CN105274192A - Nucleic acid amplification reaction mixture particle and application thereof - Google Patents
Nucleic acid amplification reaction mixture particle and application thereof Download PDFInfo
- Publication number
- CN105274192A CN105274192A CN201410360188.0A CN201410360188A CN105274192A CN 105274192 A CN105274192 A CN 105274192A CN 201410360188 A CN201410360188 A CN 201410360188A CN 105274192 A CN105274192 A CN 105274192A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- amplification reaction
- acid amplification
- freeze
- reaction mixture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleic acid amplification reaction mixture. The nucleic acid amplification reaction mixed liquor is prepared by freeze drying and is in freeze-drying granular shape, and the water content is 0.1-3%; the nucleic acid amplification reaction mixed liquor at least contains necessary components for a nucleic acid amplification reaction and a freeze-drying protective agent; the freeze-drying protective agent is a mixture containing one or more substances from sucrose, trehalose, glucose, glucan, saccharosan, bovine serum albumin, collagen, polyvinyl pyrrolidone, polyethylene glycol and carboxymethylcellulose sodium, and the weight volume ratio concentration of the freeze-drying protective agent and the nucleic acid amplification reaction mixed liquor is 5%-20%. The nucleic acid amplification reaction mixture freeze-drying particles have compact and tight particle and smooth appearance, and fine apertures are contained in the particles and can be observed under microscopic amplification state; the freeze-drying particles can be stored at room temperature for more than one year with unchanged reaction activity, the biological reaction activity is not changed at 40-45 DEG C, and during usage, a reconstitution fluid is added for rapidly dissolving the particles and recovering the reaction activity.
Description
Technical field
The invention belongs to nucleic acid amplification reaction field, particularly relate to a kind of at room temperature can preservation for a long time and easily molten nucleic acid amplification reaction mixture particle and application thereof.
Background technology
A lot of compositions in nucleic acid amplification reaction system are all unstable under the condition of relatively-high temperature, as nucleic acid polymerase, primer, dNTP etc., all need under the condition of refrigeration, such as preserve at-20 DEG C.These reaction compositions are generally independent or deposit under cryogenic in the mode of part mixing, even if but freezing conditions also can not keep the biological activity of these compositions for a long time, and need to thaw for a long time when using, or carry out packing on ice chest, the multigelation of reagent can affect the biological activity of effective constituent further.And long-term frozen is preserved to be needed to consume a large amount of energy, adds transport and storage cost.
The United States Patent (USP) that publication number is " 2008/0182312A1 " uses a kind of method of drying, loop-mediated isothermal amplification (LAMP) reagent is dry in reaction tubes lid with translucent membranaceous form, enable loop-mediated isothermal amplification reaction reagent 4 DEG C of preservations, but this reaction reagent still need refrigerated condition.
Application number for " 200810159414.3 " and Chinese patent application disclose a kind of store method of loop-mediated isothermal amplification reaction reagent mixture; the method adds specific drying protectant in loop-mediated isothermal amplification reaction reagent mixture; then the loop-mediated isothermal amplification reaction reagent mixture that with the addition of drying protectant is carried out vacuum-drying or Quick-air-drying under lower than the condition of 80 DEG C, achieve the long-term preservation of loop-mediated isothermal amplification reaction reagent mixture under normal temperature or room temperature condition.The shortcoming that the method exists is: because in the course of processing, temperature is too high, nucleic acid polymerization enzymic activity can be made to lose, and the time that the loop-mediated isothermal amplification reaction reagent mixture normal temperature of gained is preserved is also shorter, meanwhile, the loop-mediated isothermal amplification reaction reagent mixture of gained can not be deposited at too high a temperature.
In addition, the product after the method processing that above two patents are mentioned is the semisolid of compact shape, when being used for carrying out nucleic acid amplification reaction, after adding water, not easily dissolving completely in the short period of time, have impact on test operation efficiency.
Summary of the invention
The object of the invention is to the problem solving existing nucleic acid amplification reaction reagent normal temperature shelf time its effective constituent easy in inactivation shorter, and the processed products solving nucleic acid amplification reaction reagent are when using, not easily dissolve completely in the short period of time, affect the problem of test operation efficiency.
Therefore, the invention provides a kind of nucleic acid amplification reaction mixture, described nucleic acid amplification reaction mixture is formed through lyophilize by nucleic acid amplification reaction mixed solution, and in freeze-drying particulate state, its water content is 0.1 ~ 3%; Described nucleic acid amplification reaction mixed solution at least contains required composition and the lyophilized vaccine of nucleic acid amplification reaction; Wherein, described lyophilized vaccine be in sucrose, trehalose, glucose, dextran, ficoll, bovine serum albumin, collagen protein, polyethylene pyrrole network alkane ketone, polyoxyethylene glycol, Xylo-Mucine in the mixture of one or more, and the bulking value specific concentration of described lyophilized vaccine and described nucleic acid amplification reaction mixed solution is 5% ~ 20%.
In technique scheme, lyophilized vaccine can reduce the infringement to the required composition of nucleic acid amplification reaction in freezing dry process, keeps freeze-drying particle reactive behavior at high temperature, makes nucleic acid amplification reaction mixture can at room temperature standing storage.
