Fecha de publicación: 26/10/2022 Fuente: WIPO Meat FIELD: veterinary medicine.
SUBSTANCE: invention relates to veterinary microbiology, in particular to laboratory diagnostics of pathogens of infectious diseases, namely to means of diagnosing infection in animals using polymerase chain reaction. A method for detecting the DNA of the causative agent of cryptosporidiosis in biological material of animals, in water from reservoirs and feed using a polymerase chain reaction in real time includes isolation of the DNA of the causative agent of an infectious disease from the biological material of an infected animal by sorption method, setting up a one-stage polymerase chain reaction with fluorescent detection with 40 cycles of amplification in real time using oligonucleotide primers specific to the site of the genome of the DNA of the causative agent of an infectious disease of an animal, probes, fluorescent dyes, an internal control sample in the form of a suspension of bacteriophage T4 with a concentration of 5×103 phage particles per 1 mcl and a positive control sample in the form of a mixture containing fragments of the genomes of the causative agent of an infectious disease of an animal and bacteriophage T4 in the same volume ratio, measuring the accumulation of fluorescent signals through the channels of the corresponding fluorescent dyes for a specific signal testing the presence of the causative agent of an infectious disease of an animal and through the FAM/Green channel. for an internal control sample, interpretation of the results based on the presence or absence of intersection of the fluorescence curve with the threshold line, if the accumulation curves of the fluorescent signal come out before the 35th cycle, then the reaction result is considered positive, and if the curves do not cross the threshold line or cross it after the 35th cycle, then the reaction result is negative, according to the invention, the DNA of the pathogen of cryptosporidiosis is isolated from the material selectively: water from reservoirs, feed, feces, parenchymal organs, intestines and small intestine, meat and raw meat products, raw milk, and for a positive control sample, a fragment of the genome of the pathogen of cryptosporidiosis with the following nucleotide sequence is used:
Crypto-F: 5'-AAGGGTTGTATTTATTAGATAA-3';Crypto-R: 5'-GTCAGAAACTTGAATGATA-3';
Crypto-Z: HEX-5'-TCCGTAAAGTTATTATGAGTCACCAAT-3'-BHQ1, while a fluorescent dye - JOE(HEX)/Yellow is used for the DNA of the pathogen of cryptosporidiosis.
EFFECT: method makes it possible to expand functionality and increase accuracy in identifying the causative agent of cryptosporidiosis.
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