Summary of the invention
Technical problem to be solved by this invention is: provide a kind of lyophilized vaccine being applied to nucleic acid amplification system.Lyophilized vaccine of the present invention is that the freeze drying technology being applied to fluorescent quantitation diagnostic nucleic acid reagent provides a kind of effective, freeze-drying system that can realize.Diagnostic reagent is by cryodesiccated technology, and liquid reagent becomes lyophilized powder, with the preservation that the form of lyophilized powder can be more stable, reach present stage diagnostic nucleic acid reagent be beyond one's reach normal temperature transport and normal temperature preservation object, decrease reagent multigelation.Only need a step aquation to add sample again during detection and can realize testing goal, simple to operate.
For solving the problems of the technologies described above, the invention provides a kind of nucleic acid amplification system, comprising: damping fluid, enzyme mixation, nucleic acid amplification reaction liquid, and, low-temperature freeze drying protective material.
Described damping fluid comprise in following component further one or more: dNTP, Mgcl2, Thris-Hcl, KCl.
Described enzyme mixation comprise in following component further one or more: RNA reversed transcriptive enzyme, RAN inhibitor, archaeal dna polymerase.
Described nucleic acid amplification reaction liquid comprise in following component further one or more: upstream primer, downstream primer, probe.
Described lyophilized vaccine, comprising: trehalose.
The final concentration that in described lyophilized vaccine, trehalose uses is 1 ~ 10mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.2 ~ 5mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.5 ~ 2.5mmol/L.
Described lyophilized vaccine comprise in following carbohydrate/polyvalent alcohol further one or more: sucrose, Glycerose, pectinose, lyxose, pentose, ribose, wood sugar, semi-lactosi, glucose, hexose, seminose, talose, heptose, glucose, fructose, glyconic acid, sorbyl alcohol, lactose, methyl α pyranoside, maltose, saccharosonic acid, xitix, lactone, sorbose, saccharic acid, erythrose, threose, pectinose, allose, altrose, sucrose, gulose, idose, talose, erythrulose, ribulose, psicose, tagatose, Dextran 40.
Described lyophilized vaccine comprise in following polymkeric substance further one or more: HES (hydroxyethylsulfonic acid), PVP (polyvinylpyrrolidone), PEG (polyoxyethylene glycol), dextran, albumin, 30 POVIDONE K 30 BP/USP 24.
Described lyophilized vaccine comprise in following solvent further one or more: ethylene glycol, glycerine, DMSO (dimethyl sulfoxide (DMSO)), DMF (dimethyl formamide).
Described lyophilized vaccine comprises tensio-active agent further.
Described tensio-active agent is Tween80.
Described lyophilized vaccine comprises amino acids further.
Described lyophilized vaccine comprise in following salt further one or more: phosphoric acid salt, acetate, Citrate trianion.
The lyophilized vaccine of described nucleic acid amplification system does not comprise N.F,USP MANNITOL.
For solving the problems of the technologies described above, present invention also offers a kind of lyophilized vaccine being applied to nucleic acid amplification system, it is characterized in that, comprising: trehalose.
The final concentration that in described lyophilized vaccine, trehalose uses is 1 ~ 10mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.2 ~ 5mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.5 ~ 2.5mmol/L.
Described lyophilized vaccine, comprise in following carbohydrate/polyvalent alcohol further one or more: sucrose, Glycerose, pectinose, lyxose, pentose, ribose, wood sugar, semi-lactosi, glucose, hexose, seminose, talose, heptose, glucose, fructose, glyconic acid, sorbyl alcohol, lactose, methyl α pyranoside, maltose, saccharosonic acid, xitix, lactone, sorbose, saccharic acid, erythrose, threose, pectinose, allose, altrose, sucrose, gulose, idose, talose, erythrulose, ribulose, psicose, tagatose, Dextran 40.
Described lyophilized vaccine, comprise in following polymkeric substance further one or more: HES (hydroxyethylsulfonic acid), PVP (polyvinylpyrrolidone), PEG (polyoxyethylene glycol), dextran, albumin, 30 POVIDONE K 30 BP/USP 24.
Described lyophilized vaccine, comprise in following solvent further one or more: ethylene glycol, glycerine, DMSO (dimethyl sulfoxide (DMSO)), DMF (dimethyl formamide).
Comprise tensio-active agent further.
Described tensio-active agent is Tween80.
Comprise amino acids further.
Comprise in following salt further one or more: phosphoric acid salt, acetate, Citrate trianion.
The lyophilized vaccine of described nucleic acid amplification system does not comprise N.F,USP MANNITOL.
For solving the problems of the technologies described above; present invention also offers a kind of preparation method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one; it is characterized in that, comprise the following steps: by described component according to the concentration ratio mixing used, namely obtain lyophilized vaccine.
For solving the problems of the technologies described above, invention further provides a kind of preparation method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, comprising the following steps:
Step 1: by described component according to the concentration ratio mixing used;
Step 2: lyophilize, namely obtains lyophilized vaccine.