In technique scheme, Freeze Drying Technique is frozen into solid-state at lower temperature (-10 DEG C ~-50 DEG C) by wet stock or solution, then under vacuum (1.3 ~ 13 handkerchief), make moisture wherein be directly sublimed into gaseous state without liquid state, finally make the dry technology of material dewatering.In freezing dry process, the structure of the required composition of nucleic acid amplification reaction can not be destroyed, because solids component is support by the black ice on its position, space can be left when ice distils in the surplus materials of drying, so just remain the biological and chemical structure of the required composition of nucleic acid amplification reaction and the integrity of activity thereof; And freezing dry process carries out under low-temperature condition, the destructiveness of this technological process to component is little, and thermal distoftion is extremely faint, and the biological activity of nucleic acid polymerase etc. can not be caused to reduce; Meanwhile, obtained freeze-drying particle is vesicular structure, and more crisp, rehydration performance is good, can repeat completely rapidly to dissolve again, accelerates the operation efficiency of nucleic acid amplification reaction; And shape and constancy of volume before and after lyophilized products drying, dry final vacuum sealing or nitrogen-filled seal, eliminate the impact of outer bound pair freeze-drying particle.Lyophilize can get rid of the moisture of more than 97%, makes the water content of freeze-drying particle below 3%, and lower water content can improve the highest temperature of standing of nucleic acid amplification reaction frozen granules, and can extend the shelf time of freeze-drying particle.
In technique scheme, the bulking value specific concentration of described lyophilized vaccine and described nucleic acid amplification reaction mixed solution is 5% ~ 20% (w/v).The grams of contained solute in bulking value specific concentration and 100ml solution.When frozen-dried protective agent concentration is too low, can affect the physical appearance of freeze-drying prods, the freeze-drying particle of gained is shaky, frangible; When frozen-dried protective agent concentration is too high, solute concentration increases, and hinders distillation, affect freeze-drying efficiency, and too high protective material has a certain impact to nucleic acid amplification reaction.
In technique scheme, nucleic acid amplification reaction must may not comprise all components needed for nucleic acid amplification reaction system by composition, only containing wherein several component needing cryopreservation, or severally need the component of cryopreservation and the mixture of component can be preserved by normal temperature.Need the component of cryopreservation to be generally primer, nucleic acid polymerase, dNTPs, nucleotide fluorescent dye, nucleic acid amplification reaction damping fluid (buffer), front four usually-20 DEG C of preservations, and buffer can 4 DEG C of preservations.For loop-mediated isothermal amplification (LAMP), general loop-mediated isothermal amplification system comprises LAMP primer, dNTPs, has the functional nucleic acid polymerase of strand displacement, buffer, when carrying out the preparation of loop-mediated isothermal amplification freeze-drying particle, amplification reaction reagent used can only contain LAMP primer, dNTPs or have the functional nucleic acid polymerase of strand displacement, also can contain nucleotide fluorescent dye and buffer further.
As the further improvement to technique scheme, described lyophilize process comprises the following steps:
(1) control temperature is-50 DEG C ~-40 DEG C, and pressure is 1atm, maintains 10 ~ 40min;
(2) control temperature is-50 DEG C ~-40 DEG C, and pressure is 2 ~ 2.4pa, maintains 8 ~ 12h;
(3) control temperature is-5 DEG C ~ 5 DEG C, and pressure is 2 ~ 2.4pa, maintains 2 ~ 4h;
(4) control temperature is 20 DEG C ~ 25 DEG C, and pressure is 2 ~ 2.4pa, maintains 4 ~ 6h, namely completes described lyophilize process.
Wherein, described step (1) is the pre-freeze stage, described step (2) ~ (4) are drying phase under vacuum, and wherein step (2) is the sublimation drying stage, and step (3) and step (4) are the adsorption stripping and dry stage.
In technique scheme, pre-freeze is the process of setting, it can be given drying products and have identical form before drying, prevent the contraction on material surface during vacuum-drying simultaneously, and pre-freeze process water build-ups ice, produced mechanical effect and solute effect cause the deactivated important factor of nucleic acid amplification reaction reagent, cooling rate during pre-freeze, temperature and time directly can affect the quality of drying rate and freeze-drying particle, in order to ensure the good rehydration of freeze-drying particle, the loss of the nucleic acid amplification reaction agent of activity preventing from solute from concentrating causing, improve rate of sublimation as far as possible, guarantee that vacuumizing front nucleic acid amplification reaction mixing liquid reagent is frozen reality simultaneously.Pre-freezing temperature is at-50 ~-40 DEG C in the present invention, and the hold-time is 10 ~ 40min.
In technique scheme, the process of the first drying of vacuum lyophilization is exactly the process of distillation, the free water distillation effusion will freezed in nucleic acid amplification reaction mixed solution by sublimation drying.In order to reach rate of drying faster, prevent the fusing of nucleic acid amplification reaction mixed solution, sex change or avalanche, first dry temperature is set as-50 ~-40 DEG C simultaneously.In primary drying process, suitable pressure is conducive to the transmission of heat and the carrying out of distillation, and in the present invention, pressure controls at 2 ~ 2.4pa, if pressure is too low, then unfavorable to heat transfer, nucleic acid amplification reaction mixture not easily obtains heat, rate of sublimation is low, also can increase cost simultaneously; When pressure is too high, the rate of sublimation of ice slows down, and nucleic acid amplification reaction mixture own temperature can rise, and when higher than temperature of eutectic point, generation fusing is caused freeze-drying failure by nucleic acid amplification reaction mixture.
In technique scheme, redrying mainly removes part Bound moisture, this part water is adsorbed on nucleic acid amplification reaction composite reagent primarily of the weak molecular linkage such as Van der Waals force, hydrogen bond, needs more energy to be removed, in order to not destroy the biological activity of nucleic acid amplification reaction mixture, temperature is set as-5 DEG C ~ 5 DEG C by the present invention, control pressure is 2 ~ 2.4pa, keeps 2 ~ 4h, then temperature is risen to 20 ~ 25 DEG C, control pressure 2 ~ 2.4pa, keep 4 ~ 6h.
