Recent advances in quantitative PCR (qPCR) applications in food microbiology.
TL;DR: Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression.
About: This article is published in Food Microbiology.The article was published on 2011-08-01. It has received 378 citations till now.
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TL;DR: The DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses are reviewed and it is highlighted how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.
Abstract: The microbial profile of cheese is a primary determinant of cheese quality. Microorganisms can contribute to aroma and taste defects, form biogenic amines, cause gas and secondary fermentation defects, and can contribute to cheese pinking and mineral deposition issues. These defects may be as a result of seasonality and the variability in the composition of the milk supplied, variations in cheese processing parameters, as well as the nature and number of the non-starter microorganisms which come from the milk or other environmental sources. Such defects can be responsible for production and product recall costs and thus represent a significant economic burden for the dairy industry worldwide. Traditional non-molecular approaches are often considered biased and have inherently slow turnaround times. Molecular techniques can provide early and rapid detection of defects that result from the presence of specific spoilage microbes and, ultimately, assist in enhancing cheese quality and reducing costs. Here we review the DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses and, in the process, highlight how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.
1,437 citations
Cites background or methods from "Recent advances in quantitative PCR..."
...Relative quantification is based on gene expression vs. that of a “housekeeping gene,” a gene that is expressed at ubiquitous levels within the cell (Postollec et al., 2011a)....
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...In contrast, qRT-PCR quantifies the number of specific microorganisms or gene copies present in a sample and represents the “gold standard” in quantifying genes and gene expression (Bustin, 2005; Bustin et al., 2005; Postollec et al., 2011b)....
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...the number of cycles needed to reach saturation and from this the cycle threshold (Ct) is calculated (Bustin, 2005; Kubista et al., 2006; Postollec et al., 2011b)....
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...Fluorescence is then recorded vs. the number of cycles needed to reach saturation and from this the cycle threshold (Ct) is calculated (Bustin, 2005; Kubista et al., 2006; Postollec et al., 2011b)....
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...…or gene copies present in a sample and represents the “gold standard” in quantifying genes and gene expression (Bustin, 2005; Bustin et al., 2005; Postollec et al., 2011b). qPCR is rapid, extremely sensitive and has been applied in food microbiology, genomics, medicine, and environmental…...
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TL;DR: It is important to understand the basic principles of PCR and how its use can be modified to provide for sophisticated analysis of genes and the genome.
538 citations
Cites background from "Recent advances in quantitative PCR..."
...For example, in a study to link distinct taxa within the microbial community to specific metabolic processes, stable isotope probing was combined with qPCR (Postollec et al., 2011; Smith and Osborn, 2009)....
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TL;DR: The workflow of applying culture-independent HTS to food matrices is described, the current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described.
Abstract: Following recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described.
400 citations
TL;DR: In this paper, the authors proposed an operational definition of live probiotic bacteria that includes this range of metabolic states is needed for reliable enumeration, and validated these alternative techniques to strengthen the accuracy and reliability of probiotic strain enumeration.
255 citations
Cites background from "Recent advances in quantitative PCR..."
...Using this technique allows microbial populations to be quantified by measuring the abundance of a target sequence in DNA samples extracted from food products (Postellec et al., 2011)....
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TL;DR: Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course.
Abstract: The composition of the lung microbiome contributes to both health and disease, including obstructive lung disease. Because it has been estimated that over 70% of the bacterial species on body surfaces cannot be cultured by currently available techniques, traditional culture techniques are no longer the gold standard for microbial investigation. Advanced techniques that identify bacterial sequences, including the 16S ribosomal RNA gene, have provided new insights into the depth and breadth of microbiota present both in the diseased and normal lung. In asthma, the composition of the microbiome of the lung and gut during early childhood development may play a key role in the development of asthma, while specific airway microbiota are associated with chronic asthma in adults. Early bacterial stimulation appears to reduce asthma susceptibility by helping the immune system develop lifelong tolerance to innocuous antigens. By contrast, perturbations in the microbiome from antibiotic use may increase the risk for asthma development. In chronic obstructive pulmonary disease, bacterial colonisation has been associated with a chronic bronchitic phenotype, increased risk of exacerbations, and accelerated loss of lung function. In cystic fibrosis, studies utilising culture-independent methods have identified associations between decreased bacterial community diversity and reduced lung function; colonisation with Pseudomonas aeruginosa has been associated with the presence of certain CFTR mutations. Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course. Whether we can manipulate bacterial communities to improve clinical outcomes remains to be seen.
197 citations
References
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TL;DR: The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which opens up the possibility of studying the biological relevance of small expression differences.
Abstract: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
18,261 citations
"Recent advances in quantitative PCR..." refers methods in this paper
...be/genorm) (Vandesompele et al., 2002; Derzelle et al., 2009)....
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...Themost appropriate genes can be selected frommicroarray results using computer tools such as geNorm (http://medgen.ugent. be/genorm) (Vandesompele et al., 2002; Derzelle et al., 2009)....
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TL;DR: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency.
Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.
Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.
Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
12,469 citations
"Recent advances in quantitative PCR..." refers background in this paper
...In spite of the growing use of real-time PCR and of the striking rise in publications on the subject, there is an obvious lack of consensus on how best to perform experiments and interpret data (Bustin et al., 2009)....
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TL;DR: Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays.
Abstract: We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.
6,367 citations
TL;DR: The results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.
Abstract: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.
2,406 citations
"Recent advances in quantitative PCR..." refers methods in this paper
...A RNA integrity number (RIN) can be calculated (Schroeder et al., 2006) to determine suitability of samples for RT-qPCR analysis (Fleige and Pfaffl, 2006)....
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TL;DR: A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed and provides a convenient and high-throughput format for QC RT- PCR.
Abstract: A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.
2,278 citations
"Recent advances in quantitative PCR..." refers methods in this paper
...…food microbiology, essentially two detection chemistries are commonly used: the DNA binding dye assay using SYBR Green as a fluorophore (Wittwer et al., 1997), and the hydrolysis probe method (or 50 nuclease assay) (Gibson et al., 1996) mostly employing the TaqMan probe (Applied Biosystems) assay....
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..., 1997), and the hydrolysis probe method (or 50 nuclease assay) (Gibson et al., 1996) mostly employing the TaqMan probe (Applied Biosystems) assay....
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