CN114854833A - Constant-temperature amplification reagent normal-temperature storage method - Google Patents
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Abstract
The invention relates to the technical field of amplification reagents, in particular to a method for preserving a constant-temperature amplification reagent at normal temperature. A constant-temperature amplification reagent normal-temperature storage method comprises the following operation steps: the method comprises the following steps: preparing a reaction mixed solution; step two: preparing a buffer solution; step three: processing the reaction mixed solution; step four: cutting into blocks; the invention can provide a method for preparing and drying the constant-temperature amplification reagent, and solves the problem that the constant-temperature amplification reagent can not be stored at normal temperature; the constant-temperature amplification reagent normal-temperature storage method is used in nucleic acid constant-temperature detection, so that the constant-temperature amplification reagent can be stored for a long time at normal temperature, the transportation and storage cost of the constant-temperature amplification reagent is reduced, and a reliable technical means is provided for nucleic acid POCT detection; the invention adopts an intermittent feeding mode to evenly distribute the reaction mixed liquid on the glass fiber membrane.
Description
Technical Field
The invention relates to the technical field of amplification reagents, in particular to a method for preserving a constant-temperature amplification reagent at normal temperature.
Background
PCR nucleic acid detection technology is widely used in various fields such as disease detection, gene detection, virus and pathogen detection, food and environment detection, and the like. The traditional PCR detection reagent generally needs to be stored and transported at about-20 ℃ to ensure the biological activity of the effective components of the reagent. Not only is the cost high, but also the performance and the quality guarantee period of the detection reagent are directly influenced by repeated freezing and thawing of the reagent caused by the change of the environmental temperature. The liquid reagent can be transported and stored at normal temperature after being dried, the problem of cold chain transportation can be well solved, and the advantages of the dried reagent also include longer quality guarantee period and higher detection sensitivity due to more flexible sample loading amount.
However, the current research on the drying process of the nucleic acid detection reagent is less, the research on the freeze-drying process of the nucleic acid detection reagent is still imperfect, only a few foreign companies realize the commercial application of the nucleic acid detection freeze-drying reagent, and the field is basically blank at home, which greatly limits the development of the nucleic acid POCT detection technology. Therefore, a method for storing a nucleic acid amplification reagent at room temperature is desired.
In addition, in most nucleic acid detection kits, each component of the nucleic acid amplification reagent is often independently stored and mixed according to corresponding requirements before use, and in clinical application, in order to simplify operation steps and avoid operation errors, cross contamination and the like, the components are often prepared into a premixed solution for storage and use.
Disclosure of Invention
The invention aims to provide a method for preserving a constant-temperature amplification reagent at normal temperature, which aims to solve the problems in the process.
In order to achieve the purpose, the invention provides the following technical scheme: a constant-temperature amplification reagent normal-temperature storage method comprises the following operation steps:
the method comprises the following steps: preparing a reaction mixed solution; preparing an enzyme mixture, an antibody, a primer, a probe, polyethylene glycol 8000, dNTP and 4-hydroxyphthalic acid, and then mixing the enzyme mixture, the antibody, the primer, the probe, the polyethylene glycol 8000, dNTP and 4-hydroxyphthalic acid to prepare a reaction mixed solution;
step two: preparing a buffer solution; preparing 4mM magnesium chloride, 50mM potassium chloride, and 20mM tris, followed by mixing 4mM magnesium chloride, 50mM potassium chloride, and 20mM tris to prepare a buffer;
step three: processing the reaction mixed solution; adding the reaction mixed solution prepared in the step one onto a glass fiber membrane by adopting an on-membrane liquid adding device, so that the reaction mixed solution is immersed on the glass fiber membrane;
step four: cutting into blocks; and (3) conveying the glass fiber membrane prepared in the third step into a drying device, drying the glass fiber membrane in a drying mode at 42 ℃, then cutting the dried glass fiber membrane into blocks, fixing the size of the blocks, further filling the cut glass fiber membrane into a PCR tube, sealing and storing at normal temperature.
