凹凸棒土作为食源性致病菌核酸提取材料的可行性研究

Feasibility study of attapulgite as a nucleic acid extraction material for foodborne pathogens

目的研究凹凸棒土作为一种低成本、高效的食源性致病菌核酸提取材料的可行性。方法利用X射线衍射(X-ray diffraction,XRD)、傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)、Zeta电位分析、扫描电子显微镜(scanning electron microscope,SEM)和比表面积及孔径分析(brunauer-emmett-teller,BET)对凹土样品进行表征,建立凹土样品对DNA吸附和解吸附的方法和体系,并进行条件优化。利用吸附动力学模型和吸附等温模型考察其对DNA的吸附作用,以金黄色葡萄球菌为核酸提取对象,考察核酸提取的得量、纯度与完整性。结果吸附体系中最适pH为5.0,Na+最适浓度为300 mmol/L,准一级动力学模型和Freundlich模型更好地模拟吸附结果,凹凸棒土对DNA饱和吸附量理论值达到18.8972 ng/μg,选取25 mmol/L三聚磷酸钠为解吸剂,解吸率可达95.80%±0.44%。将凹凸棒土应用于金黄色葡萄球菌核酸的提取可获得完整、得量为(4.74±0.20)μg的基因组DNA。结论凹凸棒土对DNA有较强的吸附效果,且选取合适的解吸剂DNA易洗脱。以凹凸棒土为核酸固相提取材料,有望实现食源性致病菌基因组DNA的低成本、高效提取。

Objective To explore the feasibility of attapulgite as a low-cost and efficient nucleic acid extraction material for foodborne pathogenic bacteria.Methods The attapulgite from was characterized by X-ray diffraction(XRD),Fourier transform infrared spectroscopy(FTIR),Zeta potential analysis,scanning electron microscope(SEM)and brunauer-emmett-teller(BET)specific surface area and pore size analysis,and then the method and system of DNA adsorption and desorption of attapulgite were established,and conditions were optimized.The adsorption kinetics model and adsorption isotherm model were used to investigate its adsorption on DNA.The amount,purity and integrity of nucleic acid extraction were investigated with Staphylococcus aureus as the object of nucleic acid extraction.Results The experimental results showed that the optimum pH of the adsorption system was 5.0,and the optimum concentration of Na+was 300 mmol/L.The Pseudo-first-order kinetic model and Freundlich model could better simulate the adsorption results,and the saturated adsorption amount of attapulgite to DNA reached 18.8972 ng/μg.With 25 mmol/L sodium tripolyphosphate as the desorption agent,the desorption rate could reach 95.80%±0.44%.Attapulgite was applied to the extraction of nucleic acid from Staphylococcus aureus to obtain complete and yield(4.74±0.20)μg of genomic DNA.Conclusion Attapulgite has a strong adsorption effect on DNA,and DNA can be easily eluted by selecting appropriate desorption agent.Using attapulgite as the solid phase extraction material of nucleic acid can realize the low-cost and efficient extraction of the genomic DNA of foodborne pathogenic bacteria.