METHOD FOR ASSESSING INTRASPECIFIC COMPETITION OF TOXIGENIC AND NON-TOXIGENIC STRAINS OF VIBRIO CHOLERAE O1 AND O139 SEROGROUPS USING REAL-TIME PCR Russian patent published in 2023 - IPC C12Q1/68 C12N15/00 

FIELD: medical microbiology; genetic engineering.

SUBSTANCE: method for assessing toxigenic and nontoxigenic strains of Vibrio cholerae O1 and O139 serogroups using real-time PCR is described. DNA is isolated from the material under study, PCR is carried out with specific primers and subsequent analysis of the amplification reaction is carried out. Amplification of the DNA under study is carried out using two pairs of primers and two probes: ctxA5; GTTCCTCTTGCATGATCATAAAG, ctxA6; TATCGGGCAGATTCTAGACC, ctxA5Z; /ROX/-ACTCTGTCCTCTTGGCATAAGACCACCT-/BHQ2/ and cold1 ACAGGTTCGGTTAAGTGGTTT, cold2 ACCTGAAGTGATCGAATTGAAGT, coldZ_12 /R6G/ACTCCTGATAGTGGCGGCTCTGA /BHQ1/. In one sample, the concentration of toxigenic and atoxigenic Vibrio cholerae is simultaneously determined. The first pair of primers and the ctxA5Z probe detect toxigenic vibrios by presence of the ctx gene, and the second pair uses the cold shock gene csh1 as a marker gene for nontoxigenic strains. Intraspecific competition is assessed through the HEX and ROX channels of the amplifier relative to the number of targets of standard DNA samples, the quantitative result is entered into the corresponding columns of the protocol, which is expressed as the decimal logarithm of the number of cells; the higher the concentration at low temperatures, the higher the competition of the strain in the possibility of survival. Before performing PCR, standard preparations are prepared containing a known amount of DNA of toxigenic and non-toxigenic Vibrio cholerae; for this, a mass of toxigenic and non-toxigenic strains are grown for 24 hours on Marten agar with pH 7.4 at a temperature of 37°C, then 1 ppb bacterial suspension in a 0.9% sodium chloride solution at pH 7.2 is prepared using standard turbidity samples, which are diluted 10-fold to 10³, then the resulting suspensions are mixed in different dilutions containing 10⁸, 10⁷ and 10⁶ m.c./ml of toxigenic and non-toxigenic strains, then DNA is isolated from the three samples obtained, which are stored at a temperature of 4°C up to 30 days for use when performing PCR. In addition, PCR is carried out in a volume of 25 mcl, and the reaction mixture contains: 2.5 mcl of buffer, 1 unit of Taq DNA polymerase, 0.2 mcM dNTP mixture, 1.0 mcM each of the four primers ctxA5, ctxA6 and ROX-labeled ctxA5Z probe to detect toxigenic vibrios by the presence of the ctx, cold1, cold2 gene and probe coldZ_12, labeled with R6G, 0.5 mcM of each of the two primers, 5 mcl of DNA-DNA template, the remaining volume is water. PCR reactions are carried out in compliance with the following regimes: denaturation at 95°C - 2 min (1 cycle); then 30 cycles: denaturation at 95°C - 20 s, annealing and monitoring via FAM channel at 67°C - 20 s.

EFFECT: invention can be used in laboratory diagnostics when monitoring cholera and conducting research related to the study of the biology of the pathogen, including the identification of cholera pathogen strains that can successfully compete with non-toxigenic crops.

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