Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification

Wu, Guo Ping; Chen, Su Hua; Levin, Robert E.

doi:10.1016/j.mimet.2015.07.012

Cited by 29 publications

(13 citation statements)

References 25 publications

(17 reference statements)

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“…Moreover, a standard curve with a high correlation coefficient was obtained in this study, as shown in Fig. 3B , indicating that except for microorganism quantification 32 33 34 , the RT-LAMP assay can also be applied in human DNA quantification. Therefore, the RT-LAMP method can be used for the determination of trace amounts of DNA of interest among copious background DNA, such as specific mutation detection in circulating tumour DNA 24 35 , and can be applied for complex gene quantification, which is clinically meaningful 36 37 .…”

Section: Discussionsupporting

confidence: 58%

Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

Zhang

Yao²,

Zhu³

et al. 2016

Sci Rep

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Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approximately 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amounts of product and quantitatively detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine.

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Section: Discussionsupporting

confidence: 58%

Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

Zhang

Yao²,

Zhu³

et al. 2016

Sci Rep

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“…Therefore, selecting the right-sized amplicons for qPCR is a key factor to successfully detect viable foodborne pathogens (Li and Chen, 2013). Additionally, in the last few years, several more studies addressed the technical issues with the PMA-qPCR for detection of viable cells of Salmonella in food (Nocker et al, 2009; Liang et al, 2011; Banihashemi et al, 2012; Elizaquivel et al, 2012; Yang Y. et al, 2013; Barbau-Piednoir et al, 2014; Wu et al, 2015). …”

Section: Ema/pma In Differentiation Of Viable Cells Of Salmonella In mentioning

confidence: 99%

“…Wu et al (2015) combined the EMA with LAMP to detect viable cells of Salmonella . They found that the concentration EMA is critical in effecting the DNA amplification of viable cells, i.e., if the EMA concentration used was <8.0 μg/ml, the DNA amplification of viable cell was not affected, otherwise, it would be significantly affected (Wu et al, 2015). …”

Section: Ema/pma In Differentiation Of Viable Cells Of Salmonella In mentioning

confidence: 99%

Advances and Challenges in Viability Detection of Foodborne Pathogens

et al. 2016

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Foodborne outbreaks are a serious public health and food safety concern worldwide. There is a great demand for rapid, sensitive, specific, and accurate methods to detect microbial pathogens in foods. Conventional methods based on cultivation of pathogens have been the gold standard protocols; however, they take up to a week to complete. Molecular assays such as polymerase chain reaction (PCR), sequencing, microarray technologies have been widely used in detection of foodborne pathogens. Among molecular assays, PCR technology [conventional and real-time PCR (qPCR)] is most commonly used in the foodborne pathogen detection because of its high sensitivity and specificity. However, a major drawback of PCR is its inability to differentiate the DNA from dead and viable cells, and this is a critical factor for the food industry, regulatory agencies and the consumer. To remedy this shortcoming, researchers have used biological dyes such as ethidium monoazide and propidium monoazide (PMA) to pretreat samples before DNA extraction to intercalate the DNA of dead cells in food samples, and then proceed with regular DNA preparation and qPCR. By combining PMA treatment with qPCR (PMA-qPCR), scientists have applied this technology to detect viable cells of various bacterial pathogens in foods. The incorporation of PMA into PCR-based assays for viability detection of pathogens in foods has increased significantly in the last decade. On the other hand, some downsides with this approach have been noted, particularly to achieve complete suppression of signal of DNA from the dead cells present in some particular food matrix. Nowadays, there is a tendency of more and more researchers adapting this approach for viability detection; and a few commercial kits based on PMA are available in the market. As time goes on, more scientists apply this approach to a broader range of pathogen detections, this viability approach (PMA or other chemicals such as platinum compound) may eventually become a common methodology for the rapid, sensitive, and accurate detection of foodborne pathogens. In this review, we summarize the development in the field including progress and challenges and give our perspective in this area.

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“…To combat this, one group used PMA with deoxycholate to successfully improve the penetration of PMA into ghost cells (Laidlaw et al ., ), but this method was not tested on natural water sources. Others have shown that VBNC bacteria are poorly detected with PMA, which is problematic because VBNC have the potential to recover and cause illness (Wu et al ., ; Laidlaw et al ., ). Additional problems with using PMA are more easily recognized when the pathogen levels in the sample are very low.…”

Section: Dna Amplification Methodsmentioning

confidence: 99%

New strategies for the enumeration of enteric pathogens in water

Gorski

Rivadeneira

Cooley

2019

Environmental Microbiology Reports

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Water quality standards for drinking water and recreational waters have long been based on the enumeration of faecal coliforms in the various water supplies, with 0 CFU Escherichia coli/100 ml for drinking water and <126 CFU generic E. coli/100 ml for recreational waters. Irrigation water will soon undergo the same scrutiny in the United States. For over 50 years the most probable number method has been used by laboratories to estimate the level of viable bacteria in a sample, but this method is labour intensive and slow, especially if large numbers of samples need to be tested. In this review, we describe some recent innovations in methods to enumerate enteric pathogens in water. These methods are based on different reasoning schemes that can be categorized as biosensors and nucleic acid-based methods. All the methods described here used natural water sources. Several were also used to survey the bacterial levels in naturally contaminated samples. The different methods vary in their limits of detection, ease of use, and potential portability. Some combine very good limits of detection with the ability to overcome technical challenges; however, there is considerable room for improvement, as none of the methods are without shortcomings.

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Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification

Cited by 29 publications

References 25 publications

Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms

Advances and Challenges in Viability Detection of Foodborne Pathogens

New strategies for the enumeration of enteric pathogens in water

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