FIELD: medicine.
SUBSTANCE: present invention relates to medical microbiology and genetic engineering and can be used in laboratory diagnostics in detecting toxigenic strains of O1 Vibrio cholerae of "Haitian" group. Essence of the proposed invention consists in the fact that amplification of the analyzed DNA is carried out using the designed primers: 1095-1-(5’-CCATCAGTCTGCCTCTGACAC-3’), 1095-2-(5’-TTCGACAATCGTCAGTAGCG-3’), in the presence of two oligonucleotide probes: 1095ZW (5’-FAM-TCAACTGGTCAAAGTGGCCGAT-BHQ1-3’), 1095ZH (5’-HEX-TTGGATGGTCAAAGTGGCCGAT-BHQ1-3’), wherein analysis of products of amplification by an end point is carried out in a PCR detector, placing the tubes into the appropriate sockets, taking into account the amplification results visually on the computer monitor according to the FAM and HEX channels, where the glow fluorescence level is reflected, wherein the FAM channel used as the background (internal control) is the O1 strain of Vibrio cholerae strain 19191 and the positive result is determined from the length of the blue band which reflects the fluorescence level in the test samples greater than 2.5 times the length negative (background) sample strips, confirming correctness of DNA extraction and absence of inhibitors in samples, and HEX channel used negative DNA backgrounds of strains O1 Vibrio cholerae 5879 and the result is considered to be positive, if the fluorescence level of the analyzed sample a green band exceeds the background (negative control) of more than 2.5, then a toxic strain of the "Haitian" group is fixed in the given sample. PCR is taken in amount of 25 mcl and the reaction mixture contains: primers 1055-1, 1095-2 and probes 1095 ZW and 1095 ZH each 1.0 mcM, 1.5 mM Mg - buffer, 0.2 mM dNTF mixture, 0.25 units DNA polymerase, analyzed by DNA - 25 ng, remaining volume - water. Besides, PCR reactions proceed with observance of modes: denaturation 95 °C - 2 min (1 cycle); 35 cycles: denaturation 95 °C - 20 s; annealing at 65 °C - 20 s; synthesis at 72 °C - 20 s; synthesis at 72 °C is 3 minutes (1 cycle).
EFFECT: disclosed is a method for detecting toxigenic strains of o1 vibrio cholerae of the Haitian group by PCR in real time on an end point.
3 cl, 2 dwg, 3 tbl
