Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food

Peano, Clelia; Lesignoli, Francesca; Gullì, Mariolina; Corradini, Roberto; Samson, Maria Cristina; Marchelli, Rosangela; Marmiroli, Nelson

doi:10.1016/j.ab.2005.04.009

Cited by 22 publications

(9 citation statements)

References 17 publications

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“…The approach has been found to be efficient when designed to overlap with the primer‐binding site, thus competitively preventing PCR primers from annealing [von Wintzingerode et al. 1997; Peano et al. 2005; Vestheim & Jarman 2008; Shehzad et al 2012].…”

Section: What Barcode To Choose?mentioning

confidence: 99%

Who is eating what: diet assessment using next generation sequencing

et al. 2011

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The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

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Section: What Barcode To Choose?mentioning

confidence: 99%

Who is eating what: diet assessment using next generation sequencing

et al. 2011

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“…[ 30 ]], environmental microbiology [e.g. [ 31 ]], parasitology [ 32 ], and to detect genetically modified organism content in food [ 33 ], but not to our knowledge in diet studies. However, synthesis time for clamping probes is several weeks and these probes are also quite expensive.…”

Section: Introductionmentioning

confidence: 99%

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

2008

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BackgroundIdentification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.ResultsWe have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.ConclusionWe present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.

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“…The protocol for attaching the PNA probes to the slide was modified from that used for oligonucleotides, with spacers added to distance the features from the slide surface. The combination of this array platform with multiplex PCR appears to represent a reliable analytical means for GMO detection in the food chain (Germini et al 2005, Peano et al 2005b). …”

Section: Genetically Modified Organism Detection In Foodmentioning

confidence: 99%