| 孔宝华1;蔡红1;刘进元2;陈海如1;常胜军2;李文君2;段永嘉1.香石竹斑驳病毒的鉴定和RT-PCR检测[J].植物保护,2002,28(1):5-8. |
| 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 |
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| DOI： |
| 中文关键词: 香石竹 香石竹斑驳病毒 鉴定 RTPCR检测 |
| 英文关键词:Dianthus caryophyllus L. CarMV identification RT-PCR detection |
| 基金项目: |
| 孔宝华1;蔡红1;刘进元2;陈海如1;常胜军2;李文君2;段永嘉1 |
| 1.云南农业大学云南省植物病理重点实验室;2.清华大学生物科学与技术学院 |
| 摘要点击次数: 621 |
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| 中文摘要: |
| 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28～33nm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 ,OD260/OD280=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(carnation mottle virus ,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600bp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JM109,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-PCR检测香石竹斑驳病毒是可行的 |
| 英文摘要: |
| 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. OD260/OD280=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. |
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