CN102703429B - Nucleic acid isothermal amplification reaction reagent suitable for being stored and transported at normal temperature - Google Patents
Nucleic acid isothermal amplification reaction reagent suitable for being stored and transported at normal temperature Download PDFInfo
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- CN102703429B CN102703429B CN2012101798645A CN201210179864A CN102703429B CN 102703429 B CN102703429 B CN 102703429B CN 2012101798645 A CN2012101798645 A CN 2012101798645A CN 201210179864 A CN201210179864 A CN 201210179864A CN 102703429 B CN102703429 B CN 102703429B
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Abstract
The invention relates to nucleic acid isothermal amplification reaction reagent suitable for being stored and transported at normal temperature, and a kit containing the nucleic acid isothermal amplification reaction reagent. The nucleic acid isothermal amplification reaction reagent is prepared through embedding one or more components of nucleic acid isothermal amplification reaction liquid in gel. The nucleic acid isothermal amplification reaction reagent and the isothermal amplification reagent kit both provided by the utility model can be stored and transported at normal temperature, and can be directly used without special treatment. As being in solid state after treatment, the treated nucleic acid isothermal amplification reaction reagent mixture can be transported by air conveniently, and the difficulty that the reaction reagent in the prior art can not be transported conveniently for long distance by air as being in liquid state. And meanwhile, the kit disclosed by the invention has the characteristics of simple structure and low cost, greatly lowers the storage and transportation cost of the isothermal amplification reaction reagent and brings great conveniences to actualoperation of isothermal amplification reaction at the same time.
Description
Technical field
The invention belongs to the biochemical reagents technical field, be specifically related to a kind of nucleic acid isothermal amplification reaction reagent, namely a kind of can be at the nucleic acid isothermal amplification reaction reagent of normal temperature storage and transport.
Background technology
Nucleic acid isothermal amplification (isothermal nucleic acid amplification) is a class of nucleic acid amplification technologies, and they can enlarge the copy number of specific DNA or RNA fragment under a certain specific temperature.Produced a series of novel nucleic acid isothermal amplification techniques recent years, and these technology or based on the new discovery of DNA/RNA biosynthesizing Mechanism Study perhaps utilize the polysaccharase with specific function to realize under the constant temperature amplification to target nucleic acid; No matter in actually operating still aspect the instrument requirement, these isothermal amplification techniques are all simpler and cheap than round pcr, thereby are developed and utilize easilier.Especially at detection of nucleic acids and diagnostic field, isothermal amplification technique becomes the alternative method of round pcr just gradually and is widely used.Utilize all kinds of detections and the diagnostic products of isothermal amplification technique exploitation to have round pcr product incomparable advantage at aspects such as specificity, ease for use, cost and accessibility, market outlook are very wide.
Though isothermal amplification technique itself is with the obvious advantage, in the large-scale application process, face the puzzlement of some problems based on the reagent of isothermal amplification technique, wherein the most outstanding is that reagent needs the cryogenic freezing storage and transport.A kind of as nucleic acid amplification technologies, isothermal duplication also need be by the participation of the enzyme of biologically active, and the long-term maintenance of enzyme bioactivity needs the cryogenic freezing condition, required oligonucleotide primer, triphosphate deoxy-nucleotide (dNTP) and the reaction buffer etc. of isothermal amplification all need under freezing conditions to store simultaneously, and multigelation can make these compositions lose efficacy.So the product based on isothermal amplification technique also is to require to transport and preserve in the cryogenic freezing condition.This has increased transportation cost on the one hand, has also limited the application of these test kits in the environment that does not possess the cryopreservation condition or area on the other hand.
For scale operation, commercial test kit based on isothermal amplification technique, in order to guarantee consistency of product, stability and reliability, also require as far as possible the disposable interpolation of various reaction reagents and packing, and make product in storage and transportation, possess the long as far as possible quality guarantee period.Therefore, a kind of method that can preserve isothermal amplification reaction reagent mixture easily and effectively of invention has significant values.Patent of invention " store method of loop-mediated isothermal amplification reaction reagent mixture (200810159414.3) " discloses a kind of method that drying protectant carries out vacuum-drying or quick air-dry realization loop-mediated isothermal amplification reaction reagent mixture normal temperature prolonged preservation then of adding in loop-mediated isothermal amplification reaction reagent mixture.But this method needs complex apparatus such as special vacuum lyophilization instrument or quick air dryer, also needs to consume a large amount of energy, is not a kind of very economic method.