Preferably, in the first step, the concentration of the primer is preferably 1 μmol/L, the concentration of the probe is preferably 0.5 μmol/L, the concentration of polyethylene glycol 8000 is preferably 2%, the concentration of dNTP is preferably 1mM, the concentration of 4-hydroxyphthalic acid is preferably 0.01%, the pH value of the reaction mixture is 7.5, the enzyme mixture consists of 2U of reverse transcriptase, 0.5U of DNA recombinase, 0.5U of DNA polymerase and 0.25U of single-chain DNA binding protein, and the antibody consists of 0.1mg/mL antibody of reverse transcriptase and 0.1mg/mL antibody of DNA polymerase.
Preferably, the pH value of the buffer solution in the second step is 8.7.
Preferably, the on-film liquid adding device comprises a mounting base, a middle penetrating rotating shaft, two wing power gears, reciprocating staggered blocks, an upper end conveying frame, a lower pressing roller, a positioning connecting frame, a reaction mixed liquid storage cylinder, an upper cylinder penetrating shaft and an intermittent liquid supply rod, wherein the middle part penetrates through the rotating shaft and is arranged at the middle part of the mounting base, the two wing power gears are symmetrically sleeved at the two ends of the middle part penetrating through the rotating shaft, wherein the reciprocating staggered blocks are uniformly fixed at one end of the two wing power gears, the upper end conveying frame is positioned at the upper end of the mounting base, wherein the lower press roll is fixed at one side of the upper end conveying frame, the positioning connecting frames are symmetrically fixed at two sides of the middle part of the upper end conveying frame, wherein the reaction mixed liquid storage cylinder is fixed between two positioning connection frames, the lower end of the reaction mixed liquid storage cylinder is penetrated through by a shaft on the cylinder, and the intermittent liquid supply rod is positioned at the lower side in the reaction mixed liquid storage cylinder.
Preferably, settle and run through between the pivot to be connected through the pivot in base and the middle part, wherein two wings power gear is in the lower extreme of upper end carriage, two wings power gear and middle part run through and adopt screw connection between the pivot, wherein stagger each other between the piece on two wings power gear, the piece that staggers reciprocal is the hemisphere piece, wherein the both ends of reaction mixed liquid storage cylinder respectively with two location connection frames between carry out screw connection, wear to be connected through the pivot between axle and the reaction mixed liquid storage cylinder on the section of thick bamboo, wherein wear the inside that two location connection frames were inserted respectively at the both ends of axle on the section of thick bamboo, wear to carry out buckle fixed connection between axle and the intermittent type liquid supply pole on the section of thick bamboo.
Preferably, the lower side of the middle part of the upper end conveying frame is symmetrically provided with fixedly connected bottom distance rods, the bottom distance rods are arranged in an L shape, one side of the lower end of each bottom distance rod is provided with a fixedly connected jacked block, the jacked block is positioned at one side of the two wing power gears at the same side, the frame of the upper end conveying frame is uniformly provided with movably connected conveying rollers, two ends of one conveying roller are symmetrically provided with fixedly connected linkage side columns, the linkage side columns penetrate through the upper end conveying frame, one end of each linkage side column is sleeved with a fixedly connected follow-up gear, the follow-up gear and the two wing power gears are arranged in a meshing manner, the outer side of the follow-up gear is provided with a distance increasing disc fixedly connected with the linkage side columns, one end of the distance increasing disc is externally provided with a rotating driving rod connected through a rotating shaft, and the other end of the rotating driving rod is connected to a through a rotating shaft, one end of the rotation driving rod is provided with a transverse sliding groove in a penetrating way.
Preferably, the lower extreme intermediate position of reaction mixture liquid storage cylinder runs through the play liquid guide frame of arranging fixed connection, and wherein it is in the lower extreme of intermittent type liquid feed pole to go out the liquid guide frame, the lower extreme of going out the liquid guide frame arranges fixed connection's liquid frame that gathers, and wherein the lower extreme intermediate position of gathering the liquid frame runs through and sets up the liquid outlet, it is in the top of conveying roller to gather the liquid frame, and wherein the middle part bilateral symmetry of gathering the liquid frame runs through and sets up the guide spout, it has placed the liquid distribution board to gather the inside downside of liquid frame, and wherein the upper end bilateral symmetry of liquid distribution board arranges fixed connection's the frid that covers.
