Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods

Mayer, Zsuzsanna; Bagnara, A.; Färber, Paul Lawrence; Geisen, Rolf

doi:10.1016/s0168-1605(02)00250-7

Cited by 129 publications

(69 citation statements)

References 20 publications

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“…Although the actual relationship between the copy number of a particular gene and CFU depends on the growth stage of the cells, our findings suggest that an accurate correlation of gene copy number to CFU is possible. In addition, several previous studies have developed that correlation for different matrices (12,18,24).…”

Section: Discussionmentioning

confidence: 99%

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Taşkın

Gözen

Duran

2011

Appl Environ Microbiol

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Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with Escherichia coli ATCC 25922 as the model organism and the uidA gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg ⅐ liter ؊1 .

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Section: Discussionmentioning

confidence: 99%

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Taşkın

Gözen

Duran

2011

Appl Environ Microbiol

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“…Real-time PCR can be employed for quantifying both DNA and RNA abundance. It is used in a huge number of research applications such as monitoring of viral load in infected plants [9,10] or detection of genes involved in toxinogenesis [11,12]. Today, in response to compulsory requirements for food quality and safety, real-time PCR is also being employed in testing food adulteration or contamination with transgenic DNA [13].…”

Section: Introductionmentioning

confidence: 99%

Real-time PCR for the detection of precise transgene copy number in durum wheat

Gadaleta

Giancaspro

Cardone

et al. 2011

Cellular and Molecular Biology Letters

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Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

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“…From the viewpoint of human health, in particular food is a very suitable and potentially high-risk substrate for the colonisation, growth, and reproduction of toxigenic micromycetes and, consequently, for the production of mycotoxins. Aflatoxins are secondary metabolites produced especially by A. parasiticus and A. flavus (Bennett & Papa 1988;Mayer et al 2003) strains, and also by A. nomius and A. tamarii (Goto et al 1996). They can be found in various kinds of food and feedstuffs such as barley, wheat flour, maize, cereals (Villa & Markaki 2009), rice, beans, nuts and nut products, spices, and beer (Yang et al 2004).…”

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confidence: 99%

Comparison of methods for isolating fungal DNA

Moťková¹,

Vytřasová²

2011

Czech J. Food Sci.

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Moťková P., Vytřasová J. (2011): Comparison of methods for isolating fungal DNA. Czech J. Food Sci., 29 (Special Issue): S76-S85.In this study methods of fungal DNA isolation were optimised and compared. The aim of the isolation processes was to obtain DNA of sufficient quality and quantity necessary for its amplification, as most detection techniques require DNA amplification before the proper DNA detection itself. For this purpose, classic methods of DNA extraction were compared and optimised while isolations using commercial kits were also done. The methods were evaluated from several perspectives, with focus especially laid on the isolated DNA not contain PCR inhibitors which would prevent DNA amplification, thus inhibiting the detection itself. For optimising the individual methods, collection strains of the genus Aspergillus were used. After the evaluation, two most suitable methods were selected and chosen for isolating potentially aflatoxigenic moulds taken from food samples. These methods were the commercially supplied kit for isolating DNA from plant leaves from Sigma and a classic method according Cenis in combination with the cell wall disruption by means of liquid nitrogen.

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Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods

Cited by 129 publications

References 20 publications

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

Real-time PCR for the detection of precise transgene copy number in durum wheat

Comparison of methods for isolating fungal DNA

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