Method of polymerase chain reaction (PCR) is one the most promising for determination of species affiliation of tissues in foodstuff composition including one that has undergone thermal treatment. At the present time numerous modifications of this method have been developed which use DNA- and RNA-matrix, define point mutations, assess gene expression and perform quantitative analysis. One of modifications – Real-Time PCR – can accelerate research by eliminating the phase of electrophoresis.

Despite foreign experts’ high estimation of PCR-method for determination of meat products falsification, it has not gained wide practical application in the field of veterinary-sanitary expertise in our country yet. One of the possible reasons is the lack of efficient, competitive test-systems of domestic production adapted to the specific material and technical equipment of Ukrainian diagnostic laboratories.

The aim of the study was to develop domestic diagnostics based on PCR-RT to identify the species origin of meat, meat and plant ingredients in the composition of meat products, including those that have undergone thermal treatment.

The material for the isolation of genomic DNA samples was samples of raw meat (pork, beef, lamb, horse, rabbit, chicken, goose and duck), meat products that have been subjected to thermal treatment, feed for animals, foodstuffs with different plant species in their content.

As targets for determination of biological material affiliation to Sus scrofa, Gallus gallus and Bos taurus gene sequences of cytochrome b (Cytb) ot pig mitochondrial DNA (GenBank registration №X56295) and chicken mtDNA (AY235571), and also the sequence of satellite DNA IV family of cattle (AF446392 ) have been used. Lectin gene sequence (Lec) of soybean (K00821) was used to determine Glycine max.

The test system was developed based on the TaqMan PCR technology in real-time. PCR amplification was performed with the help of the device CFX96 (BioRad). For the amplification optimal conditions were selected.

Oligonucleotide probes labeled with fluorescent dyes FAM, HEX and ROX fluorescence quencher and BHQ1 and BHQ2 (Metabion, Germany) were used.

The test-system to determine the species origin of meat was produced in the multiplex format that allowed simultaneous holding of three independent reactions in a test tube. The course of each of the three reactions was monitored using a specific probe labeled with a fluorescent dye given. To detect mtDNA of a pig and a chicken the probes labeled with FAM and HEX dyes respectively have been used; to determine the DNA sequence of cattle a probe labeled with ROX have been used.

To identify components of plant origin, including soybean, multiplex test system that allows analyzing of two targets (soybean lectin gene and chloroplast DNA sequence of the plant) was developed. To identify these sequences the probes labeled with fluorescent dyes FAM and HEX respectively have been used. Applying of this test system makes it possible to detect any impurities of plant origin both in simple and in multicomponent mixtures as well as to identify the presence of soy in foodstuffs and raw materials.

Optimization of the conditions for PCR-amplification was carried out according to such parameters as primer annealing temperature, concentration of MgCl₂, concentration and ratio of primers and probes.

Evaluation of the effectiveness of the developed test-systems was performed according to such analytical characteristics as specificity, sensitivity, limit of detection, repeatability and reproducibility analysis.

A wide range of foodstuffs including meat and meat products, canned and convenience foods, pies, dumplings, as well as animal feed was used to test the developed test-systems. The results showed high efficiency of test-systems for the determination of species belonging to three kinds of meat and soy in the foodstuffs and feed, even those subjected to thermal treatment.

Key words: food safety, Real-Time PCR, test-systems, tissue species origin, foodstuffs.