Described cryodesiccated step, comprises following sub-step further:
Sub-step 1: dry step;
Sub-step 2: the step of freeze-drying;
In described sub-step 1, freeze-dried and volume ratio that is nucleic acid amplification reaction reagent is 3.6-6.8:13.2-16.3, and described nucleic acid amplification reaction reagent is RNA or DNA real-time fluorescence quantitative PCR reaction reagent;
In described sub-step 1, the step of described drying adopts vacuum freezedrying;
In described step 2, the method for described freeze-drying comprises with the next stage:
The pre-freeze stage: baffle temperature drops to-45 DEG C--55 DEG C, hold-time 3-5h;
A sublimation stage: keep 2-4h;
Secondary sublimation stage: keep 2-4h;
Whole drying stage: keep 2-3h.
For solving the problems of the technologies described above, the present invention has reoffered a kind of using method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, it is characterized in that, comprises the following steps:
Step 1: by being used for the enzyme mixation of nucleic acid amplification system, the concentration ratio according to correspondence mixes by nucleic acid amplification reaction liquid, low-temperature freeze drying protection liquid, namely obtains the composition for nucleic acid amplification system;
Step 2: lyophilize, namely obtains the composition for nucleic acid amplification system of lyophilised state.
Described cryodesiccated step, comprises following sub-step further:
Sub-step 1: dry step;
Sub-step 2: the step of freeze-drying;
In described sub-step 1, freeze-dried and volume ratio that is nucleic acid amplification reaction reagent is 3.7-6.7:13.5-16.1, and described nucleic acid amplification reaction reagent is RNA or DNA real-time fluorescence quantitative PCR reaction reagent;
In described sub-step 1, the step of described drying adopts vacuum freezedrying;
In described step 2, the method for described freeze-drying comprises with the next stage:
The pre-freeze stage: baffle temperature drops to-45 DEG C--55 DEG C, hold-time 3-5h;
A sublimation stage: keep 2-4h;
Secondary sublimation stage: keep 2-4h;
Whole drying stage: keep 2-3h.
For solving the problems of the technologies described above, the present invention separately provides a kind of detection kit comprising the lyophilized vaccine being applied to nucleic acid amplification system as described in aforementioned any one, and described detection kit can be transported at normal temperatures and/or preserve.
For solving the problems of the technologies described above, invention further provides a kind of application of lyophilized vaccine in the preparation and use of nucleic acid amplification system being applied to nucleic acid amplification system as described in aforementioned any one.
For solving the problems of the technologies described above, present invention also offers a kind of application of preparation method in the preparation and use of nucleic acid amplification system being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one.
For solving the problems of the technologies described above, present invention also offers the using method being applied to the lyophilized vaccine of nucleic acid amplification system described in a kind of aforementioned any one, in the preparation of nucleic acid amplification system and the application in using.
For solving the problems of the technologies described above, the present invention has reoffered a kind of using method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, the steps include: that described lyophilized vaccine prepares and puts into PCR pipe freeze-drying preservation; Take out the PCR pipe that freeze-drying is preserved in use, directly add sample 4 ~ 10ul, remove RNA enzyme water 10 ~ 20ul, mixing gets final product examination with computer.
The technique effect that the present invention is useful is:
After by fluorescent quantitation diagnostic nucleic acid reagent system of the present invention freeze-drying, normal temperature carries out clinical sample detection after placing again, compares with contrast product, no matter detecting in accuracy rate, or on the change of CT value, all there is no significant difference, and sensitivity is also relatively good.The present invention can the fluorescent quantitation diagnostic nucleic acid reagent system of detection RNA/DNA of freeze-drying, lyophilize can be realized, compare with the like product of listing, simple to operate during detection, only need a step aquation just can realize testing goal, and present stage diagnostic nucleic acid reagent can be reached to be beyond one's reach the object of normal temperature transport and preservation.
Embodiment
Describe embodiments of the present invention in detail below with reference to embodiment, to the present invention, how utilisation technology means solve technical problem whereby, and the implementation procedure reaching technique effect can fully understand and implement according to this.
It should be noted that, write length for saving specification sheets, avoid unnecessary repetition and waste, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.
The invention discloses a kind of lyophilized vaccine being applied to nucleic acid amplification system.Described content comprises damping fluid, enzyme mixation, nucleic acid amplification reaction liquid, lyophilized vaccine.The present invention is that the freeze drying technology being applied to fluorescent quantitation diagnostic nucleic acid reagent provides a kind of effective, freeze-drying system that can realize.Diagnostic reagent is by cryodesiccated technology, and liquid reagent becomes lyophilized powder, with the preservation that the form of lyophilized powder can be more stable, reach present stage diagnostic nucleic acid reagent be beyond one's reach normal temperature transport and normal temperature preservation object, decrease reagent multigelation.Only need a step aquation to add sample again during detection and can realize testing goal, simple to operate.
