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

Abstract

Sovová T., Křížová B., Drábková L., Ovesná J. (2017): Detection of PCR inhibition in food and feed with a synthetic plasmid. Czech J. Food Sci., 35: 160-164.We present a successful use of the plasmid inhibition detection and DNA isolation protocol optimisation for four food/feed samples in qPCR analysis of the sequence coding for chloroplast tRNA-Leu: two meat meal samples and two samples made of cranberries (jam and dried fruit). The quantitative real-time polymerase chain reaction (qPCR) can be inhibited by various substances and the DNA content in the sample can be underestimated. It is necessary to identify the PCR inhibition and choose an optimal DNA isolation protocol to correctly evaluate the sample. In a previous study, we have developed an assay using plasmid DNA carrying a non-homologous random sequence identifying possible inhibitors in qPCR in food/feed samples. The plasmid assay allowed to effectively reveal the PCR inhibition in all of the different sample matrices and to choose an optimal DNA isolation protocol.

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] .…”