Preferably, cover the frid and cover the spout of guiding in the inside of gathering the liquid frame, wherein cover the one end downside of frid and arrange fixed connection's the slotted plate that wears, wherein wear the frid and run through and guide the spout and arrange, the one end of wearing the frid is run through and is arranged upper and lower inserted bar, and wherein the cover has reset spring on the inserted bar from top to bottom, fixed connection's lamina tecti is arranged to the upper end of upper and lower inserted bar, wherein carries out fixed connection between lamina tecti and the play liquid guide frame, swing joint's adjusting lever is arranged to the other end of wearing the frid, wherein the other end of adjusting lever inserts the inside in horizontal sliding tray.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention can provide a method for preparing and drying the constant-temperature amplification reagent, and solves the problem that the constant-temperature amplification reagent can not be stored at normal temperature; the constant-temperature amplification reagent normal-temperature storage method is used in nucleic acid constant-temperature detection, so that the constant-temperature amplification reagent can be stored for a long time at normal temperature, the transportation and storage cost of the constant-temperature amplification reagent is reduced, and a reliable technical means is provided for nucleic acid POCT detection;
2. the invention adopts an intermittent feeding mode to evenly distribute the reaction mixed liquid on the glass fiber membrane, and can lead the glass fiber membrane to shake in a reciprocating way in the distribution process, thereby not only ensuring the even coverage rate of the reaction mixed liquid, but also shortening the time for the reaction mixed liquid to be immersed into the glass fiber membrane.
Drawings
FIG. 1 is a perspective view of the present invention;
FIG. 2 is a schematic semi-sectional perspective view of an upper end carriage according to the present invention;
FIG. 3 is a schematic right side view of a half section of an upper end carriage according to the present invention;
FIG. 4 is a schematic semi-sectional perspective view of a reaction mixture storage cylinder according to the present invention;
FIG. 5 is a perspective view of the conveyor roll of the present invention;
FIG. 6 is a schematic front view of a reaction mixture storage cylinder in half section according to the present invention;
FIG. 7 is an enlarged view taken at A in FIG. 4 according to the present invention.
In the figure: the device comprises a mounting base 1, a middle penetrating rotating shaft 11, two wing power gears 12, a reciprocating staggering block 13, an upper end conveying frame 14, a lower pressing roller 15, a bottom distance pulling rod 1401, a jacked block 1402, a conveying roller 1403, a linkage side column 1404, a follow-up gear 1405, a distance increasing disc 1406, a rotary driving rod 1407, a transverse sliding groove 1408, a positioning connecting frame 16, a reaction mixed liquid storage cylinder 17, a liquid outlet guide frame 1701, a liquid collecting frame 1702, a liquid outlet 1703, a guide sliding groove 1704, a liquid separating plate 1705, a covering plate 1706, a groove penetrating plate 1707, an upper and lower inserting rod 1708, a return spring 1709, a top cover plate 1710, an adjusting rod 1711, a cylinder penetrating shaft 18 and an intermittent liquid supply rod 19.
Detailed Description
Features and exemplary embodiments of various aspects of the present invention will be described in detail below. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the present invention. The present invention is in no way limited to any specific configuration and algorithm set forth below, but rather covers any modification, replacement or improvement of elements, components or algorithms without departing from the spirit of the invention. In the drawings and the following description, well-known structures and techniques are not shown in order to avoid unnecessarily obscuring the present invention.