For solving the problems of the technologies described above, the invention provides a kind of nucleic acid amplification system, comprising: damping fluid, enzyme mixation, nucleic acid amplification reaction liquid, and, low-temperature freeze drying protective material.
Described damping fluid comprise in following component further one or more: dNTP, Mgcl2, Thris-Hcl, KCl.
Described enzyme mixation comprise in following component further one or more: RNA reversed transcriptive enzyme, RAN inhibitor, archaeal dna polymerase.
Described nucleic acid amplification reaction liquid comprise in following component further one or more: upstream primer, downstream primer, probe.
Described lyophilized vaccine, comprising: trehalose.
The final concentration that in described lyophilized vaccine, trehalose uses is 1 ~ 10mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.2 ~ 5mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.5 ~ 2.5mmol/L.
Trehalose final utilization concentration (mmol/L) in lyophilized vaccine of the present invention can be: 1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 or 10.
Described lyophilized vaccine comprise in following carbohydrate/polyvalent alcohol further one or more: sucrose, Glycerose, pectinose, lyxose, pentose, ribose, wood sugar, semi-lactosi, glucose, hexose, seminose, talose, heptose, glucose, fructose, glyconic acid, sorbyl alcohol, lactose, methyl α pyranoside, maltose, saccharosonic acid, xitix, lactone, sorbose, saccharic acid, erythrose, threose, pectinose, allose, altrose, sucrose, gulose, idose, talose, erythrulose, ribulose, psicose, tagatose, Dextran 40.
Described lyophilized vaccine comprise in following polymkeric substance further one or more: HES (hydroxyethylsulfonic acid), PVP (polyvinylpyrrolidone), PEG (polyoxyethylene glycol), dextran, albumin, 30 POVIDONE K 30 BP/USP 24.
Described lyophilized vaccine comprise in following solvent further one or more: ethylene glycol, glycerine, DMSO (dimethyl sulfoxide (DMSO)), DMF (dimethyl formamide).
Described lyophilized vaccine comprises tensio-active agent further.
Described tensio-active agent is Tween80.
Described lyophilized vaccine comprises amino acids further.
Described lyophilized vaccine comprise in following salt further one or more: phosphoric acid salt, acetate, Citrate trianion.
The lyophilized vaccine of described nucleic acid amplification system does not comprise N.F,USP MANNITOL.
For solving the problems of the technologies described above, present invention also offers a kind of lyophilized vaccine being applied to nucleic acid amplification system, it is characterized in that, comprising: trehalose.
The final concentration that in described lyophilized vaccine, trehalose uses is 1 ~ 10mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.2 ~ 5mmol/L.
The final concentration that in described lyophilized vaccine, trehalose uses is 1.5 ~ 2.5mmol/L.
Described lyophilized vaccine, comprise in following carbohydrate/polyvalent alcohol further one or more: sucrose, Glycerose, pectinose, lyxose, pentose, ribose, wood sugar, semi-lactosi, glucose, hexose, seminose, talose, heptose, glucose, fructose, glyconic acid, sorbyl alcohol, lactose, methyl α pyranoside, maltose, saccharosonic acid, xitix, lactone, sorbose, saccharic acid, erythrose, threose, pectinose, allose, altrose, sucrose, gulose, idose, talose, erythrulose, ribulose, psicose, tagatose, Dextran 40.
Described lyophilized vaccine, comprise in following polymkeric substance further one or more: HES (hydroxyethylsulfonic acid), PVP (polyvinylpyrrolidone), PEG (polyoxyethylene glycol), dextran, albumin, 30 POVIDONE K 30 BP/USP 24.
Described lyophilized vaccine, comprise in following solvent further one or more: ethylene glycol, glycerine, DMSO (dimethyl sulfoxide (DMSO)), DMF (dimethyl formamide).
Comprise tensio-active agent further.
Described tensio-active agent is Tween80.
Comprise amino acids further.
Comprise in following salt further one or more: phosphoric acid salt, acetate, Citrate trianion.
The lyophilized vaccine of described nucleic acid amplification system does not comprise N.F,USP MANNITOL.
For solving the problems of the technologies described above; present invention also offers a kind of preparation method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one; it is characterized in that, comprise the following steps: by described component according to the concentration ratio mixing used, namely obtain lyophilized vaccine.
For solving the problems of the technologies described above, invention further provides a kind of preparation method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, comprising the following steps:
Step 1: by described component according to the concentration ratio mixing used;
Step 2: lyophilize, namely obtains lyophilized vaccine.
Described cryodesiccated step, comprises following sub-step further:
Sub-step 1: dry step;
Sub-step 2: the step of freeze-drying;
In described sub-step 1, freeze-dried and volume ratio that is nucleic acid amplification reaction reagent is 3.6-6.8:13.2-16.3, and described nucleic acid amplification reaction reagent is RNA or DNA real-time fluorescence quantitative PCR reaction reagent;
In described sub-step 1, the step of described drying adopts vacuum freezedrying;
In described step 2, the method for described freeze-drying comprises with the next stage:
The pre-freeze stage: baffle temperature drops to-45 DEG C--55 DEG C, hold-time 3-5h;
A sublimation stage: keep 2-4h;
Secondary sublimation stage: keep 2-4h;
Whole drying stage: keep 2-3h.
