全自动核酸高效提取纯化工作站及应用
首发时间:2020-07-02
摘要:随着当今临床诊断医学快速发展,分子诊断技术已成为研究及开发的热点及趋势,高质量的核酸提取技术是进行分子诊断关键。本文设计开发了8通道的全自动核酸高效提取纯化工作站,开发了基于特殊工艺制备的硅质滤芯核酸提取tip头,建立了全自动核酸高效提取纯化方法,并模拟了复杂的痰液中结核分枝杆菌基因组DNA的提取方法,进行了效果验证。 实验结果表明,利用自主开发的全自动核酸高效提取纯化工作站,在人工模拟的痰液加入高浓度(100ng)和低浓度(0.01ng)的结核杆菌基因组DNA进行体外抽提和纯化。高浓度组中8通道抽提的CV平均为1.84%,低浓度组中8通道抽提的CV平均为1.39%,均小于5%。进行抽提污染实验,8通道交叉抽提阴性与阳性实验组,实验结果中阴性与阳性结果无干扰。进行全阴性样本抽提,实验结果正常,不存在污染情况。以上实验说明不存在交叉污染问题。 灵敏度:在该试验方法中最高检出限为100ng,最低检出限为0.01ng,远远满足实际痰液样本中可抽提的含量。本文获得的研究成果为研究开发先进的国产化核酸高效提取纯化设备,建立高效提取纯化方法,推动了分子诊断技术进步意义重大。
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Establishment of rapid extraction and purification methods for nucleic acids in clinical samples
Abstract:With the rapid development of clinical diagnosis medicine, molecular diagnosis technology has become a research and development hotspot and trend. High quality nucleic acid extraction technology is the key to molecular diagnosis. In this paper, an 8-channel fully automatic nucleic acid extraction and purification workstation is designed and developed, and a tip head for nucleic acid extraction from siliceous filter element based on special technology is developed. A fully automatic high-efficiency nucleic acid extraction and purification method is established, and the extraction method of Mycobacterium tuberculosis genomic DNA in complicated sputum is simulated, and the effect is verified. The experimental results showed that the genome DNA of Mycobacterium tuberculosis was extracted and purified in vitro by adding high concentration (100ng) and low concentration (0.0lng) of Mycobacterium tuberculosis genomic DNA into the artificial sputum. The average CV of 8-channel extraction was 1.84% in high concentration group and 1.39% in low concentration group. In the 8-channel cross extraction negative and positive groups, there was no interference between the negative and positive results. All negative samples were extracted and the results were normal without contamination. The above experiments show that there is no cross contamination. Sensitivity: in this test method, the highest detection limit is 100ng, and the lowest detection limit is 0.01ng, which is far enough to meet the actual content of sputum samples. The research results obtained in this paper are of great significance for the research and development of advanced domestic equipment for efficient extraction and purification of nucleic acids, the establishment of efficient extraction and purification methods, and the advancement of molecular diagnosis technology.
Keywords: Nucleic acid extraction workstation Filter element Artificial sputum Mycobacterium tuberculosis
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