Referring to fig. 1 to 7, the present invention provides a technical solution: the method comprises the following operation steps:
the method comprises the following steps: preparing a reaction mixed solution; preparing an enzyme mixture, an antibody, a primer, a probe, polyethylene glycol 8000, dNTP and 4-hydroxyphthalic acid, and then mixing the enzyme mixture, the antibody, the primer, the probe, the polyethylene glycol 8000, the dNTP and the 4-hydroxyphthalic acid to prepare a reaction mixed solution;
step two: preparing a buffer solution; preparing 4mM magnesium chloride, 50mM potassium chloride, and 20mM tris, followed by mixing 4mM magnesium chloride, 50mM potassium chloride, and 20mM tris to prepare a buffer;
step three: processing the reaction mixed solution; adding the reaction mixed solution prepared in the step one onto a glass fiber membrane by adopting an on-membrane liquid adding device, so that the reaction mixed solution is immersed on the glass fiber membrane;
step four: cutting into blocks; and (3) conveying the glass fiber membrane prepared in the third step into a drying device, drying the glass fiber membrane in a drying mode at 42 ℃, then cutting the dried glass fiber membrane into blocks, fixing the size of the blocks, further filling the cut glass fiber membrane into a PCR tube, sealing and storing at normal temperature.
The reaction mixture is dried sufficiently to remove excess water without affecting the performance of the enzyme by excessive drying. Through a large number of tests, the method of the invention ensures the stability of the enzyme and can be quickly reconstituted and activated. The reaction performance of the dried reagent and the liquid reagent keep consistent, and the reagent can be stored for at least 18 months at normal temperature.
In the first step, the concentration of the primer is preferably 1 mu mol/L, the concentration of the probe is preferably 0.5 mu mol/L, the concentration of polyethylene glycol 8000 is preferably 2%, in the drying process, the polyethylene glycol 8000 can block non-specific sites of the glass fiber membrane, the activity reduction caused by non-specific adsorption of the enzyme on the glass fiber membrane is prevented, the concentration of dNTP is preferably 1mM, the concentration of 4-hydroxyphthalic acid is preferably 0.01%, the 4-hydroxyphthalic acid can chelate divalent metal ions, the stability of the enzyme mixture is improved, and meanwhile hydrolysis of the enzyme mixture by hydrolase in the environment is avoided. Furthermore, the 4-hydroxyphthalic acid also has the function of corrosion prevention, prevents the reaction mixed liquor from generating microbial pollution in the preservation process, further improves the long-term stability of the reaction mixed liquor, and the pH value of the reaction mixed liquor is 7.5.
The enzyme mixture consists of 2U reverse transcriptase, 0.5U DNA recombinase, 0.5U DNA polymerase and 0.25U single-stranded DNA binding protein, and the inventor finds that the enzyme mixture consisting of the components and the concentration has the advantages of high sensitivity and high specificity in the constant-temperature amplification process through a large number of experiments.
The antibody is composed of reverse transcriptase antibody 0.1mg/mL and DNA polymerase antibody 0.1mg/mL, and the antibody is combined with reverse transcriptase and DNA polymerase in the drying process, so that the enzyme can still maintain its activity under the conditions of high temperature and dehydration, and meanwhile, the long-term stability of the enzyme can be increased. In addition, after the antibody is combined with the enzyme, the catalytic performance of the enzyme can be blocked, non-specific amplification in the drying process is avoided, and the biological activity of the enzyme is recovered after dissociation.
The pH value of the buffer solution in the second step is 8.7, so that the sensitivity and the specificity of the nucleic acid amplification reaction can be further increased.
The on-film liquid adding device comprises a mounting base 1, a middle through rotating shaft 11, two-wing power gears 12, a reciprocating staggered block 13, an upper end conveying frame 14, a lower pressure roller 15, a positioning connecting frame 16, a reaction mixed liquid storage barrel 17, a barrel upper through shaft 18 and an intermittent liquid supply rod 19, wherein the middle through rotating shaft 11 is arranged at the middle position of the mounting base 1, the two-wing power gears 12 are symmetrically sleeved at the two ends of the middle through rotating shaft 11, the reciprocating staggered block 13 is uniformly fixed at one end of the two-wing power gears 12, the upper end conveying frame 14 is arranged at the upper end of the mounting base 1, the lower pressure roller 15 is fixed at one side of the upper end conveying frame 14, the positioning connecting frames 16 are symmetrically fixed at the two sides of the middle of the upper end conveying frame 14, the reaction mixed liquid storage barrel 17 is fixed between the two positioning connecting frames 16, and the barrel upper through shaft 18 is arranged through the lower end of the reaction mixed liquid storage barrel 17, wherein the intermittent feed rod 19 is located at the lower side of the interior of the reaction mixture reservoir tank 17.
