Abstract
Objective To optimize the experimental conditions for in-capillary DNA extraction through orthogonal test and to evaluate the optimized DNA extraction.Methods By using a protocol that controls droplet with magnetic beads,the in-capillary DNA extraction method was developed which involved sample introduction,DNA binding,magnetic beads rinsing and elution of DNA sequentially processed in a polytetrafluoroethylene (PTFE) capillary.Conventional cleavage by boiling was used as control approach.The orthogonal test was used to study the experimental conditions for DNA extraction of blood hepatitis B virus (HBV),which included the effects of DNA binding time,elution time and elution temperature on the performance of the in-capillary DNA extraction.Efficiency and reproducibility of the in-capillary DNA extraction assay,as well as the dependence of Ct value of extracted DNA on input DNA concentration were also appraised.The quantitative DNA recovery and time needed for DNA extraction were compared between the capillary-based and conventional methods.Results DNA binding for 5 min and elution at 60 ℃ for 1 min was the optimal experimental condition for in-capillary method showed by the orthogonal test.Fluorescent quantitative PCR showed good repeatability of DNA recovery and positive correlation of the DNA production with the amount of HBV DNA input (r=0.9999,P＜0.01).Paired t-test analysis showed significantly higher DNA recovery using the capillary method compared to the conventional one (t=-4.263,P＜0.001).Under optimized condition,the entire process of DNA extraction could be completed in 23 min vs 45 min or longer with conventional approach.Conclusion The experimental condition of in-capillary DNA extraction is optimized successfully using orthogonal test,and a rapid and highly efficient DNA extraction mothod is established. Key words: Laboratory techniques and procedures; Polymerase chain reaction; Orthogonal test; Capillary; DNA extraction
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