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以甜樱桃(Prunus avium L.)品种红灯的叶片为材料,提取总RNA,选用随机六聚体引物进行反转录合成cDNA,根据李矮缩病毒外壳蛋白基因设计2对特异引物,分别从感病样品中扩增出与预期片段大小相符的目的片段。通过对RT-PCR反应体系中引物、模板浓度和退火温度的优化,改进了现有的李矮缩病毒的RT-PCR检测方法,并成功用于山东泰安地区甜樱桃果园的病毒调查。另外,还可以扩增18 sRNA,实现对李矮缩病毒外壳蛋白基因表达的相对定量分析。
The total RNA was extracted from leaves of red varieties of Prunus avium L. The cDNAs were synthesized by reverse transcriptase using random hexamer primers. Two pairs of primers were designed according to the coat protein gene of Lentinus edodes. In the susceptible sample, a target fragment corresponding to the expected fragment size is amplified. Through the optimization of primers, template concentration and annealing temperature in the RT-PCR reaction system, the existing RT-PCR detection method of Li-dwarf virus was improved and the virus was successfully used in the virus investigation of sweet cherry orchard in Tai’an, Shandong. In addition, it is also possible to amplify 18 sRNA to achieve a relative quantification of the gene expression of the coat protein gene of the dwarf virus.