The middle position of the upper end of the reaction mixed liquid storage cylinder 17 is inserted with a liquid adding pipe which is fixedly connected, and the reaction mixed liquid can be added into the reaction mixed liquid storage cylinder 17 through the liquid adding pipe.
The glass fiber film is laid on the upper end conveying frame 14, and the conveying rollers 1403 on the upper end conveying frame 14 can convey the glass fiber film at the position, so that the glass fiber film passes through the lower part of the liquid outlet 1703.
The placing base 1 is connected with the upper end conveying frame 14 in a sliding way.
Settle and run through between the pivot 11 to be connected through the pivot in base 1 and the middle part, wherein both wings power gear 12 is in the lower extreme of upper end carriage 14, both wings power gear 12 runs through between the pivot 11 with the middle part and adopts the screw connection, wherein stagger each other between the piece 13 on two wings power gear 12 and arrange, the piece 13 that staggers reciprocally is the hemisphere piece, wherein the both ends of reaction mixture liquid storage cylinder 17 respectively with two location connection frames 16 between carry out the screw connection, wear to be connected through the pivot between axle 18 and the reaction mixture liquid storage cylinder 17 on the section of thick bamboo, wherein wear the inside that two location connection frames 16 were inserted respectively at the both ends of axle 18 on the section of thick bamboo, wear to carry out buckle fixed connection between axle 18 and the intermittent type liquid supply pole 19 on the section of thick bamboo. The middle part penetrates through the rotating shaft 11 and is driven by a motor to rotate.
The middle lower side of the upper end conveying frame 14 is symmetrically provided with fixedly connected bottom distance rods 1401, wherein the bottom distance rods 1401 are arranged in an L shape, one side of the lower end of the bottom distance rods 1401 is provided with fixedly connected jacked blocks 1402, the jacked blocks 1402 are positioned at one side of the wing power gears 12 at the same side, movably connected conveying rollers 1403 are uniformly arranged in the frame of the upper end conveying frame 14, both ends of one conveying roller 1403 are symmetrically provided with fixedly connected linkage side columns 1404, the linkage side columns 1404 penetrate through the upper end conveying frame 14, one end of each linkage side column 1404 is sleeved with a fixedly connected follow-up gear 1405, the follow-up gear 1405 is meshed with the wing power gears 12, the outer side of the follow-up gear 1405 is provided with a distance-increasing disc 1406 fixedly connected with the linkage side columns 1404, one end of the distance-increasing disc 1406 is arranged outside and is provided with a rotating driving rod 1407 connected through a rotating shaft, the other end of the rotating driving rod 1407 is connected to a through a rotating shaft 18, one end of the rotation driving rod 1407 is penetrated by a transverse sliding groove 1408.
The middle part penetrates through the rotating shaft 11 to rotate and drives the two wing power gears 12 to rotate, at the moment, the wing power gears 12 rotate through the following gear 1405, the distance increasing disc 1406 rotates, the further distance increasing disc 1406 drives the penetrating shaft 18 on the barrel to rotate under the action of the rotating driving rod 1407, the further intermittent liquid supply rod 19 rotates, when the intermittent liquid supply rod 19 cannot cover the liquid outlet guide frame 1701, the reaction mixed liquid in the reaction mixed liquid storage barrel 17 can enter the liquid outlet guide frame 1701 to be collected,
in addition, due to the rotation of the two wing power gears 12, the reciprocating staggered block 13 on one wing power gear 12 can firstly jack the jacked block 1402, so that the upper end conveying rack 14 is offset to one side on the installation base 1, during the offset process of the upper end conveying rack 14, the jacked block 1402 on the other side of the upper end conveying rack 14 is overlapped with the reciprocating staggered block 13 on the other wing power gear 12, and then the driven block 1402 on the two wing power gears 12 jacks the upper end conveying rack 14, so that the reciprocating enables the upper end conveying rack 14 to swing, and the reaction mixed liquid is rapidly fused in the glass fiber membrane.
