Papers by Stelios Spaniolas
Journal of Agricultural and Food Chemistry, 2012
This paper reports DNA-based food authenticity assays, in which species identification is accompl... more This paper reports DNA-based food authenticity assays, in which species identification is accomplished by the naked eye without the need of specialized instruments. Strongly colored nanoparticles (gold nanoparticles) are employed as reporters that enable visual detection. Furthermore, detection is performed in a low-cost, disposable, dipstick-type device that incorporates the required reagents in dry form, thereby avoiding multiple pipetting and incubation steps. Due to its simplicity, the method does not require highly qualified personnel. The procedure comprises the following steps: (i) PCR amplification of the DNA segment that flanks the unique SNP (species marker); (ii) a 15 min extension reaction in which DNA polymerase extends an allele-specific primer only if it is perfectly complementary with the target sequence; (iii) detection of the products of the extension reaction within a few minutes by the naked eye employing the dipstick. No purification is required prior to application of the extension products to the dipstick. The method is general and requires only a unique DNA sequence for species discrimination. The only instrument needed is a conventional thermocycler for PCR, which is common equipment in every DNA laboratory. As a model, the method was applied to the discrimination of Coffea robusta and arabica species in coffee authenticity assessment. As low as 5% of Robusta coffee can be detected in the presence of Arabica coffee.
Foodborne Pathogens and Disease
Products from Olive Tree, 2016
Journal of AOAC International
Recently, DNA-based authentication methods were developed to serve as complementary approaches to... more Recently, DNA-based authentication methods were developed to serve as complementary approaches to analytical chemistry techniques. The single nucleotide polymorphism (SNP)-based reaction chemistries, when combined with the existing detection methods, could result in numerous analytical approaches, all with particular advantages and disadvantages. The dual aim of this study was (a) to develop SNP-based analytical assays such as the single-base primer extension (SNaPShot) and pyrosequencing in order to differentiate Arabica and Robusta varieties for the authentication of coffee beans and (b) to compare the performances of SNaPshot, pyrosequencing and the previously developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using an Agilent 2100 Bioanalyzer on the basis of linearity (R2) and LOD, expressed as percentage of the adulterant species, using green coffee beans (Arabica and Robusta) as a food model. The results showed that SNaPshot analysis exhibi...
Journal of Applied Microbiology, 2003
Aims: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo wi... more Aims: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. Methods and Results: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20 000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ b-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. Conclusions: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. Significance and Impact of the Study: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.
Journal of Agricultural and Food Chemistry, 2012
A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SN... more A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL(UAA)-trnF(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca 2+ . Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation.
Journal of Agricultural and Food Chemistry, 2008
In the present study, we developed a cleaved amplified polymorphic sequence (CAPS)-based assay as... more In the present study, we developed a cleaved amplified polymorphic sequence (CAPS)-based assay as a first attempt to detect fraud in grapevine musts with a long-term objective to establish an analytical methodology to authenticate wines of Nemea denomination of origin (Agiorgitiko). The analytical assay makes use of a single nucleotide polymorphism that discriminates Agiorgitiko and Cabernet Sauvignon varieties. The latter grape variety is one of the major adulterants for Nemea wines. Agiorgitiko grapevine must was spiked with Cabernet Sauvignon in several ratios (v/v) from 50 down to 10%, and the subsequent mixes were subjected to alcoholic microfermentation. DNA was extracted from all mixture samples up to the end of the fermentation process and was subjected to the CAPS assay. Both standard agarose gel and lab-on-a-chip capillary electrophoresis illustrated the ability of the method to detect the presence of Cabernet Sauvignon down to 10% throughout the whole fermentation process.
Journal of Agricultural and Food Chemistry, 2008
Methods to discriminate plant oils facilitate the detection of either deliberate or accidental ad... more Methods to discriminate plant oils facilitate the detection of either deliberate or accidental adulteration. To this direction, the variability in length among plant species of the chloroplast trnL intron was exploited for the authentication of edible and cosmetic plant oils, with an extra emphasis on olive oil. The methodology was based on the combinatorial use of a PCR assay with a capillary electrophoresis system such as the lab-on-a-chip technology. Application of the assay on DNA extracted from different oil producing plant species, including olive oil and sesame oil, indicated the ability of the trnL intron to be used as an analytical target. Furthermore, this assay could be used for the detection of adulteration of olive oil with various other plant oils, with the exception of avocado and sesame oil.
Journal of Agricultural and Food Chemistry, 2006
Coffee is one of the most important world food commodities, commercial trade consisting almost en... more Coffee is one of the most important world food commodities, commercial trade consisting almost entirely of Arabica and Robusta varieties. The former is considered to be of superior quality and thus attracts a premium price. Methods to differentiate these coffee species could prove to be beneficial for the detection of either deliberate or accidental adulteration. This study describes a molecular genetics approach to differentiate Arabica and Robusta coffee beans. This employs a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism to monitor a single nucleotide polymorphism within the chloroplastic genome. Samples were analyzed with a lab-on-a-chip capillary electrophoresis system. Coffee powder mixtures were analyzed with this technique, displaying a 5% limit of detection. The plastid copy number was found to be relatively constant across a wide range of bean samples, suggesting that this methodology can also be employed for the quantification of any adulteration of Arabica with Robusta beans.