As the further improvement to technique scheme, described lyophilize process comprises the following steps:
(1) control temperature is-45 DEG C, and pressure is 1atm, maintains 25min;
(2) control temperature is-45 DEG C, and pressure is 2.2pa, maintains 10h;
(3) control temperature is 0 DEG C, and pressure is 2.2pa, maintains 3h;
(4) control temperature is 25 DEG C, and pressure is 2.2pa, maintains 5h, namely completes described lyophilize process.
Through the freeze-drying particle of this improving technique process, its form is more fine and close solid, is not easy because being subject to external force impact loose, thus affects the reactive behavior of described nucleic acid amplification reaction mixture in transport and use procedure.
As the further improvement to technique scheme; described lyophilized vaccine is the mixture of carbohydrate and protein; wherein said carbohydrate is one or more in sucrose, trehalose, glucose, dextran, ficoll, and described albumen is one or both of bovine serum albumin or collagen protein.
As the further improvement to technique scheme, described nucleic acid amplification reaction reagent at least comprises primer, dNTP, nucleic acid polymerase and nucleic acid amplification reaction damping fluid.As further improving, also containing nucleotide fluorescent dye in described nucleic acid amplification reaction reagent.Primer, dNTP, nucleic acid polymerase, nucleotide fluorescent dye, buffer are all important component of nucleic acid amplification reaction reagent, and usually can only preserve at low temperatures.
As the further improvement to technique scheme, described nucleic acid amplification reaction is loop-mediated isothermal amplification (LAMP), and the required composition of described nucleic acid amplification reaction at least comprises primer, dNTP, nucleic acid polymerase, nucleic acid amplification reaction damping fluid and nucleotide fluorescent dye.LAMP is a kind of easy, quick, accurate, gene amplification method at a low price, is widely used in the rapid detection of various pathogenic agent, is particluarly suitable for field work.When nucleic acid amplification reaction mixture of the present invention is applicable to the loop-mediated isothermal amplification of field operation, can avoid carrying ice bag, dry ice etc.
As the further improvement to technique scheme, when being applicable to loop-mediated isothermal amplification, the nucleic acid polymerase in amplification reaction reagent has strand displacement function and has 5 → 3 ' DNA polymerase activities, but does not have exonuclease activity.Wherein, described nucleic acid polymerase is preferably not glycerinated Bst nucleic acid polymerase.Business-like nucleic acid polymerase enzyme liquid contains the glycerine of 50% usually, and too much glycerine is unfavorable for cryodesiccated success.In the present invention, the preparation method of not glycerinated Bst nucleic acid polymerase can referenced patent number be " ZL201010276051.9 ", and name is called " bacstearothermophilus nucleic acid polymerase " patent of invention.And in order to increase the stability of Bst nucleic acid polymerase further, containing trehalose in Bst nucleic acid polymerase enzyme liquid, wherein, the weightmeasurement ratio of trehalose and described enzyme liquid is 2% ~ 10%.Such as, loop-mediated isothermal amplification reaction reagent can be composed of the following components: loop-mediated isothermal amplification primer, dNTP, Bst nucleic acid polymerase, buffer (Tris-HCl, KCl, (NH
4)
2sO
4, TritonX-100) and SYBRGreenI dyestuff.
In order to the person that makes test operation more convenient carry out nucleic acid amplification reaction, present invention also offers a kind of PCR pipe, in the pipe of described PCR pipe, be mounted with nucleic acid amplification reaction mixture of the present invention.Operator is when using PCR pipe of the present invention; can directly add in PCR pipe by redissolution liquid, freeze-drying particle can be rapidly dissolved in and redissolve in liquid, thus defines amplification reaction system; the reaction system of such formation just directly can carry out amplified reaction, fast very convenient.Redissolve liquid usually containing Mg
2+, trimethyl-glycine and deionized water.Described PCR pipe can be the pipe of the pipe of independent form, the pipe of eight townhouses, common PCR pipe or optical form.
Present invention also offers a kind of preparation method of PCR pipe; 10 ~ 15 μ L are at least joined in PCR pipe containing the required composition of nucleic acid amplification reaction and the nucleic acid amplification reaction mixed solution of lyophilized vaccine; then lyophilize process is carried out to the PCR pipe being added with described mixed solution; remove the moisture in described mixed solution; until its water content is 0.1 ~ 3%; it is particle stabilized in PCR pipe that described mixed solution can form freeze-drying, i.e. obtained described PCR pipe.In the present invention, application of sample amount is 10 ~ 15 μ L, and the particle densification that the application of sample amount of this volume is formed is solid, uniform pore diameter.Too much application of sample amount needs longer freeze-drying time, and the particle undertighten formed.
Relative to prior art, nucleic acid amplification reaction mixture of the present invention is formed through lyophilize by nucleic acid amplification reaction mixed solution, and water content is lower than 3%, lyophilize process ensure that the reactive behavior of nucleic acid amplification reaction reagent, and the freeze-drying particle form densification formed is solid, smooth in appearance, observes visible freeze-drying particle and contain tiny aperture under micro-magnifying state.The freeze-drying particle of this state can keep the integrity of reactive material, can be dissolved into rapidly again and redissolve in liquid.Simultaneously, nucleic acid amplification reaction mixture water content of the present invention is few, lower water content can improve the highest temperature of standing of nucleic acid amplification reaction frozen granules, and the shelf time of freeze-drying particle can be extended, nucleic acid amplification reaction mixture of the present invention can be preserved more than 1 year at ambient temperature and reactive behavior remains unchanged, and even at 40 ~ 45 DEG C, also can not lose biological respinse activity.
