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Identifying inhibitors/enhancers of quantitative real‐time PCR in food samples using a newly developed synthetic plasmid
Author(s) -
Sovová Tereza,
Křížová Barbora,
Hodek Jan,
Ovesná Jaroslava
Publication year - 2016
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.7178
Subject(s) - plasmid , plasmid preparation , tartrazine , dna , biology , polymerase chain reaction , real time polymerase chain reaction , microbiology and biotechnology , chromatography , chemistry , biochemistry , gene , pbr322
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. CONCLUSION The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition. © 2015 Society of Chemical Industry