Identifying inhibitors/enhancers of quantitative real-time PCR in food samples using a newly developed synthetic plasmid

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027006%3A_____%2F16%3A00003433" target="_blank" >RIV/00027006:_____/16:00003433 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1002/jsfa.7178" target="_blank" >http://dx.doi.org/10.1002/jsfa.7178</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/jsfa.7178" target="_blank" >10.1002/jsfa.7178</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Identifying inhibitors/enhancers of quantitative real-time PCR in food samples using a newly developed synthetic plasmid

Popis výsledku v původním jazyce

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. 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. The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition.

Název v anglickém jazyce

Identifying inhibitors/enhancers of quantitative real-time PCR in food samples using a newly developed synthetic plasmid

Popis výsledku anglicky

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. 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. The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

GM - Potravinářství

OECD FORD obor

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Projekt

<a href="/cs/project/QI101B267" target="_blank" >QI101B267: Vývoj a aplikace nových efektivních postupů pro kontrolu kvality produktů zemědělské v řetězci prvovýroba a posouzení bezpečnosti potravin</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2016

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of the Science of Food and Agriculture

ISSN

0022-5142

e-ISSN

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Svazek periodika

96

Číslo periodika v rámci svazku

3

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

5

Strana od-do

997-1001

Kód UT WoS článku

000368850500036

EID výsledku v databázi Scopus

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