RT—PCR方法扩增白蛉热病毒M片段的研究

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—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—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。

RT-PCR Methods for Amplifying M Segment of Phleboviruses

To amplify M fragment from unknown Phleboviruses, members of 7 serocomplexes and 8 no complex assigned Phleboviruses, total 42, were chosen and tested. The 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 examined by agarose gel electrophoresis, purified and sequenced directly. The amplification ratio with primer pair Ph-M-2FM and Ph-M-3RM was 81.0% (34/42), the size of the products were around 600bp. The amplification ratio with Ph-M-2FM and Ph-M-4R2M was 52.3% (22/42), the size of the products were around 1400bp. BLAST search showed that the sequences of amplicons were homologous with known sequences of Phleboviruses. In this study, partial M fragment of sequence unknown Phleboviruses were amplified and sequenced. The methods described here will be useful for genetic determination, phylogenetic analyses of Phleboviruses, and diagnosis of Phlebovirus infections.