FIELD: biotechnology; medical microbiology.
SUBSTANCE: used in laboratory diagnostics for the detection of viable Vibrio cholerae vibrios in environmental samples. A method for detecting viable Vibrio cholerae serogroup 01 of the Classical and El Tor biovars in the environment is proposed. A polymerase chain reaction is carried out in real time with detection of the DNA concentration of the M3 bacteriophage lysing Vibrio cholerae 01 serogroup of the Classica and El Tor biovars, using primers and a probe. The PCR results are recorded using the geometric method Cp by recording on a standard calibration curve for specified periods of time, more than 2 hours; if accumulation of viral particles of bacteriophage M3 is observed, then the presence of viable Vibrio cholerae 01 serogroup of biovars Classical and El Tor is detected in the samples, and in the absence of accumulation of viral particles, their non-viability is confirmed. To isolate the DNA of bacteriophage M3, before PCR, a four-hour sample in a volume of 0.2 ml is cultured in 10.0 ml of peptone broth, then 100 μl is taken into an Eppendorf with the addition of 0.3 ml of phage M3 in a titer of 102 PFU/ml, mixed, 100 µl is taken into an Eppendorf (1.5 ml), which corresponds to the zero point (T0), the mixture is placed in a thermostat at 37°C for one hour, then two hours, and then DNA standards from 108 to 103 PFU/ml are isolated with a set of reagents.
EFFECT: rapid and reliable detection of viable Vibrio cholerae in environmental samples using the M3 bacteriophage based on quantitative PCR, allowing to avoid extensive cultural research methods and leading to faster results.
4 cl, 2 dwg, 2 tbl, 2 ex
