The aim of the study was to analyze biotechnological approaches to the construction of an internal control sample (ICS) in the manufacture of gene diagnostic preparations and the development of such an ICS for PCR test systems produced by the Microbe Russian Anti-Plague Institute. Materials and methods. To create an artificial nucleotide sequence that no known organism has, the online resource Random DNA Sequence Generator was used. Specific primers for generation of artificial nucleotide sequences were selected using DNA-Works software (v3.2.4.). Oligonucleotides were synthesized using the standard solid-phase amidophosphite method on an ASM-800 automatic DNA synthesizer. Cloning of the amplification products used as the ICS was performed in the pAL2-T vector. Bacteria culturing and bacterial clone selection were carried out on Luria-Bertani (LB) agar medium containing 50 μg/mL of ampicillin as a selective factor. Results. The paper presents data on biotechnological approaches to constructing ICSs. In the first case, a 174-bp region of the green fluorescent protein gene sGFP-206 (gfp) of the jellyfish Aequorea victoria was selected to create an exogenous ICS, since the presence of this sequence in the studied samples was virtually excluded. In the second case, an artificial nucleotide sequence was created that no known organism possesses. When developing a diagnostic set of reagents for detecting V. cholerae strain DNA in the studied clinical samples using PCR with hybridization-fluorescence registration of results, one of the tasks was to construct an endogenous internal control sample. It was proposed to use human DNA genes as a DNA target for the ICS. Studies were conducted to assess the efficiency of amplification of ICS fragments with primer annealing temperatures of 49, 58, and 60°C according to the working protocols of the produced test systems and the absence of a negative impact on the efficiency of the studied gene-fragment amplification reaction. Conclusions. The employed approaches allowed three variants of ISOs to be created for implementation of control with the help of RT-PCR of produced sets of reagents to identify the DNA of pathogens of especially dangerous infectious diseases, such as cholera, plague, anthrax, brucellosis, and tularemia with hybridization-fluorescence registration of results.
Stepanov et al. (Mon,) studied this question.