For solving the problems of the technologies described above, the present invention has reoffered a kind of using method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, it is characterized in that, comprises the following steps:
Step 1: by being used for the enzyme mixation of nucleic acid amplification system, the concentration ratio according to correspondence mixes by nucleic acid amplification reaction liquid, low-temperature freeze drying protection liquid, namely obtains the composition for nucleic acid amplification system;
Step 2: lyophilize, namely obtains the composition for nucleic acid amplification system of lyophilised state.
Described cryodesiccated step, comprises following sub-step further:
Sub-step 1: dry step;
Sub-step 2: the step of freeze-drying;
In described sub-step 1, freeze-dried and volume ratio that is nucleic acid amplification reaction reagent is 3.7-6.7:13.5-16.1, and described nucleic acid amplification reaction reagent is RNA or DNA real-time fluorescence quantitative PCR reaction reagent;
In described sub-step 1, the step of described drying adopts vacuum freezedrying;
In described step 2, the method for described freeze-drying comprises with the next stage:
The pre-freeze stage: baffle temperature drops to-45 DEG C--55 DEG C, hold-time 3-5h;
A sublimation stage: keep 2-4h;
Secondary sublimation stage: keep 2-4h;
Whole drying stage: keep 2-3h.
For solving the problems of the technologies described above, the present invention separately provides a kind of detection kit comprising the lyophilized vaccine being applied to nucleic acid amplification system as described in aforementioned any one, and described detection kit can be transported at normal temperatures and/or preserve.
For solving the problems of the technologies described above, invention further provides a kind of application of lyophilized vaccine in the preparation and use of nucleic acid amplification system being applied to nucleic acid amplification system as described in aforementioned any one.
For solving the problems of the technologies described above, present invention also offers a kind of application of preparation method in the preparation and use of nucleic acid amplification system being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one.
For solving the problems of the technologies described above, present invention also offers the using method being applied to the lyophilized vaccine of nucleic acid amplification system described in a kind of aforementioned any one, in the preparation of nucleic acid amplification system and the application in using.
For solving the problems of the technologies described above, the present invention has reoffered a kind of using method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, the steps include: that described lyophilized vaccine prepares and puts into PCR pipe freeze-drying preservation; Take out the PCR pipe that freeze-drying is preserved in use, directly add sample 4 ~ 10ul, remove RNA enzyme water 10 ~ 20ul, mixing gets final product examination with computer.
For solving the problems of the technologies described above, the invention provides a kind of lyophilized vaccine being applied to nucleic acid amplification system, comprising one or more in following component: trehalose, tetra-sodium diethyl ester treated water.
The lyophilized vaccine of described nucleic acid amplification system comprises following component further: sucrose and/or lactose.
The lyophilized vaccine of described nucleic acid amplification system does not comprise N.F,USP MANNITOL.In the test of nucleic acid amplification system of the present invention, add N.F,USP MANNITOL and can not show a candle to as the effect of lyophilized vaccine trehalose, sucrose and/or the lactose system that the present invention adopts.
For solving the problems of the technologies described above, the invention provides a kind of preparation method as being applied to the lyophilized vaccine of nucleic acid amplification system according to any one of aforementioned, comprising the following steps: by described component according to the concentration ratio mixing used, namely obtain lyophilized vaccine.
For solving the problems of the technologies described above, the invention provides a kind of preparation method being applied to the lyophilized vaccine of nucleic acid amplification system as described in aforementioned any one, comprising the following steps:
Step 1: by described component according to the concentration ratio mixing used;
Step 2: lyophilize, namely obtains lyophilized vaccine.
Described cryodesiccated step, comprises following sub-step further:
Sub-step 1: dry step;
Sub-step 2: the step of freeze-drying;
In described sub-step 1, freeze-dried and volume ratio that is nucleic acid amplification reaction reagent is 3.5-7:13-16.5, and described nucleic acid amplification reaction reagent is RNA or DNA real-time fluorescence quantitative PCR reaction reagent;
In described sub-step 1, the step of described drying adopts vacuum freezedrying;
In described step 2, the method for described freeze-drying comprises with the next stage:
The pre-freeze stage: baffle temperature drops to-45 DEG C--55 DEG C, hold-time 3-5h;
A sublimation stage: keep 2-4h;
Secondary sublimation stage: keep 2-4h;
Whole drying stage: keep 2-3h.
For solving the problems of the technologies described above, present invention also offers a kind of described in be applied to the using method of the lyophilized vaccine of nucleic acid amplification system, comprise the following steps:
Step 1: by being used for the enzyme mixation of nucleic acid amplification system, the concentration ratio according to correspondence mixes by nucleic acid amplification reaction liquid, low-temperature freeze drying protection liquid, namely obtains the composition for nucleic acid amplification system;
Step 2: lyophilize, namely obtains the composition for nucleic acid amplification system of lyophilised state.
