摘要：为建立扩增未知序列白蛉热病毒M 片段的RT-PCR方法，本研究选取7个血清组成员和8个未分组血清型， 共42株白蛉热病毒为RT-PCR检测对象。通过排列GenBank中已知的4型白蛉热病毒M片段氨基酸序列，选择 保守区设计引物。根据保守区各已知病毒的cDNA特异序列合成寡核苷酸，将相同区的寡核苷酸等量混合成为“鸡 尾酒”引物，用于RT-PCR。扩增产物经电泳检测，纯化后直接测序。引物对Ph—M一2FM 和Ph．M-3RM扩增产物 长度约为600bp，从42株病毒中扩增出34株，阳性率为81．0％。另一引物对Ph-M一2FM 和Ph．M．4R2M扩增产物 长度约为1400bp，扩增出22株病毒，阳性率为52．3％。测出序列经BLAST检索，与GenBank中已知白蛉热病毒 同源。本研究首次成功地应用RT-PCR扩增不同血清型未知序列白蛉热病毒的部分M 片段，并测出扩增产物序列， 为白蛉热病毒属成员的基因鉴定和种系发生关系分析提供了实验手段，并将有助于白蛉热病毒感染的基因诊断。

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．