PCR检测草莓镶脉病毒的稳定性研究

Enhancing the stability of detection of Strawberry vein banding virus by PCR

利用改进的CTAB法提取了优质的草莓总DNA,可以稳定地进行草莓植株中SVBV的PCR检测。对PCR检测草莓植株中SVBV的反应体系进行了优化,结果表明,Promega公司的Taq酶与TaKaRa公司的PCR反应缓冲液配合使用效果较好,适宜引物浓度为0.5μmol/L,退火温度为53℃。20μLPCR反应液中包含0.001μL总DNA时能扩增出SVBV特异谱带,相当于能够从不足1μg的草莓新鲜叶片中检测出SVBV。基于对SVBV中国分离物外壳蛋白基因的序列分析比较,设计并筛选了检测SVBV的理想引物,提高了利用PCR检测草莓植株中SVBV方法的稳定性。

The highly pure total DNA used for stably detecting Strawberry vein banding virus （SVBV） by polymerase chain reaction （PCR） was extracted from strawberry （Fragaria spp.） with modified CTAB method. The PCR reaction system of detection of SVBV in strawberry was optimized, and the results showed： it was good that Taq DNA polymerase from Promega was combined with the PCR reaction buffer from TaKaRa, the feasible concentration of primer was 0.5μmol/L, and the feasible melting temperature was 53℃. The SVBV was detected when 20μL PCR reaction volumes contained 0.001μL total DNA, which is an amount less than 1μg of fresh material. Based on the result of sequencing and analyzing of the gene encoding coat protein of SVBV isolations in China, ideal PCR primers of detection SVBV in strawberries were designed and screened out. This research enhances the stability of detection of SVBV in strawberry by PCR.