The middle position of the lower end of the reaction mixed liquid storage cylinder 17 is provided with a fixedly connected liquid outlet guide frame 1701 in a penetrating way, wherein the liquid outlet guide frame 1701 is arranged at the lower end of the intermittent liquid supply rod 19, the lower end of the liquid outlet guide frame 1701 is provided with a fixedly connected liquid gathering frame 1702 in a penetrating way, the middle position of the lower end of the liquid gathering frame 1702 is provided with a liquid outlet 1703 in a penetrating way, the liquid gathering frame 1702 is arranged above the conveying roller 1403, the two sides of the middle part of the liquid gathering frame 1702 are symmetrically provided with guide sliding chutes 1704 in a penetrating way, a liquid distributing plate 1705 is arranged on the lower side in the interior of the liquid gathering frame 1702, and the two sides of the upper end of the liquid distributing plate 1705 are symmetrically provided with fixedly connected covering groove plates 1706.
The dispensing plate 1705 may be curved or V-shaped, wherein the shroud 1706 seals against the indexing chute 1704, with the lower end of the indexing chute 1704 being angled inwardly.
During the process of rotating the intermittent feed rod 19 driven by the upward rotation of the driving rod 1407, the rotating driving rod 1407 pushes the adjusting rod 1711 upward via the transverse sliding groove 1408, then the slot penetrating plate 1707 slides upward inside the guiding slot 1704, the liquid separating plate 1705 further moves upward, the reaction mixture liquid is separated and passes through the liquid outlet 1703 under the action of the liquid separating plate 1705, and falls on the glass fiber membrane, and further, when the rotating driving rod 1407 rotates, the intermittent feed rod 19 covers the liquid outlet guide frame 1701 again.
Example one
1. Preparation of enzyme mixture: the enzyme mixture was formulated in a centrifuge tube at the following concentrations: 2U reverse transcriptase, 0.5U DNA recombinase, 0.5U DNA polymerase, 0.25U single-stranded DNA binding protein.
2. Preparing an antibody: adding reverse transcriptase antibody 0.1mg/mL and DNA polymerase antibody 0.1mg/mL
3. Preparing a reaction mixed solution: primer concentration 1 mu mol/L, probe concentration 0.5 mu mol/L, polyethylene glycol 8000 concentration 2%, dNTP concentration 1mM, 4-hydroxy phthalic acid concentration 0.01%; the pH of the reaction mixture was adjusted to 7.5.
4. Preparing a buffer solution: the buffer solution consists of 4mM magnesium chloride, 50mM potassium chloride and 20mM tris (hydroxymethyl) aminomethane; the pH of the buffer was adjusted to 8.7.
5. And (3) drying: adding the reaction mixed solution to a glass fiber membrane, and drying at 42 ℃; cutting the dried glass fiber film into fixed size, placing into a PCR tube, sealing, and storing at room temperature.
Example 2
Verification of the stability of isothermal amplification reagents at 37 ℃
Isothermal amplification reagent 1 was prepared and dried according to example 1, and isothermal amplification reagent 2 was prepared without adding reverse transcriptase antibody 0.1mg/mL and DNA polymerase antibody 0.1mg/mL, as compared with isothermal amplification reagent 1, and the remaining components were the same. As shown in Table 1, the amplification results showed that the Tt value did not decrease significantly when the isothermal amplification reagent 2 was stored at 137 ℃ for 12 months, whereas the Tt value decreased significantly from month 4.
TABLE 137 ℃ isothermal amplification Tt values at different storage times
Example 3
Verification of accelerated stability of isothermal amplification reagents at 55 ℃
The isothermal amplification reagent 1 was prepared and dried according to example 1, and the isothermal amplification reagent 3 was not dried compared with the isothermal amplification reagent 1, and the other components were the same as those in the procedure. As shown in Table 2, the Tt value did not decrease significantly when the isothermal amplification reagent 3 was stored at 155 ℃ for 10 days, whereas the Tt value decreased significantly from day 4.
