高通量半自动细菌核酸提取与纯化体系的构建

目的 建立高通量、半自动化核酸提取与纯化体系,满足病原体网络化监测的应用。方法 使用自动液体处理器、可丢弃性机械手枪头以及96孔板型核酸提取试剂盒实现细菌核酸提取与纯化的半自动化;比较构建的体系中,不同仪器参数组合条件下核酸的产量、质量以及样品之间的交叉污染率。结果 菌液的浓度与是否能够完成核酸提取以及核酸的质量密切相关。活塞移动速度、液体混匀体积、可丢弃型机械手枪头与液面之间的位置关系与样品之间的交叉污染密切相关;与传统的单个离心柱提取DNA的方法相比,本实验建立的高通量半自动化核酸提取与纯化体系的工作效率提高2.5倍。结论 本实验建立的高通量、半自动化核酸提取与纯化体系制备的核酸能够满足绝大多数下游分子生物学应用,该体系在病原体网络化监测实验室中有较好的应用前景。

Establishment of high-throughput half-automated nucleotide extraction and purification system

Objective To establish a high throughput half-automated nucleotide extraction and purification system to be used in pathogen surveillance network. Methods We used Perkin Elmer automated workstation and disposable tips to establish a half-automated nucleotide extraction and purification system. The cross-contamination and efficiency of the system were compared with different parameters. Results Move speed of the plunger and the relationship between tips and liquid surface are key factors that influence cross-contamination. The concentration of the broth culture was the key factor that determines DNA quantity and quality. Compared with manual method, this half-automated system improved the efficiency by 2.5 folds. Conclusion We establish a high throughput half-automated nucleotide extraction and purification system, which could be integrated into the molecular subtyping working pipeline of pathogen surveillance network laboratory.