A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays

Abstract

Aims: To develop and evaluate a TaqMan‐based internal amplification control (IAC) that can be used as DNA in real‐time PCR (qPCR) or as RNA in reverse transcription real‐time PCR (qRT‐PCR) to identify the presence of assay inhibition and to evaluate its incorporation into existing qPCR and qRT‐PCR methods for bacterial detection. Methods and Results: A DNA IAC was constructed by generating a 198‐bp random sequence that was synthesized and inserted into a pZErO‐2 vector and transformed into Escherichia coli. The RNA IAC was generated through in vitro transcription of the DNA IAC. Both IAC formats were tested individually in singleplex TaqMan reactions and also included in existing multiplex assays. The DNA IAC was incorporated in a Shigella spp. detection qPCR assay (targeting ipaH). The RNA IAC was successfully evaluated in a Salmonella spp. detection qRT‐PCR (using invA mRNA as target). Conclusions: A highly versatile IAC that can be supplemented to qPCR and qRT‐PCR pathogen detection methods was developed, greatly reducing the confounding effects of false negatives because of PCR inhibitors without affecting pathogen detection. Significance and Impact of the Study: The frequency of false negatives associated with qPCR analyses is prevalent in certain matrices, particularly those involving complex foods. Hence, the IAC presented here provides a solution to unforeseen false‐negative reactions in PCR.

Detection of Allergen Markers in Food: Analytical Methods

“…Controls to detect PCR inhibition are based on detection of exogenous DNA, which is added directly to PCR reactions after ft DNA extraction. Exogenous template DNA can be cloned into a plasmid or obtained directly from a commercial supplier, and a published universal internal control based on exogenous DNA has been shown to work well in allergen detection assays [227,228,235,252]. Controls used to confirm fi suitability of extracted DNA for PCR are based on amplification of a confi served region of endogenous DNA, which is expected to amplify regardless of whether the intended allergenic target is present.…”

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

“…Food Analysis, Food Quality and Nutrition doi: 10.17221/374/2016-CJFS Food and feed samples can present as problematic in terms of DNA analysis as they can represent rich sources of PCR inhibitors (Lübeck et al 2003;Deer et al 2010). 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.…”

“…However, in certain cases, even the most careful manipulation and/or optimisation of the sample processing protocol cannot fully prevent the occurrence of inhibiting substances in analysed samples. Therefore, for a correct interpretation of the results, it is necessary to identify the presence of the inhibitors (Lübeck et al 2003;Deer et al 2010). One of the possible approaches is to perform a serial dilution of the sample, which is however quite time-consuming and requires a large quantity of PCR reagents.…”

“…Real-time PCR can be used to identify human pathogens, to quantify genetically modified organisms (GMOs), or to check correct labelling of food and feed (Renault et al 2004;Deer et al 2010). Even though qPCR is a very sensitive and precise technique, it can be hindered by the presence of the so-called PCR inhibitors or less common PCR enhancers (Hoorfar et al 2004;Hartman et al 2005;Nordstrom et al 2007).…”

“…The quantitative real-time polymerase chain reaction (qPCR) has become a fundamental method used for DNA analysis in many fields, including food science and technology, where it has become a crucial technique used in food quality and safety control (Renault et al 2004;Deer et al 2010;Rodríguez-Lázaro & Hernandéz 2013). Real-time PCR can be used to identify human pathogens, to quantify genetically modified organisms (GMOs), or to check correct labelling of food and feed (Renault et al 2004;Deer et al 2010).…”