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

Abstract

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

Detection of PCR inhibition in food and feed with a synthetic plasmid

“…A 78-bp long DNA sequence was designed in silico and cloned into a plasmid vector. The design, validation, and optimisation of the plasmid and the assay were described in Sovová et al (2016). The inhibition was identified by amplifying the sequence Results are expressed as mean ± standard deviation (SD).…”

“…It is therefore necessary to check for the presence of PCR inhibitors and, if those are present, to choose an optimal DNA isolation protocol to ensure that the content of such substances are at a level that would not hinder the subsequent analysis. In a previous study, we have developed an assay using a custom plasmid DNA carrying a non-homologous random sequence for use as an amplification control to identify and quantify possible inhibitors in qPCR in food and feed samples (Sovová et al 2016). Here we present a successful use of the assay to identify PCR inhibition in four real food and feed samples in an assay for the presence of the sequence coding for chloroplast tRNA-Leu.…”

Testing of the Effect of Food Additives on the DNA Isolation from Mackerel and Fish Products

“…Inhibovat reakci mohou rovněž polysacharidy vázající se na aktivní doménu DNA-polymeras a faktory interferující s ionty hořčíku, mezi které řadíme nukleosidtrifosfáty, primery, chelatační látky (kyselina ethylendiamintetraoctová -EDTA, kyselina egtazová -EGTA), DNA, ionty kovů a proteiny. Účinnost amplifikace tak může být snížena i vysokou koncentrací primerů či DNA v reakční směsi, která vede rovněž ke snížení specifity konkrétní reakce [6][7][8][9] .…”