METHOD FOR IDENTIFICATION OF COMMON HEDGEHOG (ERINACEUS EUROPAEUS) DNA TISSUE IN DRY FODDER AND SEMI-FINISHED MEAT

摘要

FIELD: biotechnology.;SUBSTANCE: invention is a method for identification of common hedgehog DNA (Erinaceus europaeus) in dry fodders and semi-finished meat products, involving DNA recovery from animal tissue by sorption method, polymerase chain reaction with fluorescent detection is carried out with 45 cycles of real-time amplification using oligonucleotide primers, probes, fluorescent dyes specific for the DNA genome region: for a specific signal for an animal – JOE/Yellow and Cy5/Red – for an internal control sample in the form of a suspension of bacteriophage T4 with concentration of 5×10³ phage particles per 1 mcl, a positive control sample in the form of a mixture containing fragments of animal genomes and bacteriophage T4 with a nucleotide sequence: T4F: 5'-TACATATAAATCACGCAAAGC-3' – forward primer; T4R: 5'-TAGTATGGCTAATCTTATTGG-3' – reverse primer; T4P: HEX-5'-ACATTGGCACTGACCGAGTTC-3'-BHQ1 – probe, taken in ratio of 1:1, and measurement of accumulation of fluorescent signals through channels of corresponding fluorescent dyes, interpretation of results based on the presence or absence of the intersection of the fluorescence curve with the threshold line, if the fluorescence signal accumulation curves are up to 35 cycles, the reaction result is considered to be positive, and if the curves do not cross the threshold line or cross it after 35 cycles, the result of the reaction is negative, according to the invention, DNA is recovered from common hedgehog tissue (Erinaceus europaeus) and phage antigens of bacteriophage T4 are used for the internal control sample, and for the positive control sample – fragments of genomes of native bacteriophage T4 and hedgehogs (Erinaceus europaeus) with the following nucleotide sequence: Hed-F: 5'-AGTCTATTGATTCGAATAGAGC-3' – forward primer; Hed-R: 5'-CATGAGAGGTACTAACCAGT-3' – reverse primer; Hed-P: FAM-5'-CAGGAGCTTTATTAGGTGATGATCAG-3-BHQ1 – probe.;EFFECT: invention increases identification accuracy of species identity, simplifies sample preparation process.;1 cl, 5 tbl