Summary of the invention
The purpose of this invention is to provide a kind of nucleic acid isothermal amplification reaction reagent, namely a kind of can be at the nucleic acid isothermal amplification reaction reagent of normal temperature storage and transport, thereby remedy the deficiencies in the prior art.
Nucleic acid isothermal amplification reaction reagent of the present invention is that component with one or more nucleic acid isothermal amplification reaction solutions is embedded in the gel and prepares.
Above-mentioned gel, its fusing point are lower than the deactivation temperature of nucleic acid isothermal amplification polysaccharase.
Above-mentioned gel, can also include the gel that fusing point is higher than the deactivation temperature of nucleic acid isothermal amplification polysaccharase, this dystectic gel must add simultaneously with the gel that fusing point is lower than the deactivation temperature of nucleic acid isothermal amplification polysaccharase, adding proportion be not higher than fusing point be lower than nucleic acid isothermal amplification polysaccharase the deactivation temperature gel quality affects 10%.
The component of above-mentioned nucleic acid isothermal amplification reaction solution is the polysaccharase of nucleic acid isothermal amplification.
The component of above-mentioned nucleic acid isothermal amplification reaction solution is the reaction buffer of polysaccharase of polysaccharase, the nucleic acid isothermal amplification of the amplification of primer, triphosphate deoxy-nucleotide (dNTP) and/or triphosphopyridine nucleotide (NTP), nucleic acid isothermal.
The polysaccharase of above-mentioned nucleic acid isothermal amplification is archaeal dna polymerase and/or RNA reversed transcriptive enzyme.
The polysaccharase of above-mentioned nucleic acid isothermal amplification is archaeal dna polymerase and/or RNA polymerase.
The reaction buffer of the polysaccharase of nucleic acid isothermal amplification is that reaction buffer, the chain of nucleotide sequence dependent amplification substitutes amplified reaction damping fluid or loop-mediated isothermal amplification damping fluid.
But the nucleic acid isothermal amplification reaction reagent that above-mentioned normal temperature is preserved, its a kind of preparation method is as follows: the material that at first can form gel adds in the component of nucleic acid isothermal amplification reaction solution, under the deactivation temperature of the polysaccharase that is lower than the nucleic acid isothermal amplification gel is fused then, last cooled and solidified gel is made.
Another kind of preparation method is as follows: at first can form the material of gel and water Hybrid Heating and make it to dissolve and be made into concentrated mother liquor, then the mother liquor that concentrates is joined in the component of nucleic acid isothermal amplification reaction solution, relief temperature descends, and to make mixture be that solid-state or semi-solid gel is finished preparation by settable liquid.
The interpolation concentration quality volume percent g/ml of the material of above-mentioned formed gel in the component of nucleic acid isothermal amplification reaction solution is 0.01% ~ 30%.
The above-mentioned material that can form gel is for forming polysaccharose substance and/or the gelatin class material of gel.
The above-mentioned material that can form gel is for forming the artificial polymkeric substance of gel.
The above-mentioned polysaccharose substance that can form gel and/or gelatin class material are starch, dextrin, the polysaccharide that condenses (claims to condense glue again, curdlan, hot gel, curdlan, the card Derain), gelling gum, xanthan gum, locust bean gum, flaxseed gum, konjak gum, chitosan, agar, agarose, low melting-point agarose, ultralow fusing point agarose, Furcellaria gum, Lalgine (claims alginic acid again, alginic acid, seaweeds), (as sodium alginate, alginates is otherwise known as seaweed gel, algin or phycocolloid), carrageenin, pectin, low-methoxy pectin, in gelatin and the gum arabic any or several.