Described cryodesiccated step, comprises following sub-step further:
Sub-step 1: dry step;
Sub-step 2: the step of freeze-drying;
In described sub-step 1, freeze-dried and volume ratio that is nucleic acid amplification reaction reagent is 3.5-7:13-16.5, and described nucleic acid amplification reaction reagent is RNA or DNA real-time fluorescence quantitative PCR reaction reagent;
In described sub-step 1, the step of described drying adopts vacuum freezedrying;
In described step 2, the method for described freeze-drying comprises with the next stage:
The pre-freeze stage: baffle temperature drops to-45 DEG C--55 DEG C, hold-time 3-5h;
A sublimation stage: keep 2-4h;
Secondary sublimation stage: keep 2-4h;
Whole drying stage: keep 2-3h.
For solving the problems of the technologies described above, the present invention reoffer a kind of described in be applied to the detection kit of the lyophilized vaccine of nucleic acid amplification system, described detection kit can transport at normal temperatures and/or preserve.
For solving the problems of the technologies described above; present invention also offers a kind of described in be applied to nucleic acid amplification system lyophilized vaccine and/or described in be applied to the lyophilized vaccine of nucleic acid amplification system preparation method and/or described in be applied to the using method of the lyophilized vaccine of nucleic acid amplification system, in the preparation of nucleic acid amplification system and the application in using.
Provided by the invention for lyophilized vaccine following 1) or 2) or 3):
1) described lyophilized vaccine is made up of trehalose, sucrose and tetra-sodium diethyl ester treated water (DEPC water), and described trehalose, sucrose DEPC water dissolution, proportioning is: 0.001-2000mmol/L:0.001-6000mmol/L.
2) described lyophilized vaccine is made up of trehalose, lactose and tetra-sodium diethyl ester treated water (DEPC water), and described trehalose, lactose DEPC water dissolution, proportioning is: 0.001-2000mmol/L:0.001-280mmol/L.
Described lyophilized vaccine is made up of trehalose, sucrose, lactose and tetra-sodium diethyl ester treated water (DEPC water); described trehalose, sucrose, lactose DEPC water dissolution, proportioning is: 0.001-2000mmol/L:0.001-6000mmol/L:0.001-280mmol/L.
In an embodiment of the present invention,
Above-mentioned 1), in described lyophilized vaccine, RNA amplification reaction freeze-drying system (25 μ L) of described trehalose, sucrose preparation is as following table 1:
Or 2) in described lyophilized vaccine, RNA amplification reaction freeze-drying system (25 μ L) of described trehalose, sucrose preparation is as following table 2:
Or 3) in described lyophilized vaccine, RNA amplification reaction freeze-drying system (25 μ L) of described trehalose, sucrose, lactose preparation is as following table 3:
Or 1) in described lyophilized vaccine, the DNA amplification reaction freeze-drying system (25 μ L) of described trehalose preparation is as following table 4:
Or 2) in described lyophilized vaccine, the DNA amplification reaction freeze-drying system (25 μ L) of described trehalose preparation is as following table 5:
Or 2) in described lyophilized vaccine, the DNA amplification reaction freeze-drying system (25 μ L) of described trehalose preparation is as following table 6:
In another embodiment of the invention, a kind of method preparing above-mentioned reaction system mixture is provided.
Method provided by the invention, comprises the steps: the concentration ratio of each material of said mixture according to correspondence to mix, namely obtains the composition for frozen-dried protective.
The above-mentioned application of mixture in freeze-drying nucleic acid amplification reaction reagent for frozen-dried protective is also the scope of protection of the invention.
In one more embodiment of the present invention, provide a kind of using method of freeze-drying nucleic acid amplification reaction reagent.
Using method provided by the invention, comprises the steps:
Described by the above-mentioned composition for frozen-dried protective and the mixing of nucleic acid amplification reaction reagent, dry, obtain desciccate;
Described lyophilized vaccine and described RNA nucleic acid amplification reaction volume ratio are 7:13; Lyophilized vaccine and described DNA nucleic acid amplification reaction volume ratio are 3.5:16.5;
Described nucleic acid amplification reaction reagent is real time fluorescent quantitative RNA detection reagent or real time fluorescent quantitative DNA detection reagent;
Described drying adopts vacuum freezedrying;
The condition of described freeze-drying is: in the pre-freeze stage, baffle temperature drops to-45 DEG C--55 DEG C, and hold-time 3-5h; A sublimation stage: start setting and be evacuated to 0.3mbar, simultaneous temperature starts 1 DEG C/min and rises to-35 DEG C--25 DEG C, keep 2-4h; Secondary sublimation stage: set temperature starts 1 DEG C/min and rises to-5 DEG C-0 DEG C, keeps 2-4h; Whole drying stage: set temperature 1 DEG C/min rises to 10 DEG C-25 DEG C, keeps 2-3h.