When nucleic acid amplification reaction mixture of the present invention uses, only need add redissolution liquid, just can dissolve rapidly, recover reactive behavior, the time that whole reaction consumes can not be increased, also can avoid the tedious steps of freezing, fusing, application of sample.The lyophilized vaccine used in the present invention can reduce the infringement to nucleic acid amplification reaction mixing liquid reagent in freezing dry process, keeps freeze-drying particle reactive behavior at high temperature, makes nucleic acid amplification reaction reagent can at room temperature standing storage.Nucleic acid amplification reaction mixture of the present invention may be used for food, health, the pathogenic microbes detect of the industries such as medical science.The form of frozen granules contributes to reducing shipping storage cost, and handled easily, expands the use range of detection reagent and be not limited to laboratory.
Accompanying drawing explanation
Fig. 1 is the PCR pipe containing nucleic acid amplification reaction mixture of the present invention that embodiment 1 obtains;
Fig. 2 is nucleic acid amplification reaction mixture particle of the present invention;
Fig. 3 is the Activity determination figure that in embodiment 8, normal temperature preserves the nucleic acid amplification reaction mixture of the present invention of a year;
Fig. 4 is the Activity determination figure of the nucleic acid amplification reaction mixture of the present invention heating month in embodiment 8 at 40 DEG C.
Embodiment
Below in conjunction with embodiment, the present invention is made a more detailed description; but below describe only for the explanation of making an explanation property of the present invention; any restriction is not carried out to protection scope of the present invention, all equivalents done according to content of the present invention, all in like manner within the scope of the present invention.
Embodiment 1
1. preparing nucleic acid amplified reaction mixed solution
1) prepare nucleic acid amplification reaction reagent needed for 100 loop-mediated isothermal amplification systems, totally 1000 μ L, each component of nucleic acid amplification reaction reagent and content as shown in the table:
| Component | Volume | Content |
| Bst nucleic acid polymerase (8U/ μ L) | 100μL | 800U |
| dNTPs(20mM) | 225μL | 4.5mM |
| Inner primer FIP (50 μMs) | 100μL | 5μM |
| Inner primer BIP (50 μMs) | 100μL | 5μM |
| Outer primer F3 (25 μMs) | 50μL | 1.25μM |
| Outer primer B3 (25 μMs) | 50μL | 1.25μM |
| 10xbuffer | 250μL | 2.5x |
| SYBR GREEN I nucleic acid dye (250X) | 10μL | 2.5X |
| ddH 2O | 115μL | |
| Amount to | 1000μL |
Wherein, prepared by Bst nucleic acid polymerase Guangzhou Huafeng Biotech Co., Ltd., have high activity and purity, not containing glycerine in this Bst nucleic acid polymerase enzyme liquid, in order to increase the stability of enzyme liquid, and the trehalose containing 2% (w/v) in enzyme liquid.
Wherein, the primer in table is the primer of amplification mycobacterium tuberculosis, and the sequence of each primer is:
Inner primer
FIP:GCCTCTACCAGTACTGCGGCTTTTGAGCGTAGTAGGCAGCCT；
Inner primer
BIP:GTTGAACCAGTCGACCCAGCGTTTTAACCCGGCAAGCCCT；
Outer primer F3:GGCTGGTCTCTGGCGTT;
Outer primer B3:GGCCTATACAAGACCGAGCT.
2) lyophilized vaccine is prepared
Take trehalose and the 0.03g polyoxyethylene glycol (PEG) 8000 of 0.02g, be contained in pipe.3) by step 1) in nucleic acid amplification reaction reagent join 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein, the bulking value specific concentration of trehalose and PEG8000 and nucleic acid amplification reaction mixed solution is 5%, treats nucleic acid amplification reaction mixed solution clear;
4) by step 3) in amplified reaction composite reagent according to the application of sample amount of 10ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 4 DEG C, and between pipe, difference is 1%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -45℃ | 20min | 1atm |
| 2 | -45℃ | 10h | 2.2pa |
| 3 | 0℃ | 3h | 2.2pa |
| 4 | 25℃ | 5h | 2.2pa |
| Amount to | 18.3h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, as depicted in figs. 1 and 2, record its water content is 1.5% to obtained nucleic acid amplification reaction mixture particle.
Embodiment 2
1, preparing nucleic acid amplified reaction mixed solution
1) prepare the totally 1500 μ L of the nucleic acid amplification mixture needed for loop-mediated isothermal amplification system of 100, each component of nucleic acid amplification reaction mixed solution and consumption as shown in the table:
| Component | Volume | Content |
| Bst nucleic acid polymerase (8U/ μ L) | 100μL | 800U |
| DNTPs(25mM) | 180μL | 3mM |
| Inner primer FIP (12.5 μMs) | 384μL | 3.2μM |
| Inner primer BIP (12.5 μMs) | 384μL | 3.2μM |
| Outer primer F3 (12.5 μMs) | 96μL | 0.8μM |
| Outer primer B3 (12.5 μMs) | 96μL | 0.8μM |
| 10xbuffer | 250μL | 1.7x |
| SYBR GREEN I nucleic acid dye (250 ×) | 10μL | 1.7× |
| Amount to | 1500μL |
Wherein, Bst nucleic acid polymerase and primer sequence are with embodiment 1.
2) lyophilized vaccine is prepared
Take sucrose and the 0.05g polyvinylpyrrolidone of 0.1g, be contained in pipe.
3) by step 1) in nucleic acid amplification reaction reagent join 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein the bulking value specific concentration of sucrose and polyvinylpyrrolidone and nucleic acid amplification reaction mixed solution is 10%, treats nucleic acid amplification reaction mixing liquid reagent clear.
