EXPRESS DETERMINATION OF RNA-VIRUSES OF INFLUENZA A STRAINS (H1N1) AND (H7N9) BY PCR–RFLP
NSC Institute experimental and clinical veterinary medicine of the NAAS of Ukraine
Episodic monitoring in recent years indicates that the highly pathogenic avian influenza A (H1N1) and (H7N9) virus is actively circulating in Eurasia. The issue of monitoring infected both migratory and domestic avian in places of cross-contact in Ukraine is relevant to prevent outbreaks of epizootics.
The aim of the study. 1). To develop an express method for identification and determination of avian influenza virus A H1N1 and H7N9 strains, reduce diagnostic time to 40 – 60 minutes. 2). To carry out in silico analysis of variable loci of HA (hemagglutinin), NA (neuraminidase) and NP (nucleoprotein) genes of influenza A virus of two strains, calculate primers to the most conserved sites, determine restriction sites for restriction enzymes, obtain theoretical electrophoregrams by PCR-RFLP analysis, determine conditions for PCR and RFLP analysis.
Materials and methods. Nucleotide sequences for analysis were taken from the NCBI database. The calculation of primers, annealing and landing sites, percentage of identity, PCR- RFLP reaction conditions, and theoretical electrophoregrams were performed using Alignment Service, Lasergene (version 6.0), BioEdit 7.0 and Amplify 1.06.
Results and discussion. The in silico analysis of amplicons of HA, NA, and NP genes allowed in silico to calculate primers to variable loci of studied genes, to calculate reaction conditions, to determine restriction sites to selected restriction enzymes to obtain theoretical electrophoregrams of PCR – RFLP analysis. The percentage of identity, is at least 95% for the three genes. The virus is determined if, after isolating part of the reaction mixture in an agarose gel in the test sample, RNA fragments of the size: for NP5 (H1N1) 166 and 163, 166 for H7N9 using primers specific for hemagglutinin, neuraminidase and nucleoprotein, indicate the presence of viruses. Samples in which the bands are located in the gel at the level of the positive control band are considered positive. Visualization and identification of RFLP products – analysis was performed by their electrophoretic distribution. In the absence of restriction by RFLP analysis in the polymorphic site on electrophoregrams will be found one large fragment corresponding to the length of the RNA sequence between two adjacent constant restriction sites for the same endonuclease. In the presence of a restriction at the polymorphic locus, a smaller fragment equal to the distance between the polymorphic restriction site and one of the nearby constant restriction sites will be present on the electrophoregram. The exception is the identification of the H1N1 strain, some samples of which form in RFLP analysis using selected restriction enzymes to the NP gene unique products of sizes 49-50, 348-350, 592-599 bp, others – a fragment of amplification sizes 21, 39, 201 – 203, 471 – 480 BP, identical to the products of RFLP analysis using selected restriction enzymes with strain H7N9. Express- method for detection and identification of influenza virus AH1N1 and H7N9 and for differentiation of them from samples of other pathogens of viral infections have been developed.
Conclusions. The developed method of express – identification on the basis of PCR combined with RFLP analysis makes it possible to significantly simplify the method of determination due to the specific amplification of the RNA region having a polymorphic restriction site. Before the implementation of the method in diagnostics an in vitro confirmation of in silico predicted results is required.