Lyophilized vaccine of the present invention can freeze, with in drying process, to prevent the material of active ingredient generation sex change at reagent mixed liquor.
In yet another embodiment of the invention, carry out quality evaluation by design double blind experiment to lyophilized vaccine of the present invention, the concrete method of inspection is as follows:
Trehalose available from Sigma;
Lactose, sucrose are bought from Chemical Reagent Co., Ltd., Sinopharm Group;
Primed probe is our company oneself design, and Shanghai hundred Li Ge Bioisystech Co., Ltd synthesizes;
Enzyme required for amplification and other raw materials are bought from Promega company;
Carry out preparation RNA and DNA reaction system according to table 1 in the embodiment of the present invention and table 4, after preparation end nucleic acid reaction system mixed solution, carry out vacuum lyophilization according to the freeze drying process of the embodiment of the present invention.
In another embodiment of the present invention, the method for the nucleic acid amplification reaction reagent freeze-dried products of the embodiment of the present invention is described in detail.
The preparation of lyophilized vaccine:
The selection of lyophilized vaccine component and proportioning have significant impact to the shaping of diagnostic reagent and preservation effect.
In order to make the outward appearance of this nucleic acid amplification reaction reagent freeze-dried products and better shaping; and extend its storage life; long-term analysis and research have been carried out to the component of lyophilized vaccine; through a series of test; optimize kind and the content of various component, select following component: trehalose, sucrose.
(1) preparation of lyophilized vaccine
Take trehalose 0.3g, sucrose 0.07g, joined by above reagent in 100mL volumetric flask, after fully dissolving, mixing, degerming with the membrane filtration of 0.22 μm, obtains lyophilized vaccine, be placed in 4 DEG C of refrigerators and save backup.
(2) preparation of RNA reaction buffer:
Take MgCl20.0038g, Tris-HCl (pH8.8) 0.1454g, KCl0.4473g, then measure 250 μ LdNTP (100mM), add in 70mL tri-heavy water, be settled to 100mL mixing ,-20 DEG C save backup.
(3) preparation of primed probe mixed solution:
The sequence of each primer is provided for the amplimer of enterovirns type 71 (EV71):
EV71-upstream primer (5 ' ATGATGGGYACRTTCTCAGT-3 ')
EV71-downstream primer (5 ' TTRCGCATMGGSCGAGGT-3 ')
EV71-probe (5 ' TGCTTCATYCTCATGCC3 ')
Primed probe is mixed with the mother liquor of 100 μm of ol, puts 4 DEG C and saves backup.During use, prepare (25 μ L system/person-portion) by the concentration of 300nmol:300nmol:150nmol.
(4) preparation of enzyme mixation: RNA reversed transcriptive enzyme 50U/ μ L2 μ L, RAN inhibitor 20U/ μ L1 μ L, HotStartTaqDNA polysaccharase 2.5U/ μ L1 μ L (25 μ L system/person-portion).
(5) preparation (25 μ L/ person-portion) of freeze-drying mixed solution:
RNA reaction buffer: 4 μ L
Enzyme mixation: 5 μ L
Primed probe mixed solution 4 μ L
Lyophilized vaccine 7 μ L
Above-mentioned reagent preparation mixing, centrifugal, be dispensed in PCR8 connecting leg.
(6) vacuum lyophilization:
In the pre-freeze stage, baffle temperature drops to-50 DEG C, hold-time 4h; A sublimation stage: start setting and be evacuated to 0.3mbar, simultaneous temperature starts 1 DEG C/min and rises to-25 DEG C, keeps 2h; Secondary sublimation stage: set temperature starts 1 DEG C/min and rises to 0 DEG C, keeps 2h; Whole drying stage: set temperature 1 DEG C/min rises to 15 DEG C, keep 2h, freeze-drying whole process is 10h.
Freeze-drying same day, dissolved freeze-dried powder, carries out sensitivity technique, and contrast product is:
" Enterovirus 71 nucleic acid detection kit (PCR-fluorescence probe method) " state tool note accurate 20153401611 that Aipuyi Biotech Co., Ltd., Beijing produces;
Detect: take out required freeze-drying pipe, centrifugal 1-3 minute, directly add DEPC water 20 μ L, add the RNA template that 5 μ L extract, contrast product by specification operates, and ABI7500 carries out upper machine testing, and program is as follows: reverse transcription program 50 DEG C: 25min:95 DEG C of denaturation 5min; 95 DEG C of sex change 10sec; 55 DEG C of annealing 40sec, 55 DEG C gather fluorescence, return denaturation stage, totally 45 circulations.Sensitivity technique as shown in Figure 1.
The source of sample
The routine sample of Enterovirus 71 (RNA) 100 is from Hospital Group's hospital general, east, Huainan (affiliated hospital of Anhui University of Science and Technology);
Real-time stability is tested
Normal temperature places 1 month, and detected result statistics is as following table 7.
In an alternative embodiment of the invention, a kind of preparation method being applied to DNA lyophilized vaccine is provided.