4) by step 3) in nucleic acid amplification reaction mixed solution according to the application of sample amount of 15ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 4 DEG C, and between pipe, difference is 1%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -50℃ | 10min | 1atm |
| 2 | -42℃ | 8h | 2.4pa |
| 3 | -5℃ | 2h | 2.4pa |
| 4 | 20℃ | 4h | 2.4pa |
| Amount to | 14.2h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, after testing, the water content of obtained nucleic acid amplification reaction mixture particle is 3%.
Embodiment 3
1, preparing nucleic acid amplified reaction mixed solution
1) with the step 1 of embodiment 1);
2) lyophilized vaccine is prepared
Take sucrose and the 0.08gBSA (bovine serum albumin) of 0.12g, be contained in pipe.
3) by step 1) amplifying nucleic acid amplification reaction reagent joins 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein the bulking value specific concentration of sucrose and BSA and nucleic acid amplification reaction mixed solution is 20%, treats nucleic acid amplification reaction mixed solution clear.
4) by step 3) in nucleic acid amplification reaction mixed solution according to the application of sample amount of 10ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 4 DEG C, and between pipe, difference is 1%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -40℃ | 40min | 1atm |
| 2 | -40℃ | 12h | 2pa |
| 3 | 5℃ | 2h | 2pa |
| 4 | 25℃ | 6h | 2pa |
| Total | 20.6h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, after testing, the water content of obtained nucleic acid amplification reaction mixture particle is 0.1%.
Embodiment 4
1, preparing nucleic acid amplified reaction mixed solution
1) with the step 1 of embodiment 1);
2) lyophilized vaccine is prepared
Take the trehalose of 0.05g, 0.03g bovine serum albumin and 0.02gPEG8000, be contained in pipe.
3) by step 1) amplifying nucleic acid amplification reaction reagent joins 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein the bulking value specific concentration of trehalose, bovine serum albumin and PEG8000 and nucleic acid amplification reaction mixed solution is 10%, treats nucleic acid amplification reaction mixed solution clear.
4) by step 3) in nucleic acid amplification reaction mixed solution according to the application of sample amount of 10ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 4 DEG C, and between pipe, difference is 1%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -45℃ | 30min | 1atm |
| 2 | -40℃ | 10h | 2.2pa |
| 3 | 3℃ | 3h | 2.2pa |
| 4 | 20℃ | 5h | 2.2pa |
| Total | 18.5h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, after testing, the water content of obtained nucleic acid amplification reaction mixture particle is 2.2%.
Embodiment 5
1, preparing nucleic acid amplified reaction mixed solution
1) with the step 1 of embodiment 2);
2) lyophilized vaccine is prepared
Take 0.05g glucose and 0.035g collagen protein, be contained in pipe.
3) by step 1) amplifying nucleic acid amplification reaction reagent joins 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein the bulking value specific concentration of glucose and bovine serum albumin and nucleic acid amplification reaction mixed solution is 8.5%, treats nucleic acid amplification reaction mixed solution clear;
4) by step 3) in nucleic acid amplification reaction mixed solution according to the application of sample amount of 15ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 10 DEG C, and between pipe, difference is 2%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -45℃ | 20min | 1atm |
| 2 | -45℃ | 10h | 2.2pa |
| 3 | 0℃ | 3h | 2.2pa |
| 4 | 25℃ | 5h | 2.2pa |
| Total | 18.3h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, after testing, the water content of obtained nucleic acid amplification reaction mixture particle is 2%.
Embodiment 6
1, preparing nucleic acid amplified reaction mixed solution
1) with the step 1 of embodiment 1);
2) lyophilized vaccine is prepared
Take 0.15g Xylo-Mucine, be contained in pipe.
3) by step 1) amplifying nucleic acid amplification reaction reagent joins 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein the bulking value specific concentration of collagen protein and nucleic acid amplification reaction mixed solution is 15%, treats nucleic acid amplification reaction mixed solution clear.
4) by step 3) in nucleic acid amplification reaction mixed solution according to the application of sample amount of 10ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 10 DEG C, and between pipe, difference is 2%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -45℃ | 20min | 1atm |
| 2 | -45℃ | 10h | 2.2pa |
| 3 | 0℃ | 3h | 2.2pa |
| 4 | 25℃ | 5h | 2.2pa |
| Total | 18.3h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, after testing, the water content of obtained nucleic acid amplification reaction mixture particle is 1.8%.
Embodiment 7
1, preparing nucleic acid amplified reaction mixed solution
1) with the step 1 of embodiment 1);
2) lyophilized vaccine is prepared
Take 0.25g trehalose, be contained in pipe.
3) by step 1) amplifying nucleic acid amplification reaction reagent joins 2) in pipe in, shake up mixing, form nucleic acid amplification reaction mixed solution, wherein the bulking value specific concentration of bovine serum albumin and nucleic acid amplification reaction mixed solution is 25%, treats nucleic acid amplification reaction mixed solution clear.
4) by step 3) in nucleic acid amplification reaction mixed solution according to the application of sample amount of 10ul, use automatic sample equipment to be sub-packed in PCR pipe, during application of sample, pipe should be placed on freezing pallet and carry out, and temperature of tray is at 10 DEG C, and between pipe, difference is 2%.
2, the preparation of nucleic acid amplification reaction mixture particle
Point PCR pipe installed is put into freeze drier, and freeze-drying program is as follows:
| Step | Temperature | Time | Pressure |
| 1 | -45℃ | 20min | 1atm |
| 2 | -45℃ | 10h | 2.2pa |
| 3 | 0℃ | 3h | 2.2pa |
| 4 | 25℃ | 5h | 2.2pa |
| Total | 18.3h |
After freeze-drying terminates, material is taken out rapidly from Freeze Drying Equipment, and fast by the process of pipe sealer, after testing, the water content of obtained nucleic acid amplification reaction mixture particle is 1.2%.
