Virology, 2012Co-Authors: Gloria Sánchez, Patricia Elizaquivel, Rosa AznarAbstract:The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, Propidium Monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log_10 units. Results showed that combining 50 μM of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity.
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Application of Propidium Monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water.
Food Microbiology, 2011Co-Authors: Patricia Elizaquivel, MarÍa Victoria Selma, G. Sánchez, Rosa AznarAbstract:Abstract The efficacy of sanitizing technologies in produce or in vegetable wash water is generally evaluated by plate count in selective media. This procedure is time consuming and can lead to misinterpretations because environmental conditions and sanitizing processes may affect bacterial growth or culturable capability. Thus, the aim of this study was to determine the applicability of a Propidium Monoazide real-time PCR (PMA-qPCR) method to monitor the inactivation by ultrasound treatment of foodborne bacteria in fresh-cut vegetable wash water. To this aim, lettuce wash water was artificially inoculated with Escherichia coli O157:H7 (10 6 CFU/mL) and treated by means of a continuous ultrasonic irradiation with a power density of 0.280 kW/L. Quantification data obtained by PMA-qPCR and plate counts were statistically similar during the viability reduction of 99.996% which corresponds to 4.4 log reductions. Further reductions of E. coli O157:H7 were not detected by the PMA-qPCR method due to the limit of detection of this technique (20 CFU/mL). Inactivation data obtained by both techniques successfully fitted a linear model, giving no significant differences in kinetic parameters. These results indicate that the PMA-qPCR method is a suitable technique for evaluating ultrasonic disinfection of vegetable wash water, being able to distinguish between live and dead bacteria.
C Zotti - One of the best experts on this subject based on the ideXlab platform.
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reduction of turnaround time for non tuberculous mycobacteria detection in heater cooler units by Propidium Monoazide real time polymerase chain reaction
Journal of Hospital Infection, 2020Co-Authors: Savina Ditommaso, Monica Giacomuzzi, Gabriele Memoli, Rossana Cavallo, Antonio Curtoni, Maria Avolio, Carlo Silvestre, C ZottiAbstract:SUMMARY Background Invasive non-tuberculous mycobacteria (NTM) infections are emerging worldwide in patients undergoing open-chest cardiac bypass surgery exposed to contaminated heater–cooler units (HCUs). Although this outbreak has been investigated by culturing bacteria isolated from HCU aerosol and water samples, these conventional methods have low-analytic sensitivity, high rates of sample contamination, and long turnaround time. Aim To develop a simple and effective method to detect NTM in HCUs by real-time polymerase chain reaction (PCR), with a short laboratory turnaround time and reliable culture results. Methods A total of 281 water samples collected from various HCUs at seven Italian hospitals were simultaneously screened for NTM by a Propidium Monoazide (PMA)–PCR assay and by conventional culture testing. The results were analysed with culture testing as the reference method. Findings (i) The agreement between culture testing and PMA–PCR was 85.0% with a cycle threshold (CT) cut-off value of Conclusion The use of PMA–PCR for fast detection of NTM from environmental samples is highly recommended in order to ascertain whether HCUs may represent a potential source of human exposure to NTM. This reliable and simple method reduces laboratory turnaround time compared to conventional methods (one to two days vs eight weeks, respectively), thereby improving control strategies and effective management of HCUs.
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overestimation of the legionella spp load in environmental samples by quantitative real time pcr pretreatment with Propidium Monoazide as a tool for the assessment of an association between legionella concentration and sanitary risk
Diagnostic Microbiology and Infectious Disease, 2014Co-Authors: Savina Ditommaso, Elisa Ricciardi, Monica Giacomuzzi, Susan Arauco R Rivera, Adriano Ceccarelli, C ZottiAbstract:Abstract Quantitative polymerase chain reaction (qPCR) offers rapid, sensitive, and specific detection of Legionella in environmental water samples. In this study, qPCR and qPCR combined with Propidium Monoazide (PMA-qPCR) were both applied to hot-water system samples and compared to traditional culture techniques. In addition, we evaluated the ability of PMA-qPCR to monitor the efficacy of different disinfection strategies. Comparison between the quantification obtained by culture and by qPCR or PMA-qPCR on environmental water samples confirms that the concentration of Legionella estimated by GU/L is generally higher than that estimated in CFU/L. Our results on 57 hot-water-system samples collected from 3 different sites show that: i) qPCR results were on average 178-fold higher than the culture results (Δ log 10 =2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (Δ log 10 =1.43), iii) Propidium Monoazide–induced signal reduction in qPCR were nearly 10-fold (Δ log 10 =0.95), and that iv) different degrees of correlations between the 3 methods might be explained by different matrix properties, but also by different disinfection methods affecting cultivability of Legionella . In our study, we calculated the logarithmic differences between the results obtained by PMA-qPCR and those obtained by culture, and we suggested an algorithm for the interpretation of PMA-qPCR results for the routine monitoring of healthcare water systems using a commercial qPCR system (iQ-check real-time PCR kit; Bio-Rad, Marnes-la-Coquette, France).