The preparation of lyophilized vaccine
Take trehalose 0.6g, sucrose 0.14g, above reagent is joined in 100mL volumetric flask, mixing after fully dissolving, with degerming with the membrane filtration of 0.22 μm, obtain lyophilized vaccine, be placed in 4 DEG C of refrigerators and save backup.
The preparation of RNA reaction buffer:
Take MgCl20.0038g, Tris-HCl (pH8.8) 0.1454g, KCl0.4473g, then measure 250 μ LdNTP (100mM), add in 70mL tri-heavy water, be settled to 100mL mixing ,-20 DEG C save backup.
The sequence of each primer is provided for the amplimer of B race suis (GBS):
GBS-upstream primer (5 ' CAAATTGAACCTCTTGTAGCTGAA3 ')
GBS-downstream primer (5 ' CCGCTGTTGATAGGGCATC3 ')
GBS-probe (5 ' CAAAGCTGCTATGGCAATCCTTCAACA ')
The preparation of primed probe mixed solution: primed probe is mixed with the mother liquor of 100 μm of ol, puts 4 DEG C and saves backup.During use, prepare (25 μ L system/person-portion) by the concentration of 300nmol:300nmol:150nmol.
The preparation of enzyme mixation: HotStartTaqDNA polysaccharase 2.5U/ μ L1 μ L (25 μ L system/person-portion).
The preparation (25 μ L/ person-portion) of freeze-drying mixed solution:
DNA reaction buffer 11.5 μ L
Enzyme mixation 1 μ L
Primed probe mixed solution 4 μ L
Lyophilized vaccine 3.5 μ L
Above-mentioned reagent preparation mixing, centrifugal, be dispensed in PCR8 connecting leg.
Vacuum lyophilization
In the pre-freeze stage, baffle temperature drops to-50 DEG C, hold-time 4h; A sublimation stage: start setting and be evacuated to 0.3mbar, simultaneous temperature starts 1 DEG C/min and rises to-25 DEG C, keeps 2h; Secondary sublimation stage: set temperature starts 1 DEG C/min and rises to 0 DEG C, keeps 2h; Whole drying stage: set temperature 1 DEG C/min rises to 15 DEG C, keep 2h, freeze-drying whole process is 10h.
Freeze-drying same day, dissolved freeze-dried powder, carries out sensitivity technique, and contrast product is:
No. 3400105th, tool (standard) word 2013 supervised by Bo Er " B race suis (GBS) kit for detecting nucleic acid (PCR-fluorescence probe method) " the state food medicine that really (Beijing) Science and Technology Ltd. produces
Detect: take out required freeze-drying pipe, carry out centrifugal 1-3 minute, directly add DEPC water 20 μ L, add the RNA template that 5 μ L extract, contrast product by specification operates, and ABI7500 carries out upper machine testing, and program is as follows: 95 DEG C of denaturation 5min; 95 DEG C of sex change 10sec; 55 DEG C of annealing 40sec, 55 DEG C gather fluorescence, return denaturation stage, totally 40 circulations.Sensitivity technique as shown in Figure 2.
The source of sample
The routine sample of B race suis (RNA) 100 is from Hospital Group's hospital general, east, Huainan (affiliated hospital of Anhui University of Science and Technology);
Real-time stability is tested
Normal temperature places 1 month, and detected result statistics is as following table 1.
The result
The clinical sample selected is as follows:
(1) enterovirns type 71 clinical sample is from Hospital Group's hospital general, east, Huainan (affiliated hospital of Anhui University of Science and Technology)
(2) B race suis clinical sample is from Hospital Group's hospital general, east, Huainan (affiliated hospital of Anhui University of Science and Technology)
Each 200 parts of above-mentioned every class clinical sample, the system wherein after freeze-drying of the present invention detects 100 parts of clinical samples, contrast product detects 100 parts of clinical samples.
Contrast product is originated:
" Enterovirus 71 nucleic acid detection kit (PCR-fluorescence probe method) " state tool note accurate 20153401611 that Aipuyi Biotech Co., Ltd., Beijing produces;
No. 3400105th, tool (standard) word 2013 supervised by Bo Er " B race suis (GBS) kit for detecting nucleic acid (PCR-fluorescence probe method) " the state food medicine that really (Beijing) Science and Technology Ltd. produces.
Assessment result:
(1) accuracy rate and CT value are analyzed
Freeze-drying system of the present invention respectively carries out the detection of 100 times to 2 class clinical samples, and compare analysis with the detected result of contrast product, accuracy rate result is as following table 7 simultaneously:
Table 7 stability experiment result
A: system of the present invention;
B: contrast product;
The CT value significance (P value) of C:A and B
(2) sensitivity analysis
Apply system of the present invention and carry out sensitivity technique to enterovirns type 71 and B race suis, enterovirus type 71 viruses nutrient solution and B race streptococcus culture fluid are carried out 10 times of gradient dilutions, and detected result is as Fig. 2.
