快速提取肉类基因组DNA的裂解液的研究

Study on rapid extraction lysate of meat genomic DNA

中文关键词:  重组酶聚合酶扩增技术  现场检测  肉类  裂解液  基因组DNA

英文关键词:recombinase polymerase amplification  on-site detection  meat  lysate  genomic DNA

中文摘要:

      目的 配制一种快速提取肉类DNA且满足重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)要求的裂解液。方法 分别配制3种裂解液: NaOH裂解液、Chelex裂解液与PBS裂解液, 取200 μL裂解液与20 mg肉类样品进行混合, 振荡5 s, 取上清液稀释10倍直接作为扩增模板。结果 3种裂解液中NaOH裂解液提取DNA在稀释与未稀释条件下均有较好的扩增, Chelex裂解液提取DNA在稀释与未稀释下均无扩增, PBS裂解液提取DNA在稀释作用下有较好的扩增, 在未稀释作用下无扩增。裂解液提取法和试剂盒方法提取的纯度、产物的大小几乎一致, 所扩增的为同一产物, 裂解液所需成本与试剂盒方法相比较低。结论 NaOH裂解液可以在5 min内提取肉类中的DNA, 浓度与纯度均较高, 所需时间短, 成本低, 且满足分子检测要求。

英文摘要:

      Objective To prepare a lysate rapidly extractingmeat DNA and meetingthe requirements of recombinase polymerase amplification (RPA). Methods The 3 kinds of lysates including NaOH lysate, Chelex lysate and PBS lysate were prepared. 200 μL of lysate was mixed with 20 mg of meat sample, shaken for 5 s, and the supernatant was diluted 10 times directly as an amplification template. Results The DNA extracted from the NaOH lysate in the 3 kinds of lysates was well amplified under both diluted and undiluted conditions. The DNA extracted from the Chelex lysate was not amplified in both diluted and undiluted, and the DNA extracted from the PBS lysate was diluted. There was better amplification and no amplification without dilution. The purity and product size extracted by the lysate extraction method and the kit method are almost the same, and the same product is amplified, and the cost of the lysate is lower than that of the kit method. Conclusion The NaOH lysate can extract DNA with high concentration and purity in the meat within 5 min, the time required is short, and the cost is low, which can meet the molecular detection requirements.