重庆辣椒上番茄斑萎病毒的血清学检测及分子鉴定

为明确重庆地区辣椒上番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的发生情况及其外壳蛋白基因(cp)序列变异特征,采用斑点免疫杂交法(Dot-ELISA)和反转录PCR(RT-PCR)技术对采自重庆的298份辣椒病毒病样品进行了检测和分子鉴定。Dot-ELISA检测结果表明,有40份辣椒样品检测到TSWV,检出率为13.42%。通过电子显微镜在TSWV检测呈阳性的样品中观察到具包膜的球状病毒粒体。设计扩增TSWV cp的引物,选取6个TSWV阳性样品进行RT-PCR扩增,均扩增到大小约750 bp的目的片段。序列分析表明,重庆辣椒分离物TSWV-CQ(KX611497)与已报道的22个TSWV分离物cp基因的核苷酸相似性为96.1%~99.4%,与已报道的TSWV中国分离物亲缘关系较近,表现出明显的地理相关性。

Serological Detection and Molecular Identification of Tomato spotted wilt virus in Pepper in Chongqing

To identify the occurrence and variation of the coat protein gene(cp)of Tomato spotted wilt virus(TSWV)in pepper in Chongqing,a total of 298 diseased pepper samples were collected and subjected to the detection of Dot-ELISA with specific antiserum of TSWV and RT-PCR with specific primers.Results of Dot-ELISA detection revealed that 40 samples were positive with a detection ratio of 13.42％.Typical enveloped spherical particles were observed in the TSWV-positive samples through electron microscopy.Six positive samples were randomly selected and amplified by RT-PCR with primers specific for the cp gene of TSWV,and results revealed that an approximate 750 bp fragment was obtained in each sample.Sequencing showed that cp gene of the isolate TSWV-CQ (KX611497) shared 96.1％ to 99.4％ identities with those of the other 22 previously reported TSWV isolates.Phylogenetic analysis showed that the isolate TSWV-CQ was most closely related to TSWV isolates of China and suggested that geographical selection force played a role in virus evolution.