Sequencing of the VP1-2A Genome Segment for Hepatitis A Subtyping.

MATERIALS AND

methodsNucleic acids were extracted by using commercial kits for nucleic acid extraction and reverse transcription in a manual and automatic version. Primer sequences were selected using the DNASTAR software. Oligonucleotides were synthesized by the amidophosphite method followed by purification with polyacrylamide gel electrophoresis. The sought fragments of the genome were obtained with PCR. Commercial components were used to prepare the reaction mixture. The results were verified by agarose gel electrophoresis with ethidium bromide DNA staining. After amplification, the DNA fragments were purified by adsorption on silica columns. Sequence identification of the purified fragments was performed by the Sanger sequencing method. The obtained sequences of the VP1-2A fragment were analyzed by the MEGA 6 software package. The optimized technique for sequencing of the VP1-2A segment was tested with 171 clinical and environmental samples containing hepatitis A virus RNA.

resultsThe sequencing technique has been optimized at the stage of amplification of the variable VP1-2A structural genome segment. To obtain this result, we created original primer sequences and selected the polymerase reaction conditions (length, localization and annealing temperature, concentration of the primers and template). The sought fragment with a length of 249 nucleotide pairs was located between the 2988

conclusionThe sequencing technique optimized at the stage of VP1-2A fragment amplification allows one to determine the hepatitis A virus genotype, identify imported virus strains, and also establish an epidemiological relationship between different cases of the disease.