The above results shows, after by fluorescent quantitation diagnostic nucleic acid reagent system of the present invention freeze-drying, normal temperature carries out clinical sample detection after placing again, compare with contrast product, no matter detecting in accuracy rate, or on the change of CT value, all there is no significant difference, and sensitivity is also relatively good.The present invention work out a kind of can the fluorescent quantitation diagnostic nucleic acid reagent system of detection RNA of freeze-drying, lyophilize can be realized, compare with the like product of listing, simple to operate during detection, only need a step aquation just can realize testing goal, and present stage diagnostic nucleic acid reagent can be reached to be beyond one's reach the object of normal temperature transport and preservation.
In one more embodiment of the present invention, for the various lyophilized vaccine combinations of RNA freeze-drying system, carry out again following experiment:
The sample processing method of this group experiment
| Solution system | Volume | Freeze-drying system | Volume |
| Nucleic acid amplification reaction liquid | 5ul | | |
| Enzyme mixation | 4ul | | |
| Virus PCR reaction solution | 4ul | | |
| Moisturizing | 7ul | Moisturizing | 20ul |
| Template | 5ul | Template | 5ul |
| Cumulative volume | 25ul | Cumulative volume | 25ul |
1., in the embodiment of the present invention, RNA amplification reaction freeze-drying system (25 μ L) of described trehalose, sucrose, lactose preparation is as follows:
1. trehalose and sucrose, experimental result as shown in Figure 3.
2. trehalose and lactose, experimental result as shown in Figure 4.
3. trehalose and semi-lactosi, experimental result as shown in Figure 5.
4. trehalose and seminose, experimental result as shown in Figure 6.
5. trehalose and glucose, experimental result as shown in Figure 7.
6. trehalose and fructose, experimental result as shown in Figure 8.
7. trehalose, sucrose and lactose, experimental result as shown in Figure 9.
8. trehalose, sucrose and semi-lactosi, experimental result as shown in Figure 10.
9. trehalose, sucrose and fructose, experimental result as shown in figure 11.
10. trehalose, glucose and lactose, experimental result as shown in figure 12.
The above results shows, after by fluorescent quantitation diagnostic nucleic acid reagent system of the present invention freeze-drying, normal temperature carries out clinical sample detection after placing again, compare with contrast product, no matter detecting in accuracy rate, or on the change of CT value, all there is no significant difference, and sensitivity is also relatively good.The present invention work out a kind of can the fluorescent quantitation diagnostic nucleic acid reagent system of detection RNA of freeze-drying, lyophilize can be realized, compare with the like product of listing, simple to operate during detection, only need a step aquation just can realize testing goal, and present stage diagnostic nucleic acid reagent can be reached to be beyond one's reach the object of normal temperature transport and preservation.
In one more embodiment of the present invention, for the various lyophilized vaccine combinations of DNA freeze-drying system, carry out again following experiment:
1., in the embodiment of the present invention, described DNA amplification reaction freeze-drying system (25 μ L) is as follows:
2. lyophilized vaccine combination
1. trehalose and sucrose, experimental result as shown in figure 13.
2. trehalose and lactose, experimental result as shown in figure 14.
3. trehalose and semi-lactosi, experimental result as shown in figure 15.
4. trehalose and seminose, experimental result as shown in figure 16.
5. trehalose and glucose, experimental result as shown in figure 17.
6. trehalose and fructose, experimental result as shown in figure 18.
7. trehalose, sucrose and lactose, experimental result as shown in figure 19.
8. trehalose, sucrose and semi-lactosi, experimental result as shown in figure 20.
9. trehalose, sucrose and fructose, experimental result as shown in figure 21.
10. trehalose, glucose and lactose, experimental result as shown in figure 22.
3. sample processing method
| Solution system | Volume | Freeze-drying system | Volume |
| Enzyme mixation | 12.5ul | | |
| Virus PCR reaction solution | 4ul | | |
| Moisturizing | 3.5ul | Moisturizing | 20ul |
| Template | 5ul | Template | 5ul |
| Cumulative volume | 25ul | Cumulative volume | 25ul |
The above results shows, after by fluorescent quantitation diagnostic nucleic acid reagent system of the present invention freeze-drying, normal temperature carries out clinical sample detection after placing again, compare with contrast product, no matter detecting in accuracy rate, or on the change of CT value, all there is no significant difference, and sensitivity is also relatively good.The present invention work out a kind of can the fluorescent quantitation diagnostic nucleic acid reagent system of detection DNA of freeze-drying, lyophilize can be realized, compare with the like product of listing, simple to operate during detection, only need a step aquation just can realize testing goal, and present stage diagnostic nucleic acid reagent can be reached to be beyond one's reach the object of normal temperature transport and preservation.
All above-mentioned this intellecture properties of primary enforcement, not setting restriction this product innovation of other forms of enforcement and/or novel method.Those skilled in the art will utilize this important information, and foregoing is revised, to realize similar implementation status.But all modifications or transformation belong to the right of reservation based on product innovation of the present invention.