METHOD FOR DETECTION OF DNA OF DONKEY (EQUUS ASINUS) TISSUE IN DRY FODDER AND SEMI-FINISHED MEAT PRODUCTS

摘要

FIELD: biotechnology.;SUBSTANCE: invention represents a method for DNA identification of donkey tissue (Equus asinus) in dry fodder 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 and Cy5 / Red for an internal control sample in 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 TACATATAAATCACGCAAAGC – forward primer; T4R TAGTATGGCTAATCTTATTGG – reverse primer; T4P CY5 ACATTGGCACTGACCGAGTTC – probe, taken in ratio of 1:1, measurement of accumulation of fluorescent signals via channels of corresponding fluorescent dyes, interpretation of results based on presence or absence of intersection of fluorescence curve with threshold line, if 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 donkey tissue (Equus asinus), and the internal control sample is used with phagolysate of bacteriophage T4, and for a positive control sample – fragments of genomes of native bacteriophage T4 and donkey tissue (Equus asinus) with the following nucleotide sequence: Donk F: 5'- CATATCTTAATCCCAATAATAGAC-3' – forward primer; Donk R: 5'- AGTATTCGTTCTGATATAGGC-3' – reverse primer; Donk P: PAM-5'- ATCTTATACTGGACTATTCTATCGAC-3 -BHQ1 – probe, wherein DNA detection of donkey tissue (Equus asinus) employs fluorescent dye FAM / Green.;EFFECT: invention increases accuracy of identification of species identity and simplifies the process of preparing samples.;1 cl, 5 tbl