The above-mentioned artificial polymkeric substance that can form gel be in methylcellulose gum, carboxymethyl cellulose, acrylamide, the polyacrylamide any or several.
The above-mentioned interpolation concentration quality volume percent g/ml of polysaccharide in the component of nucleic acid isothermal amplification reaction solution that condense is 0.2% ~ 3%, is preferably 0.5% ~ 2%.
The interpolation concentration quality volume percent g/ml of above-mentioned ultralow fusing point agarose in the component of nucleic acid isothermal amplification reaction solution is 0.3% ~ 3.5%, is preferably 0.5% ~ 3%.
The present invention also provides a kind of isothermal amplification kit, include above-mentioned can be at the nucleic acid isothermal amplification reaction reagent of normal temperature state prolonged preservation.
Nucleic acid isothermal amplification reaction reagent provided by the invention and isothermal amplification kit, but storage at normal temperature and transportation, need not special processing during use can directly use.Because the isothermal amplification reaction reagent mixture after handling is solid-state shape, can carry out air transportation very easily, has overcome before this reaction reagent mixture and be in a liquid state and difficultly utilize the aviation mode to carry out the difficult problem of long-distance transportation.Method of the present invention and test kit have technology characteristics simple, with low cost simultaneously, when reducing the storing cost of isothermal amplification reaction reagent greatly, also bring great convenience for the actually operating of isothermal amplification.The present invention will promote effectively isothermal amplification technique in the utilization in fields such as scientific research, medical treatment, check and quarantine with based on the diagnosis of isothermal amplification technique or the large-scale promotion application of detection reagent product.
Embodiment
In the nucleic acid isothermal amplification reaction reagent and test kit that use at present, if directly with nucleic acid isothermal amplification reaction reagent (being the component of nucleic acid isothermal amplification reaction solution) as oligonucleotide primer, triphosphate deoxy-nucleotide (dNTP) or triphosphopyridine nucleotide (NTP), polysaccharase (archaeal dna polymerase, RNA polymerase or reversed transcriptive enzyme etc.), the mixture of the reaction buffer of polysaccharase (dissimilar isothermal amplifications needs different polysaccharase and reaction reagent) etc. is kept under normal temperature or the room temperature condition, and these reaction reagent mixtures have just lost the amplification ability to purpose nucleic acid after generally 1 ~ 2 day.Discover, under the condition of normal temperature or room temperature, it is the easiest in the isothermal amplification liquid (being isothermal amplification reaction reagent mixture) that what lose reactive behavior is polysaccharase, and the major cause of these polysaccharase loss of activity is because preserve for a long time, temperature is too high or temperature variation causes enzyme active center to be kept due to the hydrogen bond of higher structure, ionic linkage, disulfide linkage equimolecular reactive force be damaged.And under normal temperature or room temperature condition, the oligonucleotide primer in the isothermal amplification liquid (being isothermal amplification reaction reagent mixture), dNTP also can very fast degradeds, can't participate in gene amplification.The applicant finds in long term studies, in isothermal amplification reaction reagent mixture, add 0.01% ~ 30%(W/V, be weightmeasurement ratio g/ml) can form the material of gel, be heated to suitable temperature and make gel molecular fusion (or dissolving), the reaction reagent mixture temperature is descended, mixture can be become the gel of solid-state (or semi-solid state) by liquid state, the polysaccharase molecule will be fixed by the tridimensional network that gel molecular forms, the space structure of enzyme molecule (three-dimensional structure or higher structure) is able to long term maintenance, and the activity of polysaccharase can keep for a long time; Simultaneously, gel also can be fixed the various compositions in the primer in the isothermal amplification reaction reagent mixture, triphosphate deoxy-nucleotide (dNTP) or triphosphopyridine nucleotide (NTP) and the polymeric enzyme reaction damping fluid, make in its micropore that is embedded in gel molecular formation, form the partition of physical property to each other, and also completely cut off air, thereby made the various compositions of isothermal amplification reaction reagent mixture be able to steady in a long-term the preservation at normal temperatures.
