Application of DNA/RNA-based technologies in monitoring food safety and quality

Abstract

DNA/RNA-based methods for food analysis have emerged from the laboratory and are becoming integrated into regular analytical usage. Problems remain in the standardisation of procedures between laboratories, and the often highly variable nature of template DNA preparation. Quality control is essential in the food industry and efficient quality assurance is becoming increasingly important. Food analytical researchers are increasingly asking for efficient control methods, in particular through up to date quality sensors, firstly to satisfy the consumer and regulatory requirements and secondly to improve the production feasibility, quality sorting, automation and reduction of production cost and production time. Therefore, all three drivers of the quality control, consumers, authorities and food producers, have great interest in the development of new sensing systems which are beyond the existing on-line technologies. The molecular based analytical technique offers the advantages of versatility and extreme sensitivity.This work is based on three main issues. These include: DNA based methods for detection of food-borne pathogens, detection of risk material in food chain and identification of various animal species in food and feed matrices. The first issue was done under cooperation with an European network-research project (Food PCR). The aim of this project was to validate and standardize the use of diagnostic PCR for the detection of foodborne pathogens. The plan of Food-PCR was to devise noncommercial and nonpatented, standardized PCR-based detection methods for five major pathogens: E. coli O157, Salmonella spp., Campylobacter spp., enterohemorrhagic Listeria monocytogenes and Yersinia enterocolitica. The role of the Institute of Veterinary Food Science, Giessen in this project was task leader for development and validation of a specific PCR assay for detection of E. coli O157 to provide a diagnostic method which will be suitable for routine adoption and future proposal as a standard. This goal was preceeded in three phases. In phase 1, researchers working in expert laboratories have developed and selected promising candidate PCR-based methods, and tested them for efficiency and selectivity against comprehensive collections of reference strain DNA. The final selected PCR assays were optimized, and taken forward into phases 2 and 3. In phase 2, an interlaboratory trial was conducted to confirm the selectivity of the PCR assays. This phase will itself be conducted in two steps: in the first all reagents will be supplied by the originating laboratory, while in the second the participants will use their own. This provided a thorough evaluation of the efficiency and robustness of the PCR assays.In phase 3, the complete procedure comprising sample pre-treatment and the PCR assay (PCR-based method) will be subjected to interlaboratory trials, to provide validated PCR-based pathogen detection protocols. The second issue was the detection of BSE risk material in food chain. For an efficient consumer s protection, European legislation prohibited several bovine tissues encompassing mainly central nervous system tissues from food chain. A quantitative real-time RT-PCR was designed to identify BSE risk material in meat and meat products. This was based on an mRNA assay that used bovine, ovine, and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as a marker. The real time RT-PCR assay included a housekeeping gene as an endogenous control. The quantitative real-time RT-PCR detection of GFAP mRNA appeared to be useful as a routine diagnostic test for the detection of illegal use of CNS tissues in meat and meat products. The stability of the specific region of GFAP mRNA allows the detection of CNS tissues also after meat processing steps. This region was protected by an international patent. The third issue is the identification of various animal species in food. Authentication of food starts at the species level of the used meat as raw material for other productions. Food authentication linked to problems of adulteration. Adulteration usually involves substituting high quality raw materials with cheaper ones. For prevention of mislabelling or undeclared admixture, either deliberate or accidental, PCR-RFLP, species specific primer and PCR sequencing have been adopted for the differentiation of snails, ostrich dog and cat species in food and feed matrices. These DNA based methods being highly sensitive, reproducible, rapid, simple and not expensive could be used even after heat treatment.