Development of a test system for detection of the rabies virus genome using real-time PCR with an endogenous control: Theoretical design and optimization

Introduction. Although rabies incidence in Russia has decreased over the past ten years, it still remains a highly relevant disease. Polymerase chain reaction (PCR) in its various modifications is one of the tools for detecting and characterizing rabies virus in pathological samples. To ensure reliable diagnosis, the current data on genetic properties of the isolates circulating in particular area should be considered when developing PCR.

Objective. Providing a theoretical foundation and optimizing the test system based on combined reverse transcription polymerase chain reaction (C-RT-PCR) for the detection of a rabies virus genome fragment using endogenous internal control.

Materials and methods. Rabies virus vaccine strain “RV-97” and laboratory fixed rabies virus strain “CVS”, brain samples from animals of various species, both rabies virus positive and negative, were used in the work. Oligonucleotide design and in silico specificity testing were performed using Primer Blast online tool. Syntol reagents (Russia) were used for C-RT-PCR.

Results. As a result of the analysis of 193 N-gene sequences of rabies virus isolates circulating in Russia and neighboring countries, six primers and two TaqMan probes were designed. Two primers and a probe, optimal for the development of the test system, were experimentally identified. During a series of experiments, C-RT-PCR parameters such as oligonucleotide and magnesium ion concentrations, as well as annealing temperature were optimized. To control the reaction conditions in each tube, an oligonucleotide system was developed for the amplification and detection of the mammalian beta-actin gene region. The possibility of simultaneous application of two PCR systems in one test tube has been experimentally demonstrated. Analysis of 193 N-gene sequences of various rabies virus isolates showed that the developed oligonucleotides can theoretically ensure detection of the vast majority of the viruses circulating in the Russian Federation and neighboring countries.

Conclusion. The main components and reaction conditions of the test system for detecting the rabies virus genome by C-RT-PCR using mammalian beta-actin gene-based endogenous internal control have been developed and optimized.

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