When needs use the isothermal amplification reaction reagent added gel or its mixture, only need add other required reagent compositions and nucleic acid-templated of isothermal amplification therein, being heated to certain temperature is incubated, gel is fused into liquid state fully under this temperature, and isothermal amplification can carry out smoothly.Isothermal amplification reaction reagent mixture behind the above-mentioned interpolation gel carries out the result of isothermal amplification gained with identical with the resulting result of freshly prepared reaction mixture.
Because of the required optimal reactive temperature difference of dissimilar isothermal duplications, so need adding the mixture of the gel of different sorts, different melting points or its different ratios, dissimilar isothermal amplification reaction reagent mixtures realizes normal temperature prolonged preservation based on gel sets.In actual the use, can be chosen in the gel that can fuse wholly or in part under this temperature to obtain preferable protection and expanding effect according to the needed optimum temperuture of isothermal duplication.Generally, the fusing point of selected gel should be lower than the deactivation temperature for the polysaccharase of nucleic acid isothermal amplification, sometimes in order to improve the intensity of gel, can addition portion divide fusing point to be higher than the gel of the deactivation temperature of the polysaccharase that increases for nucleic acid isothermal, but this dystectic gel must add simultaneously with the gel that fusing point is lower than for the deactivation temperature of the polysaccharase of nucleic acid isothermal amplification, and adding proportion is not higher than fusing point and is lower than for 10% of the gel of the deactivation temperature of the polysaccharase of nucleic acid isothermal amplification.Improve the intensity of gelatin as the mode that can utilize " 10% gelatin+0.1% gelling gum ", maybe can utilize the mode of " 2.0% agarose+0.2% polyacrylamide " to improve the intensity (above-mentioned per-cent is represented the interpolation concentration of gel in the component of nucleic acid isothermal amplification reaction solution, is quality volume percent g/ml) of sepharose.
Above-mentioned gel refers to interconnection or self the molecule water-swelling under certain condition of colloidal particle in the solution or polymer, forms the space reticulated structure, has been full of a kind of special dispersion system as the liquid of dispersion medium in the structure space.
The invention will be further described by the following examples.
Embodiment 1 is based on ring mediated isothermal amplification (loop-mediated isothermal amplification, the preservation effect analysis in test kit LAMP) behind the reaction reagent mixture interpolation gel
1. assemble following each component in the packing box of test kit:
(1) augmentation detection pipe, interior dress LAMP reaction reagent mixture and nucleic acid dye;
(2) negative control pipe, interior dress do not have white spot syndrome virus nucleic acid (White spot syndrome virus, FTA diaphragm WSSV);
(3) positive control pipe, interior dress have adsorbed the FTA diaphragm of WSSV nucleic acid;
The sample area cutting is got off on the FTA card that above-mentioned FTA diaphragm is Britain Whatman, the scraps of paper that size is 3 square millimeters.
2.LAMP reaction reagent mixture adds different concns and the different sorts gel is preserved
The mixture 6048 μ L(of the various LAMP reaction reagents except sample nucleic acid are comprised LAMP primers F IP and each 1.6 μ M of BIP, each 0.2 μ M of primers F 3 and B3, each 1.4mM of dATP, dTTP, dGTP and dCTP, MgCl
26mM, trimethyl-glycine 1M, Tris-HCl 20mM, KCl 10mM, MgSO
42mM, (NH
4)
2SO
410mM, Triton X-1000.1%, Bst archaeal dna polymerase 2016U) is sub-packed in the centrifuge tube of 14 1.5mL by every pipe 432 μ L, in the centrifuge tube of each 1.5mL, add material and the mixing that can form gel by concentration and the kind shown in table 1 the 1st row (totally 14 row), centrifuge tube with 1.5mL places on the metal bath then, according to the solvent temperature of different gels in 50 ~ 65 ℃ of insulations 5 minutes the deactivation temperature of BstDNA polysaccharase (should be lower than 80 ℃), gel is fully dissolved, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, 20 ℃ (normal temperature), preserve under 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
