METHOD OF INDICATING PATHOGENIC LEPTOSPIRA USING MULTIPLEX PCR Russian patent published in 2025 - IPC C12Q1/68 

FIELD: veterinary science; microbiology.

SUBSTANCE: invention relates to veterinary microbiology, particularly to laboratory diagnostics of infectious agents, namely to means of diagnosing infection in animals. One-step real-time PCR is performed on three specific DNA loci of the genome of the causative agent of leptospirosis. Measurement is carried out on channels of fluorescent signal accumulation FAM, ROX and HEX on lfb1, lipl32, colA gene loci. Interpretation of results is carried out on the basis of presence or absence of fluorescence curve intersection with threshold line and is determined by value of threshold cycle "C_t". If the fluorescent signal accumulation curves are up to cycle 40 (C_t<40), then DNA of leptospirosis agents is present and the reaction result is considered to be positive. If the curves do not cross the threshold line or cross it after cycle 40 (C_t>40), then pathogenic leptospira DNA is absent and the reaction result is negative. PCS is a recombinant plasmid pALT2 containing three DNA-loci of leptospirosis agents – lfb1, lipl32, colA gene sections. Effectiveness of the proposed technical solution lies in the fact that to increase sensitivity and specificity, detection of the DNA of the causative agent of leptospirosis is used at three specific loci of the lfb1, lipl32 and colA genes.

EFFECT: high specificity and sensitivity of the PCR due to the use of the method for detecting DNA of the causative agent of leptospirosis by three specific DNA loci, as well as eliminating such shortcomings as electrophoresis, and reducing the risk of contamination.

1 cl, 13 dwg