地被菊CVB病毒基因的RT-PCR检测
Development of RT-PCR method for detection of Chrysanthemum virus B in ground-cover chrysanthemums

为建立可靠、灵敏且高效的地被菊中菊花B病毒（Chrysanthemum virus B，CVB）的检测方法，利用特异性CVB基因引物，通过RT-PCR技术进行了特异性、重复性和灵敏度测试，最终利用优化的RT-PCR体系对地被菊植株感染CVB病毒情况进行了检测。结果显示，以染病地被菊植株的总RNA为模板扩增出的特异性片段编码211个氨基酸，与基因数据库中其它来源的CVB基因外壳蛋白同源性为96%~99%，可确定为菊花B病毒外壳蛋白基因；重复性和灵敏度检测中均获得了清晰的目标条带，且1 ng的总RNA即可扩增出特异性片段；对7个地被菊品种共32个植株的检测中，CVB的检出率为56.2%，检测的准确率为68.75%。表明建立的地被菊CVB基因的RT-PCR检测体系可有效用于地被菊植株感染病毒情况的鉴定。
To establish a reliable, sensitive and effective method to detect Chrysanthemum virus B (CVB) from ground-cover chrysanthemums, a method based on reverse transcription polymerase chain reaction (RT-PCR) was developed to amplify the target fragment using specific primer set for CVB. The specificity, repetitiveness and sensitivity of the method were then tested to optimize the assay. RT-PCR method was validated by surveying other chrysanthemum samples. In this study, total RNA extracted from infected chrysanthemum leaves was used as template for first-strand cDNA synthesis. The PCR product amplified by designed CVB primer pairs encoded 211 amino-acid residues and shared 96%-99% homology with other resources of CVB sequence in GenBank. The results indicated that the amplified product was CVB coat protein gene. Repetitiveness and sensitivity test indicated the specific product might be detected when the template was 1 ng total RNA. Screening 32 samples of other seven hybrids demonstrated that 56.2% of samples were positive for CVB, which showed that the rate of accuracy was 68.75%. In conclusion, the RT-PCR based method for detection of CVB was developed and could be applied effectively into the early virus survey of plant samples.