3.LAMP two kinds of reaction reagent mixture interpolations and two or more gel are preserved
The mixture 6048 μ L(of the various LAMP reaction reagents except sample nucleic acid are comprised LAMP primers F IP and each 1.6 μ M of BIP, each 0.2 μ M of primers F 3 and B3, each 1.4mM of dATP, dTTP, dGTP and dCTP, trimethyl-glycine 1M, Tris-HCl 20mM, KCl 10mM, MgSO
48mM, (NH
4)
2SO
410mM, Triton X-1000.1%, BstDNA polysaccharase 2016U) is sub-packed in the centrifuge tube of 14 1.5mL by every pipe 432 μ L, press concentration and the kind shown in table 2 the 1st row (totally 14 row) and in the centrifuge tube of each 1.5mL, add gel and mixing, centrifuge tube with 1.5mL places on the metal bath then, solvent temperature according to different gels is incubated 5 minutes in 50 ~ 65 ℃, gel is fully dissolved, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, then with 18 the every group 0.2mL that mixture is housed the centrifuge tube branch test kit of packing into, test kit is placed 4 ℃ respectively, 20 ℃, preserve under 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
4. preservation and the validity check of LAMP reaction reagent mixture in the test kit
The test kit that is stored in 4 ℃ carries out sampling inspection respectively when store expiration 2 months, 4 months and 6 months, the test kit that is stored in 20 ℃ carries out sampling inspection respectively when store expiration 1 month, 2 months and 3 months, the test kit that is stored in 37 ℃ carries out sampling inspection respectively when store expiration 10 days, 20 days and 30 days.The method of inspection is as follows:
1) according to above-mentioned 9 period of storage section, the validity that need carry out 9 batches, every batch 56 test kit altogether detects, and needs nucleic acid (30ng/ μ L) 56 μ L equivalent with the prawn WSSV after the sex change be sub-packed in being equipped with of 56 test kits in the time of each the detection and is mixed with in the centrifuge tube of 0.2mL of LAMP reaction reagent mixture of gel.Simultaneously, according to the fresh LAMP reaction solution 24 μ L of the preparation of the ratio of reagents in the present embodiment step 2, WSSV nucleic acid (30ng/ μ L) the 1 μ L after the interpolation sex change is as positive control.
2) above-mentioned centrifuge tube is carried out the LAMP reaction in 63 ° of C insulation 60min.
Behind the LAMP reaction terminating, in each centrifuge tube, add the nucleic acid dye GeneFinder of 10 times of dilutions of 2 μ L
TM, make reaction product dyeing and termination reaction in 90 ° of C insulation 5min then.
If reaction solution presents green in the centrifuge tube, illustrate that then the mixture of the LAMP reaction reagent in this centrifuge tube has normal reactive behavior, this test kit is normal; If reaction solution presents orange-yellowly in the centrifuge tube, illustrate that then the mixture of the LAMP reaction reagent in this centrifuge tube has lost normal reactive behavior, this test kit lost efficacy.Detected result shows that all colour developing is green in the land contrast that experiment arranges, and shows that this LAMP reaction system is working properly; The blank group that experiment arranges (not adding gel quav in the mixture of LAMP reaction reagent directly preserves under 4 ° of C, 20 ℃ and 37 ° of C conditions) all develops the color orange-yellow, the mixture that shows the LAMP reaction reagent do not add gel quav and directly be placed on 4 ° of C, 20 ℃ and 37 ° of C conditions under, through after the preservation of above-mentioned time, lost response capacity, its test kit can't normally use accordingly; The detected result of the preservation effect of the test kit of the LAMP reaction reagent mixture of above-mentioned interpolation different concns, different sorts gel is seen Table 1 and table 2.Product after the reaction system amplification of above-mentioned part positive control, blank and interpolation gel is carried out electrophoresis detection, the result shows: amplification poststaining reaction solution shows that the typical LADDER banding pattern of LAMP product all appears in its track of green sample, and amplification poststaining reaction solution shows that the typical LADDER banding pattern of LAMP product does not all appear in its track of orange-yellow sample.