Table Tt values for isothermal amplification at different retention times at 255 ℃
In summary, the isothermal amplification reagent prepared in example one has a longer isothermal standing time than the isothermal amplification reagents prepared in example two and example three, and the isothermal amplification reagent prepared in example one can be stored at room temperature for at least eighteen months.
Different features which are present in different embodiments may be combined to advantage. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art upon studying the drawings, the specification, and the claims. In the claims, the term "comprising" does not exclude other means or steps; the indefinite article "a" does not exclude a plurality; the terms "first" and "second" are used to denote a name and not to denote any particular order. Any reference signs in the claims shall not be construed as limiting the scope. The functions of the various parts appearing in the claims may be implemented by a single hardware or software module. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
Claims (8)
1. A constant-temperature amplification reagent normal-temperature storage method comprises the following operation steps:
the method comprises the following steps: preparing a reaction mixed solution; preparing an enzyme mixture, an antibody, a primer, a probe, polyethylene glycol 8000, dNTP and 4-hydroxyphthalic acid, and then mixing the enzyme mixture, the antibody, the primer, the probe, the polyethylene glycol 8000, the dNTP and the 4-hydroxyphthalic acid to prepare a reaction mixed solution;
step two: preparing a buffer solution; preparing 4mM magnesium chloride, 50mM potassium chloride, and 20mM tris, followed by mixing 4mM magnesium chloride, 50mM potassium chloride, and 20mM tris to prepare a buffer;
step three: processing the reaction mixed solution; adding the reaction mixed solution prepared in the step one onto a glass fiber membrane by adopting an on-membrane liquid adding device, so that the reaction mixed solution is immersed on the glass fiber membrane;
step four: cutting into blocks; and (3) conveying the glass fiber membrane prepared in the third step into a drying device, drying the glass fiber membrane in a drying mode at 42 ℃, then cutting the dried glass fiber membrane into blocks, fixing the size of the blocks, further filling the cut glass fiber membrane into a PCR tube, sealing and storing at normal temperature.
2. The method for preserving isothermal amplification reagent according to claim 1, characterized in that: in the first step, the concentration of the primer is preferably 1 mu mol/L, the concentration of the probe is preferably 0.5 mu mol/L, the concentration of polyethylene glycol 8000 is preferably 2%, the concentration of dNTP is preferably 1mM, the concentration of 4-hydroxyphthalic acid is preferably 0.01%, the pH value of the reaction mixed solution is 7.5, the enzyme mixture consists of 2U reverse transcriptase, 0.5U DNA recombinase, 0.5U DNA polymerase and 0.25U single-chain DNA binding protein, and the antibody consists of 0.1mg/mL reverse transcriptase antibody and 0.1mg/mL DNA polymerase antibody.
3. The method for preserving isothermal amplification reagent according to claim 1, characterized in that: and the pH value of the buffer solution in the second step is 8.7.
4. The method for preserving isothermal amplification reagent according to claim 1, characterized in that: the on-film liquid adding device comprises a mounting base, a middle through rotating shaft, two wing power gears, reciprocating staggered blocks, an upper end conveying frame, a lower pressing roller, a positioning connecting frame, a reaction mixed liquid storage cylinder, an upper cylinder penetrating shaft and an intermittent liquid supply rod, wherein the middle part penetrates through the rotating shaft and is arranged at the middle part of the mounting base, the two wing power gears are symmetrically sleeved at the two ends of the middle part penetrating through the rotating shaft, wherein the reciprocating staggered blocks are uniformly fixed at one end of the two wing power gears, the upper end conveying frame is positioned at the upper end of the mounting base, wherein the lower press roll is fixed at one side of the upper end conveying frame, the positioning connecting frames are symmetrically fixed at two sides of the middle part of the upper end conveying frame, wherein the reaction mixed liquid storage cylinder is fixed between two positioning connection frames, the lower end of the reaction mixed liquid storage cylinder is penetrated through by a shaft on the cylinder, and the intermittent liquid supply rod is positioned at the lower side in the reaction mixed liquid storage cylinder.
