Quantitative competitive (QC) PCR for quantification of porcine DNA

Abstract

Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios.

Introduction

Several analytical approaches have been made to identify animal species in fresh or processed meat products in order to protect consumers from fraud and adulteration. Today several methods exist for determination of animal species (Bauer & RippelRachle, 1998, Meyer & Candrian, 1996a). Besides protein based techniques like isoelectric focusing (IEF) or Elisa which suffer from inaccuracy and denaturation of soluble proteins during processing (Berger et al., 1988, Jemmi & Schlosser, 1991, Sinclair & Slattery, 1982), DNA techniques have become very important and are widely used nowadays. Advantages of DNA analysing methods are manifold: DNA is a relatively stable molecule easily allowing analysis of processed and heat treated food products (Beneke & Hagen, 1998, Unseld et al., 1995). Due to the ubiquity of DNA in every type of cells, all kinds of tissue can be analysed. Early methods based on hybridisation of specific probes (Chikuni et al., 1990, Wintero et al., 1990) were complicated, time consuming and insufficient for complex matrices.

The polymerase chain reaction (PCR) (Saiki et al., 1988) proved to be an adequate technique for detection of small amounts of DNA, specifically amplifying a target region of template DNA in a rapid and sensitive manner. Using either specific or universal primers, many sequences of mitochondrial (e.g. cytochrome b gene) (Burgener & Hubner, 1998, Matsunaga et al., 1999) or genomic DNA have been analyzed for various animals like fish (Cespedes et al., 1999, Quinteiro et al., 1998), game species (Wolf, Rentsch & Hubner, 1999), several domestic animals and from caviar (Birstein et al., 1998, Wolf et al., 1999) as well as from plants (Meyer et al., 1996, Schmidt-Puchta et al., 1989) and a variety of genetically modified organisms (GMO) (Hupfer et al., 1998, Meywe, 1995, Zimmerman et al., 1998). Universal primer systems like cytochrome b PCR allow identification of several species within one analysis but, however, PCR products have to be longer to contain sufficient DNA sequence variation for restriction analysis (RFLP) (Mayr et al., 1993, Wolf et al., 1999) or single strand conformation polymorphism (SSCP) (Rehbein et al., 1999). Especially in processed food products, DNA is often degraded to just a few hundred base pairs and is precluded from PCR amplification. Species specific PCR systems allow shorter amplicons, as amplification indicates the presence of the gene/sequence of interest. For many domestic animals like cattle (Zhang, Zheng, Zhou, Ouyang & Lu, 1999), pig (Meyer, Candrian & Lüthy, 1994), chicken (Hopwood, Fairbrother, Lockley & Bardsley, 1999), sheep and goat (Chikuni, Tabata, Kosugiyama, Monma & Saito, 1994), single PCR systems exist and recently a multiplex PCR system was reported (Matsunaga et al., 1999) using one universal and one specific primer for each species for simultaneous detection of several animals in one analysis.

PCR systems used today only allow a qualitative detection of animal species. Because even trace amounts of less than 0.1% of one species lead to positive PCR results (Behrens, Unthan, Brinkmann, Buchholz & Latus, 1999), it is necessary to differentiate between technically unavoidable contamination or intentional admixture. Therefore, adequate quantitative detection methods for small amounts of DNA must be available. PCR analysis can be used as a quantitative method if internal standards (competitors) are coamplified with the target DNA. Quantitative competitive PCR (QC-PCR) was first described in 1990 (Gilliland, Perrin, Blanchard & Bunn, 1990) and is widely used in many fields of analysis (Moller & Jansson, 1997, Siebert & Larrick, 1992, Studer et al., 1998). However, so far no QC-PCR system has been reported for detection of animal species in food products.

In our work we describe the development and evaluation of a new, porcine specific QC-PCR system based on the growth hormone gene of sus scrofa.