香石竹斑驳病毒的鉴定和RT-PCR检测

Identification and detection of carnation mottle virus on carnation by RT-PCR

从表现为叶斑驳、花碎色症状的香石竹病株上获得一病毒分离物 ,电镜负染观察到直径为 2 8～ 33nm的球状粒子。病毒提取液经紫外光测定呈典型核蛋白吸收曲线 ,OD2 60 /OD2 80 =1 70 ;血清学反应与CarMV抗血清出现明显的沉淀线。通过以上实验结果 ,确定该病毒分离物为香石竹斑驳病毒 (carnationmottlevirus ,CarMV)。根据该病毒的RNA序列设计引物 ,对病健材料进行了RT PCR检测 ,结果从感病材料中扩增出大约 6 0 0bp的特异片段 ,而健康植物无此扩增带。将PCR产物连接 pGEM-T-easy载体 ,转化大肠杆菌JM 10 9,得到了含目的片段的重组子 ,经双脱氧序列分析 ,与Guilley报道的序列对应部分的核苷酸序列基本一致 (其同源性达 96 % ) ,最低检出病毒核酸含量为 5ng ,表明应用RT

A virus isolated from carnation with mosaic leaves and broken flowers. Through TEM, the virus particle was spherical of about 28-33 nm in diameter. The purified virus from infected tissues showed typical ultraviolet absorption of nucleoprotein, i.e. OD 260 /OD 280 =1.7. Virus particles were in bar shape with antiserum of CarMV by immunodiffusion tests, which showed that the virus isolate was identical with carnation mottle virus (CarMV). A pair of primers were designed based on the sequence of CarMV RNA. RT-PCR was applied with total RNA of the purified virus, of infected tissues and healthy tissues. A fragment of about 600 bp was amplified from the infected samples, but not from healthy samples. The PCR product was cloned into pGEM-T-easy Vector, and transformed into Escherichia coli JM109. The recombinant plasmid was obtained and sequenced with Sanger's method. The result showed that two sequences were almost identical (96% identity), compaired with the sequence of CarMV RNA reported by Guilley. The lowest detected RNA content by RT-PCR was 5ng. It proved that detection of CarMV by RT-PCR is feasible.