Food Control, 2008
This paper describes a generic approach to the evaluation of DNA extraction methodology using gre... more This paper describes a generic approach to the evaluation of DNA extraction methodology using green and roasted coffee beans as a model commodity. The use of Design-Expert Ò software was used in the design of experimental work to compare and optimize yields using a variety of commercial DNA extraction kits. The quality of the extracted DNA in terms of PCR inhibitor content is assessed and a judgment made that GeneSpin represents perhaps the best methodology in this instance. Coffee is a major commercial crop and there is the potential for fraudulent adulteration of the more expensive Arabica with Robusta beans. It is demonstrated that the DNA extracted from both green and roasted beans could be used in a PCR-RFLP based analysis to differentiate between Arabica and Robusta types of coffee.
Food Chemistry, 2012
Authenticity and traceability of high quality monovarietal extra virgin olive oils is a major con... more Authenticity and traceability of high quality monovarietal extra virgin olive oils is a major concern for markets and consumers. Although analytical chemistry techniques are widely used to satisfy these needs recently developed DNA-based methods can serve as complementary approaches. A SNP database comprising 10 Greek olive varieties was constructed and five SNPs, residing in restriction sites, were selected for the development of a PCR-RFLP capillary electrophoresis method to discriminate these varieties using leaf DNA as template. An identification key was constructed indicating that five SNPs were adequate to discriminate nine out of the 10 varieties. As a proof of principle the assay was applied on DNA extracted from five of their corresponding monovarietal olive oils. Three SNPs were able to identify the varietal origin of these olive oils confirming the validity of this approach.
Food Chemistry, 2010
ABSTRACT This work describes a methodology for the identification of the botanical origin of plan... more ABSTRACT This work describes a methodology for the identification of the botanical origin of plant oils emphasising on the detection of adulteration of olive oil with sesame oil. This methodology comprises a PCR-based assay that exploits the polymorphisms found in the plastid genome combined with a capillary electrophoresis system to discriminate a range of 11 plant species commonly used for oil extraction on the basis of differential length of their corresponding PCR amplicons. The assay takes advantage of universal primers that amplify a region from the trnL (UAA) intron of every plant species. The results showed that amplicons from all species can be accurately discriminated apart from olive and avocado. Single-base primer extension was then proposed as an additional methodology to discriminate the two species and to confirm the results of the former approach. These assays were successfully applied on olive and sesame oils, thus confirming the validity of this approach. (C) 2010 Elsevier Ltd. All rights reserved.
European Food Research and Technology, 2008
Filtered olive oil samples spiked with three diVerent concentrations of DNA were stored at 25°C u... more Filtered olive oil samples spiked with three diVerent concentrations of DNA were stored at 25°C under a 12 h photoperiod for up to a year. These samples were used for DNA extraction and PCR ampliWcation of DNA amplicons of 107, 415 and 691 bp length. The ampliWcation signal was gradually decreased with longer storage periods, while the strength of the signal was related to the initial concentration of spiking DNA particularly during longer storage periods. The 107 bp amplicon was the only one successfully ampliWed from all the samples, regardless of both concentration of spiking DNA and storage period. The ampliWcation of 415 and 691 bp amplicons was not successful for samples stored longer than a threshold period of 20 and 10 days, respectively. These results suggest that detection of polymorphic markers requiring DNA templates shorter than 100 bp might have a wider range of applications in DNA Wngerprinting of olive oil. In addition, the DNA extracts were tested for the presence of inhibitors in PCR ampliWcation reactions of yeast DNA amplicons. The inhibitory eVect of olive DNA extracts was partial and gradually increased with the storage period of the olive oil samples used for the DNA extraction.
… of Agricultural and …, Jan 1, 2012
This paper reports DNA-based food authenticity assays, in which species identification is accompl... more This paper reports DNA-based food authenticity assays, in which species identification is accomplished by the naked eye without the need of specialized instruments. Strongly colored nanoparticles (gold nanoparticles) are employed as reporters that enable visual detection. Furthermore, detection is performed in a low-cost, disposable, dipstick-type device that incorporates the required reagents in dry form, thereby avoiding multiple pipetting and incubation steps. Due to its simplicity, the method does not require highly qualified personnel. The procedure comprises the following steps: (i) PCR amplification of the DNA segment that flanks the unique SNP (species marker); (ii) a 15 min extension reaction in which DNA polymerase extends an allele-specific primer only if it is perfectly complementary with the target sequence; (iii) detection of the products of the extension reaction within a few minutes by the naked eye employing the dipstick. No purification is required prior to application of the extension products to the dipstick. The method is general and requires only a unique DNA sequence for species discrimination. The only instrument needed is a conventional thermocycler for PCR, which is common equipment in every DNA laboratory. As a model, the method was applied to the discrimination of Coffea robusta and arabica species in coffee authenticity assessment. As low as 5% of Robusta coffee can be detected in the presence of Arabica coffee.
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Papers by Stelios Spaniolas