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Virologica Sinica, 18 (5) : 437-440, 2003
Research Article
RT-PCR M ethods for Amplifying M Segment of Phleboviruses
Department of Etiology,Wuhan University Medical School,Wuhan 430071,China
2.Institute of Virology,Wuhan University Medical School,Wuhan 43007 1,China
3.Department of Pathology,Wuhan University Zhongnan Hospital, Wuhan 430071,China
4.Department ofPathology,University ofTexas Medical Branch,Galveston,Texas 77555-0609,USA
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
Key Words: Phlebovirus;  M segment;  r-PCR
  Published online: 5 Oct 2003
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