5. The method for preserving isothermal amplification reagent according to claim 4, characterized in that: settle and run through between the pivot to be connected through the pivot in base and the middle part, wherein two wings power gear is in the lower extreme of upper end carriage, two wings power gear and middle part run through and adopt screw connection between the pivot, wherein stagger each other between the piece on two wings power gear, the piece that reciprocates to stagger is the hemisphere piece, wherein the both ends of reaction mixed liquid storage cylinder respectively with two positioning connection between the frame carry out screw connection, wear to be connected through the pivot between axle and the reaction mixed liquid storage cylinder on the section of thick bamboo, wherein wear the inside that two positioning connection frames were inserted respectively at the both ends of axle on the section of thick bamboo, wear to carry out buckle fixed connection between axle and the intermittent type liquid supply pole on the section of thick bamboo.
6. The method for preserving isothermal amplification reagent according to claim 5, characterized in that: the lower side of the middle part of the upper end conveying frame is symmetrically provided with fixedly connected bottom distance rods, the bottom distance rods are arranged in an L shape, one side of the lower end of each bottom distance rod is provided with a fixedly connected driven block, the driven block is positioned at one side of the two wing power gears at the same side, the frame of the upper end conveying frame is uniformly provided with movably connected conveying rollers, two ends of one conveying roller are symmetrically provided with fixedly connected linkage side columns, the linkage side columns penetrate through the upper end conveying frame and are arranged, one end of each linkage side column is sleeved with a fixedly connected follow-up gear, the follow-up gear and the two wing power gears are arranged in a meshing manner, the outer side of the follow-up gear is provided with a distance increasing disc fixedly connected with the linkage side columns, one end of the distance increasing disc is arranged outside and is provided with a rotating driving rod connected through a rotating shaft, and the other end of the rotating driving rod is connected to a through a rotating shaft on a cylinder, one end of the rotation driving rod is provided with a transverse sliding groove in a penetrating way.
7. The method for preserving isothermal amplification reagent according to claim 6, characterized in that: the lower extreme intermediate position of reaction mixture liquid storage cylinder runs through the play liquid guide frame of arranging fixed connection, and wherein play liquid guide frame is in the lower extreme of intermittent type liquid feed pole, the lower extreme of play liquid guide frame arranges fixed connection's the liquid frame that gathers, and wherein the lower extreme intermediate position of gathering the liquid frame runs through and sets up the liquid outlet, it is in the top of conveying roller to gather the liquid frame, and wherein the middle part bilateral symmetry of gathering the liquid frame runs through and sets up the guide spout, the liquid distribution board has been placed to the inside downside of gathering the liquid frame, wherein the upper end bilateral symmetry of liquid distribution board arranges fixed connection's the frid that covers.
8. The method for preserving isothermal amplification reagent according to claim 7, characterized in that: cover the frid and cover the spout of guiding in the inside of gathering the liquid frame, wherein cover the one end downside of frid and arrange fixed connection's the slotted plate that wears, wherein wear the frid and run through and guide the spout and arrange, the one end of wearing the frid runs through and arranges upper and lower inserted bar, and wherein the cover has reset spring on the inserted bar from top to bottom, fixed connection's lamina tecti is arranged to the upper end of upper and lower inserted bar, wherein carries out fixed connection between lamina tecti and the play liquid guide frame, swing joint's adjusting lever is arranged to the other end of wearing the frid, and wherein the other end of adjusting lever inserts the inside of horizontal sliding tray.
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| CN202210781547.4A CN114854833A (en) | 2022-07-05 | 2022-07-05 | Constant-temperature amplification reagent normal-temperature storage method |
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Cited By (1)
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| CN115701839A (en) * | 2023-01-05 | 2023-02-14 | 深圳无微华斯生物科技有限公司 | Constant-temperature amplification kit and normal-temperature storage method |
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