Azlin Mustapha - One of the best experts on this subject based on the ideXlab platform.
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detection of viable escherichia coli in environmental water using combined Propidium Monoazide staining and quantitative pcr
Water Research, 2018Co-Authors: Yuan Yuan, Guolu Zheng, Mengshi Lin, Azlin MustaphaAbstract:Abstract The objectives of this study were to specifically detect viable Escherichia coli in environmental waters by targeting the ycjM gene in a Propidium Monoazide (PMA)-qPCR assay. PMA is a viability dye that can inhibit the amplification of DNA from dead cells, thus allowing for the detection and quantification of only viable cells. The ycjM primers were used to target E. coli that directly originated from the feces of warm blooded animals, and avoid false positive detection caused by “naturalized” E. coli that can exist in the environment. In this study, tap water and environmental waters were inoculated with E. coli isolated from animal feces. Following cell collection, samples were treated with PMA, followed by DNA isolation and qPCR detection. For pure cultures, 5 μM PMA with a 10-min light exposure was efficient at inhibiting the amplification of DNA from 105 CFU/mL dead E. coli cells, with a detection limit of 102 CFU/100 mL viable cells. For tap and environmental waters collected in the winter, a 10 μM PMA was required and as low as 103 CFU/100 mL viable cells could be detected in the presence of 105 CFU/100 mL dead cells. For water samples collected during the summer, 102 CFU/10 mL viable cells could be detected in the presence of 104 CFU/10 mL dead cells, after a 20 μM PMA treatment. No significant differences were found among the PMA-qPCR assay and two other standard culture-based methods for detection of viable E. coli in environmental water. In conclusion, with proper pretreatment of environmental water samples, this PMA-qPCR assay that targets the ycjM gene could quantify viable E. coli cells that directly come from the feces of warm-blooded animals, and therefore effectively and accurately indicate the quality of environmental water.
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Detection of Viable but Nonculturable Escherichia coli O157:H7 in Ground Beef by Propidium Monoazide real-time PCR
International Journal of Agricultural Science and Food Technology - Peertechz Publications, 2017Co-Authors: Jehan Mahmoud Mahmoud Ouf, Yuan Yuan, Prashant Singh, Azlin MustaphaAbstract:Escherichia coli O157:H7 can enter into a viable but nonculturable (VBNC) state under stress conditions. Pathogens in this dormant state may escape detection if conventional methods are employed, and potentially pose serious threats to human health. Studies have shown that many intervention and preservation processes that are commonly used in the food industry may instead induce a VBNC state rather than kill the intended pathogens. This study aimed to detect whether E. coli O157:H7, an important and dangerous foodborne pathogen, could adapt to the stress caused by lactic acid exposure by entering the VBNC state. A Propidium Monoazide (PMA) quantitative PCR (qPCR) method was used for detection and quantifi cation of VBNC E. coli O157:H7 cells. The performance of this PMA-qPCR method was assessed using pure culture and ground beef samples inoculated with VBNC E. coli O157:H7 cells. The applied assay could detect as low as 103 CFU/mL VBNC E. coli O157:H7 in pure culture and 4 × 104 CFU/g VBNC cells in ground beef. Results indicate that PMA qPCR could accurately quantify E. coli O157:H7 in a VBNC state.
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detection of viable escherichia coli o157 h7 in ground beef by Propidium Monoazide real time pcr
International Journal of Food Microbiology, 2014Co-Authors: Yarui Liu, Azlin MustaphaAbstract:Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines Propidium Monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 10(2) CFU/mL viable E. coli O157:H7 in pure culture and 10(5) CFU/g in ground beef, in the presence of 10(6)/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 10(6) dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells.
Francesc Codony - One of the best experts on this subject based on the ideXlab platform.
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discrimination of viable acanthamoeba castellani trophozoites and cysts by Propidium Monoazide real time polymerase chain reaction
Journal of Eukaryotic Microbiology, 2011Co-Authors: Mariana Fittipaldi, Gemma Agusti, Barbara Adrados, Jordi Morato, Nancy Pino J Rodriguez, Gustavo A Penuela, Francesc CodonyAbstract:Even though the advent of quantitative polymerase chain reaction (PCR) has improved the detection of pathogen microorganisms in most of areas of microbiology, a serious limitation of this method may arise from the inability to discriminate between viable and nonviable pathogens. To overcome it, the use of real-time PCR and selective nucleic acid intercalating dyes like Propidium Monoazide (PMA) have been effectively evaluated for different microorganisms. To assess whether PMA pretreatment can inhibit PCR amplification of nonviable amoeba DNA, Acanthamoeba castellani survival was measured using cell culture and real-time PCR with and without PMA pretreatment. Autoclave and contact lens disinfecting solutions were used to inactivate amoebae. After these inactivation treatments, the results indicated that the PMA pretreatment approach is appropriate for differentiating viable A. castellani, both trophozoites and cysts. Therefore, the PMA-PCR approach could be useful as a rapid and sensitive analytical tool for monitoring treatment and disease control, assessing effective disinfection treatments, and for a more reliable understanding of the factors that contribute to the interaction amoeba– pathogenic bacteria.
