RT-PCR M ethods for Amplifying M Segment of Phleboviruses
Abstract: To amplify M fragment from unknown Phleboviruses,members of 7 serocomplexes and 8 no complex assigned Phleboviruses,total 42,were chosen and tested.Tl1e M segment amino acid sequences Of 4 Phleboviruses in GenBank were aligned.Conserved regions were selected to design primers. OligOnucleOtides were synthesized according to the cDNA sequences at the conservative regions.Then the OligOnucleOtides of the same region were mixed together as“cocktail"primers for RT-PCR.The amplification products were exam ined by agarose gel electrophoresis,purified an d sequenced directly. Th e am plification ratio with primer pair Ph—M一2FM and Ph—M一3RM was 8 1.0% (34,42),the size of the products were around 600bp.Tl1e am plification ratio with Ph—M一2FM an d Ph—M 一4R2M was 52.3% (22/42),the size of the products were around 1 400bp.BLAST search showed that the sequences of am plicons were homologous with known sequences of Phleboviruses.In this study,partial M fragment of sequence unknown Phleboviruses were amplified an d sequenced.TIle methods described here will be useful for genetic determination,phylogenetic analyses of Phleboviruses,an d diagnosis of Phlebovirus infections.