草莓斑驳病毒分子变异及PCR检测技术研究

Studies on Molecular Variations and PCR Detection of Strawberry Mottle Virus

【目的】明确草莓斑驳病毒(Strawberry mottle virus,SMoV)的分子变异特点;探索利用嵌套PCR和转录增强技术检测SMoV的方法。【方法】利用RT-PCR扩增SMoV3′非编码区(non-coding region,NCR)和大外壳蛋白(large coat protein,LCP)基因特异片段,并对扩增产物进行克隆测序。利用生物信息学软件分析不同地区分离物变异特点及系统发育关系。参考测序结果,在SMoV基因组保守区设计引物,利用嵌套PCR和转录增强技术检测SMoV。【结果】获得了SMoV中国分离物的NCR区和部分LCP基因核酸序列,不同分离物部分LCP基因核酸序列同源性为76.8%～99.7%。系统进化分析显示不同分离物呈现轻微的地理相关性。3个波兰分离物,4个中国分离物中的3个,7个荷兰分离物中的4个分别聚集成一簇;2个德国分离物与其它分离物亲缘关系较远,形成一个独立的分支。建立了利用半嵌套PCR和转录增强技术检测SMoV的技术体系,灵敏度高于常规PCR。【结论】SMoV不同分离物变异复杂,德国分离物可能是一个代表特殊株系的群体;基于定位基因组保守区引物,利用嵌套PCR和转录增强技术可有效检测SMoV。

[Objective] The study was conducted to analyze the characterization of molecular variation of Strawberry mottle virus （SMoV） and develop SMoV detection methods by nested PCR and transcriptional enhancement techniques. [Method] The 3′ non-coding region （NCR） and large coat protein （LCP） gene of SMoV were amplified by reverse transcription-polymerase chain reaction （RT-PCR）. The specific segments were cloned and sequenced. The characterization of molecular variation for some isolates of SMoV and phylogenetic analysis were studied by using some bioinformatics soflwares. The primers were designed in conserved region of genome of SMoV by analyzing the nucleotide acid sequences. SMoV was detected by nested PCR and transcriptional enhancement techniques. [Result] The nucleotide sequences of NCR and partial LCP gene of Chinese isolates were obtained. Sequence analysis of the partial LCP gene of various SMoV isolates showed nucleotide acid identities ranged from 76.8 to 99.7%. There was a slight tendency for isolates to group according to their geographical origin. All of three Polish isolates, four isolates among seven Dutch isolates, three isolates among four Chinese isolates formed a small separate branch, respectively. Two Germanic isolates had the far relationship with other isolates, formed a separate clade. The detection protocol for SMoV by semi-nested PCR and transcriptional enhancement techniques were developed, respectively. Both semi-nested PCR and transcriptional enhancement techniques were rather more sensitive than standard RT-PCR. [Conclusion] It had the enormous genetic variation for SMoV isolates. The Germanic isolates could be a group for representing of a specific strain. Based on the primers located in the conserved region of genome of SMoV, SMoV could be steadily detected by semi-nested PCR and transcriptional enhancement techniques.