Abstract:

Objective To investigate the application value of long-chain alcohols and oils as washing solution in nucleic acid extraction based on magnetic bead method. Methods Totally，10²-10⁵ copies/μL DNA and RNA mixtures were extracted by magnetic bead method，and 5 kinds of long-chain alcohols，including undecanol，decanol，octanol，1-nonanol and 2-dodecanol，and 3 kinds of oils，including dimethicone，FC-40 and paraffin oil，were determined as washing solution，with the traditional 70% ethanol as control. The extracted nucleic acids were amplified using polymerase chain reaction（PCR） or reverse transcription-polymerase chain reaction（RT-PCR），and the nucleic acid extraction efficiency of different washing solutions was evaluated by cycle threshold（Ct）. The nucleic acid extraction performance under different reagent status（dry reagents and wet reagents） and different mixing modes（vortex mixing，pipette mixing and inversion mixing） was compared using oils as washing solution，and the performance for extracting genomic nucleic acid from clinical pathogen samples was evaluated as well. Results The nucleic acid extraction performance of dimethicone and FC-40 among the 8 kinds of washing solutions was basically the same as that of traditional 70% ethanol. The 10²-10⁵ copies/μL DNA or RNA could be effectively extracted without inhibiting PCR and RT-PCR. When using 70% ethanol as control and dimethicone or FC-40 as washing solution，no statistical significance was found in Ct values of 10³ copies/μL DNA and RNA amplification，regardless of the combination of reagent status or mixing mode（P>0.05）. There was no statistical significance in Ct value of DNA extracted from Mycoplasma pneumoniae simulated clinical samples（P>0.05），and it was basically consistent with commercial nucleic acid extraction kits. Conclusions Dimethicone and FC-40 can be used as washing solutions for nucleic acid extraction by magnetic bead method and can be used for the development of microfluidic products.

Key words: Dimethicone, FC-40, Magnetic bead method, Nucleic acid extraction, Microfluidics