三种 DNA 提取方法对检测草莓镶脉病毒稳定性的研究

摘要

草莓成熟叶片中含有大量的多糖、蛋白质、多酚和色素等物质，影响高质量DNA的提取和下游PCR反应的进行。为确定适用于PCR技术检测草莓镶脉病毒（ Strawberry vein banding virus，SVBV）的DNA提取方法，从草莓叶片中提取DNA后，对样品中的SVBV进行特异性的PCR检测，比较确定不同DNA提取方法对PCR检测SVBV稳定性的影响。以感染SVBV的草莓叶片为材料，分别采用CTAB试剂盒法、高盐低pH法、改良SDS法提取草莓叶片的总DNA，将不同方法提取的DNA通过紫外分光光度法和琼脂糖凝胶电泳法进行测定，对DNA产量和质量进行分析。结果表明，3种方法均能获得较为完整的DNA片段，用CTAB试剂盒提取的DNA质量最高，改良SDS法提取的DNA质量较低。将不同方法提取的DNA经过PCR反应特异性检测SVBV后，将与目的片段大小一致的PCR扩增产物经序列测定及分析，证明与SVBV的序列相吻合，证明了方法的可靠性，其质量能够满足PCR特异性检测需要。%Different DNA isolation methods were used in researches due to the diversity of secondary metabo -lites in different plant tissues .The existing of large amounts of polysaccharides ,proteins,polyphenols and pigments in strawberry plants influenced the extraction of high quality DNA from strawberry leaves and afterwards PCR reac -tion .In order to identify the DNA extraction method which was applied to the PCR detection for Strawberry vein ban-ding virus ( SVBV ) , total DNAs extracted from strawberry leaves by three different extraction methods to detect SVBV by PCR were tested and compared in this study .Strawberry leaves infected by SVBV were used as the materi-als from which CTAB kit method,high salt and low pH method (HSLPM) and modified SDS method were used to extract DNA in this paper .The quality and quantity of DNA extracted by these three different methods were analyzed by gel electrophoresis and UV spectrophotometers .SVBV was detected by PCR from extracted DNA to determine the effect of different DNA extraction methods on the stability of PCR detection .The results showed that all the three methods could isolate complete DNA fragments .Sequencing and sequence analysis of the PCR products from straw-berry leaves infected by SVBV proved the reliability of the methods .DNA extracted by three methods from strawber-ry leaves could be detected the infection of SVBV showed the quality of DNA extracted by all the three methods could meet the specific demands of PCR detection of SVBV in strawberry leaves .Compared with the other two ex-traction methods ,results showed the quality of DNA extracted by CTAB kit was the best in these three different DNA extraction methods in this paper .