Food Authenticity and Traceability

M Lees

• Publisher: Woodhead Publishing
• Year: 2003
• Print ISBN: 9781855735262
• eBook ISBN: 9781855737181
• Topic: Technology & Engineering
• Subtopic: Food Science
• Index: Technology & Engineering

The ability to trace and authenticate a food product is of major concern to the food industry. This important topic is reviewed extensively in this authoritative text on current and emerging techniques.Part one deals with analytical techniques applied to food authentication. There are chapters on both established and developing technologies, as well as discussions of chemometrics and data handling. Part two relates these methodologies to particular food and beverage products, such as meat, dairy products, cereals and wine. In part three traceability is reviewed in detail, looking at the development of efficient traceability systems and their application in practice to such areas as animal feed and fish processing.Food Authenticity and Traceability is an essential reference for all those concerned with food safety and quality.- Outlines methods and issues in food authentication and traceability- Deals with analytical techniques applied to food authentication, with chapters on established and developing technologies, chemometrics and data handling- Explores how techniques are applied in particular sectors and reviews recent developments in traceability systems for differing food products

Part I

Methods for authentication and traceability

1

Advanced PCR techniques in identifying food components

N. Marmiroli; C. Peano; E. Maestri University of Parma, Italy

1.1 Introduction

The development of fast low-cost DNA synthesis procedures, which produce a new fragment of DNA with a specific nucleotide sequence has greatly accelerated molecular cloning and DNA characterisation. The Polymerase Chain Reaction (PCR) developed by Kary Mullis (U.S. patent 4683202) has also had a major impact. The possibility of generating great quantities of DNA by amplifying fragments of genomic or cloned cDNA has greatly increased the possibility of screening gene-banks, analysing mutation, mapping chromosomes and thousands of other applications (Saiki et al., 1985). The Polymerase Chain Reaction (PCR), the repetitive bi-directional DNA synthesis via primer extension of a region of nucleic acid, is simple in design and can be applied in seemingly endless ways. PCR amplification of a template requires two oligonucleotides primers, the four deoxynucleotides triphosphates (dNTPs), Magnesium ions in molar excess of the dNTPs, and a thermostable DNA polymerase to perform the DNA synthesis (Dieffenbach and Dveksler, 1995).

The PCR reaction has a great efficacy, but this must be measured also by its specificity, efficiency and accuracy that depend on a number of parameters.

In vitro DNA replication has been accomplished from many different sources (Saiki et al., 1985; Mullis and Faloona, 1987; Keohavong et al., 1988; Saiki et al., 1988) The initial PCR procedure described by Saiki et al. (1985) used the Klenow fragment of Escherichia coli DNA polymerase I. This enzyme was heat labile and fresh enzyme had to be added during each cycle following the denaturation and primer hybridisation steps. Introduction of the thermostable Taq polymerase, the DNA polymerase obtained from Thermus aquaticus, in PCR (Saiki et al., 1988) resolved this problem and made possible the automation of the thermal cycling in the procedure. Virtually all forms of DNA and RNA are suitable substrates for PCR. These include genomic, plasmid, and phage DNA, previously amplified DNA, cDNA, and mRNA. Samples prepared via standard molecular methodologies (Sambrook et al., 1989) are sufficiently pure for PCR, and usually no extra purification steps are required. In general the efficiency of PCR is greater for smaller-size template DNA than for high molecular weight DNA.

For many applications of PCR, primers are designed to be exactly complementary to the template. However, for other applications, such as allele specific PCR, the engineering of mutations or of new restriction endonuclease sites into a specific region of the genome, and cloning of homologous genes where sequence information is lacking, base pair mismatches are introduced either intentionally or unavoidably (Kwok et al., 1995). In either case, an ideal pair of primers should hybridise efficiently to the target sequence with negligible hybridisation to other related sequences that are present in the sample. Studies have shown that different DNA polymerases have distinct characteristics that affect the efficiency of PCR. For example Taq Polymerase does not have the 3ʹ-5ʹ exonuclease proofreading function, and as a result, it has a relatively high error rate in PCR (Lawyer et al., 1989).

1.1.1 How PCR techniques work

The PCR reaction allows the million-fold amplification of a specific target DNA fragment framed by two primers (synthetic oligonucleotides, complementary to either one of the two strands of the target sequence). The PCR is a multiple-process with consecutive cycles of three different temperatures, where the number of target sequences grows exponentially according to the number of cycles. In each cycle (Fig. 1.1) the three temperatures correspond to three different steps in the reaction (Dieffenbach and Dveksler, 1995).

Fig. 1.1 Schematic representation of the three steps in PCR amplification: denaturation of the template DNA, hybridisation of the primers, extension by Taq polymerase. Temperatures in the hybridisation (or annealing) step may vary.

In the first step the template, the DNA serving as master copy for the DNA polymerase is separated into single strands by heat denaturation at ~ 95 °C. In the second step the reaction mix is cooled down to a temperature of 50–60 °C (depending on the composition of the primers used) to allow the annealing of the primers to the target sequence. Primer hybridisation is favoured over DNA/DNA hybridisation because of the excess of primers molecules. In the third step, the annealed primers are extended using a Thermus aquaticus (Taq) polymerase at the optimum temperature of 72 °C. With the elongation of the primers, a copy of the target sequence is generated. The cycle of these three different temperatures is then repeated from 20 to 50 times, depending on the amount of DNA present and the length of the amplicon (amplified DNA fragment).

In order to use PCR, the analyst must know the exact nucleotide sequence that flank both ends of the target DNA region. Any PCR-based detection strategy will thus depend on the selection of the oligonucleotide primers and the detailed knowledge of the molecular structure and DNA sequences used. Faster cycling with better temperature control using capillaries in air heated thermal cyclers has improved PCR specificity. ‘Rapid cycle’ PCR requires amplification cycles of 20–60 sec and the whole procedure of amplification, 30 cycles, only 10–30 min. Rapid cycle is based on a ‘kinetic’ rather than an ‘equilibrium’ paradigm ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright page
• Contributor contact details
• The Humber Institute of Food & Fisheries
• Part I: Methods for authentication and traceability
• Part II: Authenticating and tracing particular foods
• Part III: Traceability
• Index