Embodiment 8: freeze-drying seed activity detects
The PCR pipe containing nucleic acid amplification reaction mixture particle prepared by above-described embodiment is respectively at storing 1 year under normal temperature (20 ~ 35 DEG C) condition and storing one month under 40 DEG C of temperature condition, freeze-drying particle prepared by each embodiment of preserving under getting two kinds of temperature condition respectively restores, redissolution liquid to 22.5 μ l is added in each PCR pipe, after abundant mixing, make freeze-drying grain dissolution, the LAMP reaction mixture restored can be obtained.
Above-mentioned 3 storage time sections are restored the LAMP reaction mixture obtained and carries out Activity determination, concrete steps are as follows:
1) measuring samples (concretion mycobacterium nucleic acid) of 2.5 μ l is joined in the PCR pipe of the LAMP reaction mixture that recovery is housed, mixing; Wherein the concentration of measuring samples is respectively 10000 copies/μ l, 1000 copy/μ l, 100 copy/μ l.
2) add paraffin oil sealed reaction tube liquid level, prevent the pollution of following reaction.
3) above-mentioned PCR pipe is positioned on fluorescence nucleic acid augmentative instrument, carries out LAMP reaction in 65 DEG C of insulation 60min, then in 90 DEG C of insulation 5min termination reactions.
4) after LAMP reaction terminating, the reactive behavior of freeze-drying particle can be judged according to the colour developing result of nucleic acid dye SYBRGREENI, if response curve is clear and legible, then illustrate that the LAMP reaction composite reagent in PCR pipe has normal reactive behavior; If unstressed configuration curve, then illustrate that the LAMP in PCR pipe reacts the reactionless activity of composite reagent, reaction result is as shown in the table, Activity determination figure as shown in Figure 3 and Figure 4, in Fig. 3, the curve at 1,2,3 places represents the curve of sample that concentration is 10000 copies/μ l, 1000 copy/μ l, 100 copy/μ l respectively, and in Fig. 4, the curve at 4,5,6 places represents the curve of sample that concentration is 10000 copies/μ l, 1000 copy/μ l, 100 copy/μ l respectively.
Note: in table, "+" number represents that LAMP reaction reagent mixture has good amplification active, "-" number represents that LAMP reaction reagent mixture does not increase activity.
Claims (10)
1. a nucleic acid amplification reaction mixture, is characterized in that: described nucleic acid amplification reaction mixture is formed through lyophilize by nucleic acid amplification reaction mixed solution, and in freeze-drying particulate state, its water content is 0.1 ~ 3%; Described nucleic acid amplification reaction mixed solution at least contains required composition and the lyophilized vaccine of nucleic acid amplification reaction;
Wherein, described lyophilized vaccine be in sucrose, trehalose, glucose, dextran, ficoll, bovine serum albumin, collagen protein, polyethylene pyrrole network alkane ketone, polyoxyethylene glycol, Xylo-Mucine in the mixture of one or more, and the bulking value specific concentration of described lyophilized vaccine and described nucleic acid amplification reaction mixed solution is 5% ~ 20%.
2. nucleic acid amplification reaction mixture as claimed in claim 1, is characterized in that: described lyophilize process comprises the following steps:
(1) control temperature is-50 DEG C ~-40 DEG C, and pressure is 1atm, maintains 10 ~ 40min;
(2) control temperature is-50 DEG C ~-40 DEG C, and pressure is 2 ~ 2.4pa, maintains 8 ~ 12h;
(3) control temperature is-5 DEG C ~ 5 DEG C, and pressure is 2 ~ 2.4pa, maintains 2 ~ 4h;
(4) control temperature is 20 DEG C ~ 25 DEG C, and pressure is 2 ~ 2.4pa, maintains 4 ~ 6h, namely completes described lyophilize process.
3. nucleic acid amplification reaction mixture as claimed in claim 2, is characterized in that: described lyophilize process comprises the following steps:
(1) control temperature is-45 DEG C, and pressure is 1atm, maintains 25min;
(2) control temperature is-45 DEG C, and pressure is 2.2pa, maintains 10h;
(3) control temperature is 0 DEG C, and pressure is 2.2pa, maintains 3h;
(4) control temperature is 25 DEG C, and pressure is 2.2pa, maintains 5h, namely completes described lyophilize process.
4. nucleic acid amplification reaction mixture as claimed in claim 1; it is characterized in that: described lyophilized vaccine is the mixture of carbohydrate and protein; wherein said carbohydrate is one or more in sucrose, trehalose, glucose, dextran, ficoll, and described albumen is one or both of bovine serum albumin or collagen protein.
5. nucleic acid amplification reaction mixture as claimed in claim 1, is characterized in that: the required composition of described nucleic acid amplification reaction at least comprises primer, dNTP, nucleic acid polymerase and nucleic acid amplification reaction damping fluid.
6. nucleic acid amplification reaction mixture as claimed in claim 1, it is characterized in that: described nucleic acid amplification reaction is loop-mediated isothermal amplification, the required composition of described nucleic acid amplification reaction at least comprises primer, dNTP, nucleic acid polymerase, nucleic acid amplification reaction damping fluid and nucleotide fluorescent dye.
7. nucleic acid amplification reaction mixture as claimed in claim 6, is characterized in that: described nucleic acid polymerase is not glycerinated Bst nucleic acid polymerase.
8. nucleic acid amplification reaction mixture as claimed in claim 7, is characterized in that: containing trehalose in described Bst nucleic acid polymerase enzyme liquid, wherein, the weightmeasurement ratio of described trehalose and described enzyme liquid is 2% ~ 10%.
