title: Identifying inhibitors/enhancers of quantitative real-time PCR in food samples using a newly developed synthetic plasmid.
abstract: BACKGROUND
  Polymerase chain reaction (PCR) has become a common technique offering fast and sensitive analysis of DNA in food/feed samples. However, many substances, either already present in the sample or introduced during sample processing, inhibit PCR and thus underestimate the DNA content. It is therefore necessary to identify PCR inhibition in order to correctly evaluate the sample.
  
  
  RESULTS
  We designed and validated a synthetic plasmid DNA that can be used to detect and quantify PCR inhibition. The DNA sequence, appropriate primers and probe, were designed in silico, synthesized and the sequence was inserted into a plasmid vector. The performance of the plasmid was verified via calibration curves and by performing the assay in the presence of various DNAs (crops, fungus, bacterium). The detection of PCR inhibition was assessed using six inhibiting substances with different modes of action, substances used in sample processing (EDTA, ethanol, NaCl, SDS) and food additives (sodium glutamate, tartrazine). The plasmid performance proved to be reproducible and there were no interactions with other DNAs. The plasmid was able to identify the presence of the inhibitors in a wide range of concentrations.
  
  
  CONCLUSIONS
  The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition.
authors: Department of Molecular Genetics, Crop Research Institute, 161 06 Prague, Czech Republic.
authors: Tereza
authors: T
authors: Sovová
authors: Department of Molecular Genetics, Crop Research Institute, 161 06 Prague, Czech Republic.
authors: Barbora
authors: B
authors: Křížová
authors: Department of Molecular Genetics, Crop Research Institute, 161 06 Prague, Czech Republic.
authors: Jan
authors: J
authors: Hodek
authors: Department of Molecular Genetics, Crop Research Institute, 161 06 Prague, Czech Republic.
authors: Jaroslava
authors: J
authors: Ovesná
mesh_terms: D017931:DNA Primers; D006801:Humans; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: PCR inhibition; plasmid DNA; food quality; food additives