Establishment Of Room-temperature-storable Quantitative Real-time PCR Mixes Based On Freeze-Drying

Quantitative real-time PCR(qPCR),as a commonly used method for nucleic acid testing,has been widely used in the prevention,diagnosis and treatment of disease.However,the preparation of liquid-form qPCR reagents is cumbersome and complicated,particularly when low quantities of each component are required,which increases the requirements for the operating environment and personnel quality.In addition,it’s hard for liquid-form qPCR reagents to maintain its bioactivity at room temperature,which limits its use outside the central laboratory.Vacuum freeze-drying technology(freeze-drying)can dehydrate qPCR reagents in a pre-mixed form and maintain their activity at room temperature for a long term.Besides,freeze-dried reagents are convenient to use because they only require reconstitution in water.This room-temperature-storable and simple to be used freeze-dried reagent is expected to be widely applied in many scenarios inside or outside central laboratories.The purpose of this study is to establish a method based on freeze-drying technology that ensures the qPCR reagents be stored at room temperature for a long term.Firstly,indexes to evaluate the performance of freeze-dried qPCR reagents were defined based on an initial freeze-drying method.And then according these indexes,optimal parameters for freeze-drying qPCR reagents for RNA or DNA detection were explored and confirmed,including the combination of lyophilized additives with appropriate concentrations,the procedures of freeze-drying suitable for qPCR reagents,and the components with suitable concentrations of qPCR reagents to be freeze-dried.Under these optimal parameters,the freeze-dried qPCR reagents for RNA target detection could be stored at room temperature for 569 days,and could be stored at 37℃,56℃,and 80℃ for 99 days,six days and one day,respectively;the freeze-dried qPCR reagents for DNA target detection could be stored at room temperature for 617 days,and could be stored at 37℃,56℃,and 80℃ for 617 days,26 days and 21 days,respectively.Moreover,this optimized freeze-drying method was universal and could be applied to different qPCR reagents,such as the qPCR reagents contained probes labeled with different fluorophores(FAM,HEX,ROX,CY5 and CY5.5),the qPCR reagents used for different targets(viruses,bacteria and cells)detection.And based on the research results,this article further clarified the key factors that might affect the activity of freeze-dried qPCR reagents during long-term storage,such as the thermal stability of bioactive material,the properties of selected lyophilized additive,the glyceryl content in qPCR reagents,the residual moisture content of the freeze-dried reagents,the storage temperature of the freeze-dried product,and so on.These in-depth studies are beneficial to prolong the shelf life of the freeze-dried qPCR reagents.In summary,this study has successfully established a universal freeze-drying method that allowed qPCR reagents to be stored at room temperature for 1-2 years,and clarified the key factors in maintaining the activity of freeze-dried qPCR reagents during long-term storage.In terms of operation,this method can simplify nucleic acid testing.In terms of storage,it can get the reagents for nucleic acid testing out of temperature restrictions.Using this method,it’s convenient for nucleic acid testing to be carried out in both central laboratories and resource-limited areas.