9. a PCR pipe, is characterized in that: be mounted with the nucleic acid amplification reaction mixture described in the arbitrary claim of claim 1 ~ 8 in the pipe of described PCR pipe.
10. the preparation method of a PCR pipe; it is characterized in that: 10 ~ 15 μ L are at least joined in PCR pipe containing the required composition of nucleic acid amplification reaction and the nucleic acid amplification reaction mixed solution of lyophilized vaccine; then lyophilize process is carried out to the PCR pipe being added with described mixed solution; remove the moisture in described mixed solution; until its water content is 0.1 ~ 3%; it is particle stabilized in PCR pipe that described mixed solution can form freeze-drying, i.e. obtained described PCR pipe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410360188.0A CN105274192A (en) | 2014-07-25 | 2014-07-25 | Nucleic acid amplification reaction mixture particle and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410360188.0A CN105274192A (en) | 2014-07-25 | 2014-07-25 | Nucleic acid amplification reaction mixture particle and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105274192A true CN105274192A (en) | 2016-01-27 |
Family
ID=55144019
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410360188.0A Pending CN105274192A (en) | 2014-07-25 | 2014-07-25 | Nucleic acid amplification reaction mixture particle and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105274192A (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463125A (en) * | 2016-02-02 | 2016-04-06 | 江苏正大天创生物工程有限公司 | Nucleic acid amplification system and freeze-drying protective agent thereof |
| CN105671171A (en) * | 2016-03-11 | 2016-06-15 | 中国人民武装警察部队后勤学院 | Protective agent for freeze drying of nucleic acid amplification reagent |
| CN106591432A (en) * | 2016-10-19 | 2017-04-26 | 珠海丽珠试剂股份有限公司 | Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent |
| CN107326094A (en) * | 2017-09-04 | 2017-11-07 | 重庆市畜牧科学院 | The kit and its detection method of chicken rhinitis type Klebsiella are detected based on ring mediated isothermal amplification |
| CN108048552A (en) * | 2017-12-19 | 2018-05-18 | 益善生物技术股份有限公司 | It is a kind of to be used to detect the micro-deleted Nucleic acid combinations of Y chromosome and kit and application |
| CN108277291A (en) * | 2018-03-05 | 2018-07-13 | 南京岚煜生物科技有限公司 | Kit and its application method for detecting multiple pathogens |
| CN108411037A (en) * | 2018-03-26 | 2018-08-17 | 郑州安图生物工程股份有限公司 | A kind of freeze-dried reagent that can distinguish three kinds of pathogen nucleic acids simultaneously |
| CN109055531A (en) * | 2018-09-10 | 2018-12-21 | 厦门为正生物科技股份有限公司 | For detecting specific primer group, kit and the detection method of people's MTHFR and MTRR gene polymorphism sites |
| CN109295182A (en) * | 2018-09-30 | 2019-02-01 | 苏州百源基因技术有限公司 | A kind of PCR dispenses the reaction tube of reagent and prepackage PCR reagent in advance |
| CN109457039A (en) * | 2018-11-26 | 2019-03-12 | 广州华峰生物科技有限公司 | A kind of freeze-drying PCR reagent detecting shrimp seedling liver sausage born of the same parents worm |
| CN109593834A (en) * | 2019-01-29 | 2019-04-09 | 百康芯(天津)生物科技有限公司 | Frozen-dried protective system needed for a kind of nucleic acid amplification agents and preparation method thereof |
| CN110452972A (en) * | 2018-05-08 | 2019-11-15 | 北京中科生仪科技有限公司 | A kind of freeze-drying microballoon of nucleic acid amplification reaction reagent and preparation method thereof |
| CN110468239A (en) * | 2019-09-22 | 2019-11-19 | 山东森芃生物科技有限公司 | A kind of quick Q-PCR detection method of freeze-dried type African swine fever virus and kit |
| WO2020173164A1 (en) * | 2019-02-28 | 2020-09-03 | 京东方科技集团股份有限公司 | Lyophilized powder of mesenchymal stem cell and preparation method therefor |
| CN113106161A (en) * | 2021-05-14 | 2021-07-13 | 公安部物证鉴定中心 | STR rapid individual recognition amplification reagent for fully integrated microfluidic chip and application thereof |
| WO2023142130A1 (en) * | 2022-01-30 | 2023-08-03 | 皮乐迪有限公司 | Application of improved enzyme pellet in target nucleic acid detection |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591703A (en) * | 2008-11-22 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The store method of loop-mediated isothermal amplification reaction reagent mixture |
| CN102084005A (en) * | 2008-07-02 | 2011-06-01 | 恩尼格马诊断有限公司 | Freeze-dried compositions for biochemical reactions |
| CN103911367A (en) * | 2014-03-20 | 2014-07-09 | 中国农业大学 | Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method |
-
2014
- 2014-07-25 CN CN201410360188.0A patent/CN105274192A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102084005A (en) * | 2008-07-02 | 2011-06-01 | 恩尼格马诊断有限公司 | Freeze-dried compositions for biochemical reactions |
| CN101591703A (en) * | 2008-11-22 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The store method of loop-mediated isothermal amplification reaction reagent mixture |
| CN103911367A (en) * | 2014-03-20 | 2014-07-09 | 中国农业大学 | Freeze-drying protective agent of nucleic acid amplification reaction reagents and freeze-drying method |
Non-Patent Citations (1)
| Title |
|---|
| 李美霞: "环介导等温扩增体系中生物分子的稳定性及其保护", 《中国优秀硕士学位论文全文数据库——基础科学辑》 * |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463125A (en) * | 2016-02-02 | 2016-04-06 | 江苏正大天创生物工程有限公司 | Nucleic acid amplification system and freeze-drying protective agent thereof |
