Study On Freeze-Drying Of Reagents For Nucleic Acid Extraction And Amplification In Point Of Care Testing

Nucleic acid extraction and amplification reagents used for nucleic acid detection contain protein active substances(proteinase K,polymerase,etc.),and these bioactive components in liquid format are difficult to maintain their biological activity while under room temperature conditions.The conditions of low temperature storage and transportation(4 ℃ and-20 0C)limit the clinical application and industrialization of nucleic acid detection in rural areas severely.Therefore,it is necessary to explore a reliable and efficient method,which could realize the room temperature preservation of extraction and amplification reagents used for nucleic acid detection.Since the stability of the bioactive raw material in the solid dry powder state is much higher than that of the liquid state,long-term storage at room temperature could be achieved by freeze-drying treatment.In this study,we pre-mixed magnetic beads,proteinase K and acryl carrier used for nucleic acid extraction;and buffer,deoxynucleotide triphosphates(dNTPs),primers,probes,and enzymes used for nucleic acid amplification,respectively,then the lyophilization(freeze-drying)treatment is carried out.A lyophilization method for the nucleic acid detection reagents were established preliminarily.For nucleic acid amplification reagents,we screened out that trehalose and sucrose as suitable freeze-drying protective additive for nucleic acid detection reagents firstly,which could prevent bioactive components from being damaged during lyophilization.Taking the price and storage temperature of the two sugars into account,we finally chose sucrose as protective additive with an optimum concentration of 200 mmol/L;Secondly,in order to facilitate the dispensing of lyophilized reagents in practical applications,it was found that polyethylene glycol 20000(PEG20000)could be used as an excipient to realize the skeleton formation of a complete freeze-dried bead and transfer of lyophilized reagents,and the added concentration of PEG20000 had been optimized:2.8 mmol/L;Further,the long-term preservation effect of lyophilized reagents were verified by accelerated stability evaluation experiments:After the lyophilized nucleic acid amplification reagents were sealed and stored at 20 0C for two weeks,the detection performance was consistent with the non-lyophilized liquid control reagents.For nucleic acid extraction reagents,the magnetic beads were separated from the proteinase K and the acryl carrier while freeze-drying.Based on the lyophilization process of nucleic acid amplification reagents,the amount of additive for the beads while freeze-drying was determined:10 μL 20%PEG20000(w/v%);the added concentration of sucrose for lyophilized proteinase K and the acryl carrier was:200 mmol/L,and the additive amount of the excipient:10 μL 20%PEG20000(w/v%).After the lyophilized nucleic acid extraction reagents were sealed and stored at 20 ℃and 37 0C for 18 days,the detection performance was consistent with the non-lyophilized liquid control reagents.In this study,lyophilized nucleic acid extraction and amplification reagents were prepared by vacuum freeze-drying method,and realizing the storage and transportation of nucleic acid detection reagents(extraction and amplification)at room temperature.The lyophilized reagent has a complete lyophilized bead form and can be transferred.The results of this study are conducive to the sinking of nucleic acid detection technology in primary health care units.