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viability determination of helicobacter pylori using Propidium Monoazide quantitative pcr
Helicobacter, 2010Co-Authors: Gemma Agusti, Francesc Codony, Mariana Fittipaldi, Barbara Adrados, Jordi MoratoAbstract:Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with Propidium Monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms. Materials and Methods: Viability, morphological distribution, and the number of live H. pylori cells were determined using a Propidium Monoazide-based quantitative PCR method, at various time points. Results: Under adverse environmental conditions was observed the conversion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with Propidium Monoazide, over the time. Conclusions: Incorporation of Propidium Monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with Propidium Monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.
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discrimination of infectious bacteriophage t4 virus by Propidium Monoazide real time pcr
Journal of Virological Methods, 2010Co-Authors: Mariana Fittipaldi, Francesc Codony, Barbara Adrados, Nancy Pino J Rodriguez, Gustavo A Penuela, Jordi MoratoAbstract:The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether Propidium Monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.
Patricia Elizaquivel - One of the best experts on this subject based on the ideXlab platform.
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application of Propidium Monoazide quantitative pcr for selective detection of live escherichia coli o157 h7 in vegetables after inactivation by essential oils
International Journal of Food Microbiology, 2012Co-Authors: Patricia Elizaquivel, Rosa Aznar, Gloria SánchezAbstract:The use of Propidium Monoazide (PMA) is enjoying increased popularity among researchers in different fields of microbiology. Its use in combination with real-time PCR (qPCR) represents one of the most successful approaches to detect viable cells. PMA-qPCR has successfully been used to evaluate the efficacy of various disinfection technologies in different microorganisms. Initially, in this study the effect of four essential oils (EOs), cumin, clove, oregano and cinnamon, was evaluated on suspensions of the enterohemorrhagic Escherichia coli O157:H7 by PMA-qPCR, LIVE/DEAD BacLight flow cytometry analysis (LIVE/DEAD-FCM), and plate count. E. coli O157:H7 cells treated with EOs at killing concentrations were permeable to PMA which was confirmed by LIVE/DEAD-FCM. However, the PMA-qPCR assay allows specific quantification among the autochthonous microbiota of food products. Therefore, the PMA-qPCR assay was used to evaluate its applicability in artificially contaminated iceberg lettuce and soya sprouts. Amplification signal was negative for the spiking tests performed with any of the EO-killed E. coli cells. It demonstrates that the PMA-qPCR assay is a suitable technique for monitoring E. coli O157:H7 inactivation by essential oils in fresh-cut vegetables.
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Discrimination of Infectious Hepatitis A Viruses by Propidium Monoazide Real-Time RT-PCR
Food and Environmental Virology, 2012Co-Authors: Gloria Sánchez, Patricia Elizaquivel, Rosa AznarAbstract:The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, Propidium Monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log_10 units. Results showed that combining 50 μM of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity.
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Application of Propidium Monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water.
Food Microbiology, 2011Co-Authors: Patricia Elizaquivel, MarÍa Victoria Selma, G. Sánchez, Rosa AznarAbstract:Abstract The efficacy of sanitizing technologies in produce or in vegetable wash water is generally evaluated by plate count in selective media. This procedure is time consuming and can lead to misinterpretations because environmental conditions and sanitizing processes may affect bacterial growth or culturable capability. Thus, the aim of this study was to determine the applicability of a Propidium Monoazide real-time PCR (PMA-qPCR) method to monitor the inactivation by ultrasound treatment of foodborne bacteria in fresh-cut vegetable wash water. To this aim, lettuce wash water was artificially inoculated with Escherichia coli O157:H7 (10 6 CFU/mL) and treated by means of a continuous ultrasonic irradiation with a power density of 0.280 kW/L. Quantification data obtained by PMA-qPCR and plate counts were statistically similar during the viability reduction of 99.996% which corresponds to 4.4 log reductions. Further reductions of E. coli O157:H7 were not detected by the PMA-qPCR method due to the limit of detection of this technique (20 CFU/mL). Inactivation data obtained by both techniques successfully fitted a linear model, giving no significant differences in kinetic parameters. These results indicate that the PMA-qPCR method is a suitable technique for evaluating ultrasonic disinfection of vegetable wash water, being able to distinguish between live and dead bacteria.
