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目的探讨一种高效提取RNA噬菌体核酸的新方法,并通过核酸分析从基因水平了解宿主谱改变对噬菌体核酸的影响。方法采用传统的聚乙二醇(PEG)沉淀蛋白酶消化酸性酚提取法、Tripure提取法以及抗体沉淀盐酸胍一步法3种方法分别提取大肠埃希菌f2噬菌体及其宽宿主谱株的RNA,通过琼脂糖凝胶电泳鉴定其纯度。采用兼并引物逆转录聚合酶链反应(RTPCR)及随机引物随机扩增多态性DNA聚合酶链反应(RAPDPCR)比较分析噬菌体宿主谱改变前后其核酸序列组成的变化。结果抗体沉淀盐酸胍一步法提取的噬菌体核酸具纯度高、条带完整等优点;f2噬菌体及其宽噬株均为6000bp左右的单链RNA噬菌体;两噬菌体基因cDNA扩增出的RAPDDNA片段差异明显,其中26条为可区分的DNA带型;噬菌体f2cDNA在450bp附近出现重复性较好的扩增片段,而其宽噬株cDNA在相同条件下未出现扩增产物。结论抗体沉淀盐酸胍一步法较其他两种RNA病毒核酸提取法具有明显的优势,具有应用价值的新的RNA噬菌体核酸的提取方法;宽宿主谱噬菌体的宿主特异性裂解效应已从基因水平发生了变化。
OBJECTIVE: To study a new method for efficient extraction of RNA phage nucleic acids and to understand the effect of changes in the host spectrum on phage nucleic acids at the gene level by nucleic acid analysis. Methods RNA extracted from Escherichia coli bacteriophage f2 and its broad-host spectrum by conventional protease-digested acidic phenol extraction method, Tripure extraction method and antibody precipitation guanidine hydrochloride method were used to separate RNA from Agarose gel electrophoresis to determine its purity. The changes of the nucleic acid sequence composition of the phage before and after the change of the phage were analyzed by RTPCR and random amplified polymorphic DNA polymerase chain reaction (RAPDPCR). Results The phage nucleic acid extracted by one-step method with guanidinium hydrochloride showed the advantages of high purity and complete band. The f2 phage and its wide-baker phage were about 6000bp single-stranded RNA phage. , Of which 26 were distinguishable DNA bands. The f2cDNA of phage displayed a repeatability amplified fragment near 450bp, whereas the cDNA of wide-breeder cDNA did not appear to be amplified under the same conditions. Conclusion The antibody-precipitated guanidine hydrochloride one-step method has obvious advantages over the other two RNA virus nucleic acid extraction methods, and has a novel method of extracting RNA phage nucleic acids. Host-specific lysis of broad-spectrum host bacteriophage has occurred at the gene level Variety.