| CN105671171A (en) * | 2016-03-11 | 2016-06-15 | 中国人民武装警察部队后勤学院 | Protective agent for freeze drying of nucleic acid amplification reagent |
| CN106591432A (en) * | 2016-10-19 | 2017-04-26 | 珠海丽珠试剂股份有限公司 | Freeze-drying protective agent and freeze-drying method for RNA amplification reaction agent |
| CN107326094A (en) * | 2017-09-04 | 2017-11-07 | 重庆市畜牧科学院 | The kit and its detection method of chicken rhinitis type Klebsiella are detected based on ring mediated isothermal amplification |
| CN108048552A (en) * | 2017-12-19 | 2018-05-18 | 益善生物技术股份有限公司 | It is a kind of to be used to detect the micro-deleted Nucleic acid combinations of Y chromosome and kit and application |
| CN108277291A (en) * | 2018-03-05 | 2018-07-13 | 南京岚煜生物科技有限公司 | Kit and its application method for detecting multiple pathogens |
| CN108411037A (en) * | 2018-03-26 | 2018-08-17 | 郑州安图生物工程股份有限公司 | A kind of freeze-dried reagent that can distinguish three kinds of pathogen nucleic acids simultaneously |
| CN110452972A (en) * | 2018-05-08 | 2019-11-15 | 北京中科生仪科技有限公司 | A kind of freeze-drying microballoon of nucleic acid amplification reaction reagent and preparation method thereof |
| CN110452972B (en) * | 2018-05-08 | 2022-08-02 | 北京中科生仪科技有限公司 | Freeze-dried microsphere of nucleic acid amplification reaction reagent and preparation method thereof |
| CN109055531A (en) * | 2018-09-10 | 2018-12-21 | 厦门为正生物科技股份有限公司 | For detecting specific primer group, kit and the detection method of people's MTHFR and MTRR gene polymorphism sites |
| CN109295182A (en) * | 2018-09-30 | 2019-02-01 | 苏州百源基因技术有限公司 | A kind of PCR dispenses the reaction tube of reagent and prepackage PCR reagent in advance |
| CN109457039A (en) * | 2018-11-26 | 2019-03-12 | 广州华峰生物科技有限公司 | A kind of freeze-drying PCR reagent detecting shrimp seedling liver sausage born of the same parents worm |
| CN109593834A (en) * | 2019-01-29 | 2019-04-09 | 百康芯(天津)生物科技有限公司 | Frozen-dried protective system needed for a kind of nucleic acid amplification agents and preparation method thereof |
| WO2020173164A1 (en) * | 2019-02-28 | 2020-09-03 | 京东方科技集团股份有限公司 | Lyophilized powder of mesenchymal stem cell and preparation method therefor |
| CN110468239A (en) * | 2019-09-22 | 2019-11-19 | 山东森芃生物科技有限公司 | A kind of quick Q-PCR detection method of freeze-dried type African swine fever virus and kit |
| CN113106161A (en) * | 2021-05-14 | 2021-07-13 | 公安部物证鉴定中心 | STR rapid individual recognition amplification reagent for fully integrated microfluidic chip and application thereof |
| WO2023142130A1 (en) * | 2022-01-30 | 2023-08-03 | 皮乐迪有限公司 | Application of improved enzyme pellet in target nucleic acid detection |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105274192A (en) | Nucleic acid amplification reaction mixture particle and application thereof | |
| Adams | The principles of freeze-drying | |
| US7169606B2 (en) | Lyophilization of human platelets | |
| Adams et al. | The principles of freeze-drying | |
| US5556771A (en) | Stabilized compositions of reverse transcriptase and RNA polymerase for nucleic acid amplification | |
| CN110452956B (en) | Freeze-dried microsphere of PCR reaction reagent and preparation method thereof | |
| US11649512B2 (en) | Freeze-dried composition | |
| AU2001268057A1 (en) | Preservation and storage medium for biological materials | |
| US20060177426A1 (en) | Method of preserving lyophilized microorganisms for transport, storage and recovery of viable microorganisms | |
| CN113652468A (en) | Freeze-drying protective agent suitable for freeze-drying of nucleic acid detection kit and freeze-drying method thereof | |
| CN204958918U (en) | Reaction tube of pre -installation fluorescence PCR reagent | |
| US20140193456A1 (en) | Method for Drying-Conservation of Natural Substances | |
| CN114752703A (en) | Novel freeze-drying detection reagent for coronavirus nucleic acid and preparation method thereof | |
| CN1261563C (en) | Method for freeze-drying and ultra low temperature storing ginseng callus cell | |
| CN111893169A (en) | Freeze-dried powder reagent for nucleic acid amplification and preparation method and use method thereof | |
| CN113025757B (en) | Freeze-drying protective agent for 2019-nCov multiplex amplification reaction reagent and application thereof | |
| CN114015758A (en) | Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process | |
| CN106967706B (en) | A kind of Taq polymerase freeze drying protectant | |
| JP3937019B2 (en) | Dispersion medium for preservation of microorganism and container for preservation of microorganism | |
| CN117535275A (en) | DNA polymerase freeze-drying protective agent and application thereof | |
| CN116549652A (en) | Freeze-drying protective agent, freeze-drying reagent and preparation method thereof | |
| Ferrando et al. | Thermophysical properties of a monolayer tissue with respect to freeze-drying | |
| CN116004780A (en) | Drying method of PCR reagent and application thereof | |
| JP2003245069A (en) | Method for preserving dna | |
| CN117265075A (en) | Freeze-drying auxiliary reagent and kit for nucleic acid isothermal amplification reaction and use method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160127 |
|
| RJ01 | Rejection of invention patent application after publication |