BACKGROUND
Subtypes H5, H7, H9 and H10 of the influenza A virus have a high pandemic potential, due to their ability to adapt to new hosts by acquiring mutations and genetic reassortment. Sporadic cases of human infection are often characterized by severe course of disease and high mortality. The development of a PCR reagent kit for in vitro diagnostics of potentially pandemic influenza A viruses subtypes is important.
OBJECTIVE
Development and implementation of a diagnostic reagent kit for the detection of influenza A virus RNA and typing of subtypes H5, H7, H9, H10 in human biological material using RT-PCR with hybridization-fluorescence detection.
MATERIAL AND METHODS
Specific primers and hybridization-fluorescent probes have been developed for the amplification of influenza A virus RNA and H5, H7, H9, and H10 virus subtypes. Optimal RT-PCR conditions were selected and the analytical and diagnostic characteristics of test kit based on selected specific oligonucleotides were evaluated. Clinical and laboratory tests of a set of reagents were carried out to confirm its effectiveness and safety.
RESULTS
The analytical sensitivity of the PCR reagent kit for in vitro diagnostics was assessed on model samples of human biomaterial. The value is 1·103 GE of the target viral RNA per ml of nasopharyngeal and oropharyngeal mucosa swabs or sputum. The absence of cross-reactions with the genetic material of influenza A virus of other subtypes, influenza viruses B, C, other viruses and bacteria was shown. To assess the diagnostic characteristics, clinical material from patients with typical clinical symptoms of acute respiratory viral infections, as well as model samples simulating biological material from patients with influenza subtypes H5, H7, H9 and H10 were used. To obtain these model samples, clinical material was contaminated with strains of H5N1, H7N3, H9N2 and H10N5 subtypes or with biological material from birds affected by influenza. As a result, 343 samples containing RNA of influenza A virus of various subtypes, including H5, H7, H9, H10, and not containing RNA of influenza A virus, were studied. Complete agreement of the results with the results of the comparison system was shown.
CONCLUSION
Based on the results of clinical laboratory tests, it was confirmed that the developed test kit has high analytical and diagnostic characteristics, is effective and safe when used as intended, and can be recommended for registration as a medical device for in vitro diagnostics.
