METHOD OF DETECTING COLD SHOCK GENE CSH1 IN STRAINS OF VIBRIO CHOLERAE O1 AND NEO1/NEO139 BY LOOP ISOTHERMAL AMPLIFICATION (LAMP) Russian patent published in 2024 - IPC C12Q1/68 C12N1/00 

FIELD: medical microbiology; biotechnology; molecular biology.

SUBSTANCE: essence of the invention consists in the fact that amplification is carried out using a set of six primers to the cshl gene of the following composition: L7F3-CAGGTTCGGTTAAGTGGTT; L7B3-ACATTCACAGCCTGTAGG; L7FIP-TGAACAAATACATCAGAGCCGCTTTTAATGAAAGTAAAGGCTTTGGC; L7BIP-TTCAATTCGATCGCTTCAGGTGTTTTCCTTACTTCCCTGTTCGATAC; L7LoopF-CCACTATCAGGAGTAATGAAG; L7LoopB-CTTTATTTGAAGATCAAAAAGTC. The reaction of loop isometric amplification is carried out using an amplifier according to the scheme of two steps—the first one: 64.5 °C, 30 sec; second: 65 °C, 30 sec, taking into account by FAM channel, total 50 cycles, and detection of results of amplification is carried out by channel of fluorophore FAM by geometrical index Cp, displayed in window of results of thermocycler. For LAMP, 10-fold mixture of primers is prepared for each of the genes, which includes: 16 mcM of each FIP- and BIP-primers, 2 mcM of each F3- and B3-primers, 4 mcM of each LoopF- and Loop-B-primers, water without nucleases to a final volume of 100 mcl. Positive control includes all components of the reaction and a DNA preparation of cshl+ strain V.cholerae. Negative control is deionised water not containing the analyzed DNA. Amplification is carried out in volume of 25 mcl and the incubation mixture contains: 12.5 mcl of BioMaster buffer LAMP SYBR (2x) (manufactured by Biolabmix, Novosibirsk, Russia), 2.5 mcl of 10-fold mixture of primers, 5 mcl of a DNA matrix, the remaining volume—water.

EFFECT: invention extends the range of molecular diagnostics means and can be used for laboratory diagnostics of cholera both in practical health care and in research institutions and laboratories.

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