基于硅质滤芯的核酸提取方法
首发时间：2020-07-01
摘要：近年随着分子生物学技术及应用的飞速发展，传统的核酸提取方法已无法满足分子检测技术要求，因此，新型的核酸提取纯化方法成为行业关注热点。本文开发了基于硅质滤芯的核酸提取纯化方法，并对硅质滤芯法和传统的柱离心法进行对比评估，建立一种回收率高、提取核酸纯度高和稳定性好的提取纯化方法。开发了基于硅质滤芯的快速提取核酸的裂解液和洗脱液试剂，以克雷伯氏菌为研究对象将两种检测方法进行对比，利用实时荧光定量PCR、紫外分光光度计等方法进行了核酸提取效果表征。硅质滤芯对核酸回收率为66%左右，柱离心提取核酸法CV值为3.1%，回收率为32%。硅质滤芯法比柱离心法回收率高2倍，纯度较好且稳定。利用硅质滤芯方法提取核酸的CV值小于5%。样本核酸提取浓度、纯度、稳定性上比离心柱的提取方法更具优势。本文研究的基于硅质滤芯的核酸提取技术为开发新型的核酸提取技术提供参考，便于结合自动化工作站实现高通量提取，满足分子生物学下游技术中对高精度核酸样本要求，提供实验技术支撑。
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Nucleic acid extraction method based on silicon filter
Abstract：With the rapid development of molecular biology technology and applications, traditional nucleic acid extraction methods can no longer meet the requirements of molecular detection technology. Therefore, new nucleic acid extraction and purification methods have become the focus of industry attention. In this paper, a nucleic acid extraction and purification method based on a silicon filter element was developed, and a comparison and evaluation of the silicon filter element nucleic acid extraction method and the traditional column centrifugation method was carried out to establish an extraction and purification method with high recovery rate, high purity of extracted nucleic acid and good stability. The lysate and eluent reagents for rapid nucleic acid extraction based on silicon filter element were developed. The two detection methods were compared with Klebsiella as the research object, and real-time fluorescence quantitative PCR and ultraviolet spectrophotometer were used. Characterization of nucleic acid extraction effect. The recovery rate of nucleic acid with silicon filter element is about 66%, the CV value of column centrifugal nucleic acid extraction method is 3.1%, and the recovery rate is 32%. The method in this paper has twice the recovery rate of column centrifugation, and the purity is better and stable. The CV value of nucleic acid extraction using silicon filter element method is less than 5%. The sample nucleic acid extraction concentration, purity, and stability are more advantageous than the spin column extraction method. The nucleic acid extraction technology based on silicon filter element studied in this paper provides a reference for the development of a new type of nucleic acid extraction technology, which is easy to combine with an automated workstation to achieve high-throughput extraction, meet the requirements of nucleic acid samples in molecular biology downstream technology, and provide experimental technical support.
Keywords： Biomedical engineering Nucleic acid extraction Silicon filter element Column centrifugation method
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