基于纳米孔测序的细菌基因组DNA提取试剂盒比较与流程优化研究OA

Objective To obtain high-quality nanopore sequencing data by comparing the performance of various commercial bacterial DNA extraction kits in extracting DNA from different bacterial species for nanopore sequencing and optimizing protocol,so as to enhance the accuracy of pathogen identification.Methods Firstly,Escherichia coli,Serratia marcescens,Pseudomonas aeruginosa,Staphylococcus aureus and Staphylococcus lentus were selected as the test samples,and then tested in combination with six commercially available kits with brands of MAGBETTER,Rebeads Biotech,TIANGEN Biotech(using the magnetic bead method),Omega Bio-tek,TIANGEN Biotech(using the centrifugal column method)and QIAGEN,so as to determine the kit suitable for nanopore sequencing by the comparison in terms of DNA yield,N/Q ratio,purity indices(A260/A280 and A260/A230)and electrophoresis bands.Secondly,the lysis parameters were optimized for the selected kits,and with Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus system optimization was carried out for lysis temperature(55,65,75℃),lysis time(10,20,40 min)and enzyme concentration to determine the optimal extraction protocol.Finally,nanopore sequencing was used to compare the extraction performance of the optimized protocol with that recommended in the kit manual.Results The TIANGEN Biotech kit(using the centrifugal column method)consistently yielded DNA concentrations exceeding 20 ng/μL in most samples,showed optimal extraction stability with N/Q ratios closest to 1 across different strains,indicated high data concentration,repeatability and alignment with the ideal reference ranges for the two purity indicators of A260/A280 and A260/A230,produced clear and distinct genomic DNA bands in all strains and ensured sufficient DNA yield from Gram-negative bacteria while maintaining efficient extraction from Gram-positive bacteria,which behaved the best and was selected for subsequent optimization experiments.By adjusting the lysis temperature,lysis time and enzyme concentration,the optimal lysis parameters for Escherichia coli were determined to be 55-65℃,10-20 min and 1×enzyme concentration,and the enzyme concentration could be reduced to 0.5×when sufficient samples were available and cost was taken into consideration;For Staphylococcus aureus,a robust lysis parameter range was identified as 55-65℃,10-20 min and 0.5-2×enzyme concentration.Both Escherichia coli and Staphylococcus aureus samples extracted under the conditions recommended in the kit manual and under optimized extraction conditions could be assembled into single,complete circular genomes,with reference genome alignment rates exceeding 90%in all the cases.When compared with the conditions recommended in the kit manual,the optimized extraction conditions made the N50 read length for Escherichia coli and Staphylococcus aureus increased significantly.Conclusion In nanopore sequencing,the TIANGEN Biotech kit(using the centrifugal column method)and optimization for its key lysis parameters contribute to increasing the amount of usable data and improving read length distribution without altering reagent composition or introducing significant exogenous contamination,thereby enhancing the completeness of subsequent genome assembly and the reliability of analytical results.[Chinese Medical Equipment Journal,2026,47(3):34-46]