The back detected result (seeing Table 1 and 2) of preservation effect of test kit of adding the LAMP reaction reagent mixture of different concns, different sorts gel shows: the shelf time is when being 1 month, 2 months, and the mixture overwhelming majority of storing under 4 ℃ and 20 ℃ of conditions all has normal reactive behavior; Shelf time, the mixture overwhelming majority of storing under 37 ℃ of conditions all had normal reactive behavior when being 10 days.
In addition, detect at the FTA diaphragm that has adsorbed WSSV nucleic acid in the positive control pipe, find that the viral nucleic acid of this FTA diaphragm absorption also still can normally increase as the template of LAMP reaction; This with Whatman company alleged this TFA card for a long time room temperature preservation nucleic acid be consistent.
Table 1. different concns, different sorts gel are to the protection effect of LAMP reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the LAMP reaction reagent mixture.
2. the contrast of table empty is not for adding the LAMP reaction reagent mixture of gel quav.
3. "+" number expression LAMP reaction reagent mixture has good amplification active in the table, and "-" represents the LAMP reaction reagent mixture activity that do not increase.
The two or more gels of table 2. are to the protection effect of LAMP reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the LAMP reaction reagent mixture.
2. the contrast of table empty is not for adding the LAMP reaction reagent mixture of gel quav.
3. "+" number expression LAMP reaction reagent mixture has good amplification active in the table, and "-" represents the LAMP reaction reagent mixture activity that do not increase.
Embodiment 2 is based on nucleotide sequence dependent amplification (Nucleic acid sequence-based amplification, the preservation effect analysis in test kit NASBA) behind the reaction reagent mixture interpolation gel
1. assemble following each component in the packing box of test kit:
(1) augmentation detection pipe, interior dress NASBA reaction reagent mixture and nucleic acid dye;
(2) negative control pipe, interior dress do not have prawn taura syndrome virus (Taura syndrome viru, TSV) the FTA diaphragm of nucleic acid;
(3) positive control pipe, interior dress have adsorbed the FTA diaphragm of prawn TSV nucleic acid;
The scraps of paper of size 3 mm square that the sample area cutting is got off on the FTA card that above-mentioned FTA diaphragm is Britain Whatman.
2.NASBA reaction reagent mixture adds different concns and the different sorts gel is preserved
Mixture according to following formulated NASBA reaction reagent: comprise 40mM Tris-HCl(pH8.5 in per 24 μ L reaction systems), 70mM KCl, 12mM MgCl
2, each 0.2 μ M of 1mM dNTP, 2mM NTP, 15%DMSO, 10mM DTT, primer, 40U t7 rna polymerase, 8UAMV reversed transcriptive enzyme, 0.2U RNase H, 20U RNasin and 2 μ gBSA, prepare 3456 μ L altogether.Be sub-packed in the centrifuge tube of 8 1.5mL by every pipe 432 μ L, in the centrifuge tube of each 1.5mL, add material and the mixing that can form gel by concentration and the kind shown in table 3 the 1st row (totally 8 row), centrifuge tube with 1.5mL places on the metal bath then, according to the solvent temperature of different gels in 40 ~ 55 ℃ of insulations 5 minutes the deactivation temperature of t7 rna polymerase and AMV reversed transcriptive enzyme (should be lower than 70 ℃), gel is fully dissolved, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, 20 ℃, preserve under 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
3.NASBA two kinds of reaction reagent mixture interpolations and two or more gel are preserved
Mixture according to following formulated NASBA reaction reagent: comprise 40mM Tris-HCl(pH8.5 in per 24 μ L reaction systems), 70mM KCl, 12mM MgCl
2, each 0.2 μ M of 1mM dNTP, 2mM NTP, 15%DMSO, 10mM DTT, primer, 40U t7 rna polymerase, 8U AMV reversed transcriptive enzyme, 0.2U RNase H, 20U RNasin and 2 μ g BSA, prepare 2592 μ L altogether.Be sub-packed in the centrifuge tube of 6 1.5mL by every pipe 432 μ L, press concentration and the kind shown in table 4 the 1st row (totally 6 row) and in the centrifuge tube of each 1.5mL, add gel and mixing, centrifuge tube with 1.5mL places on the metal bath then, solvent temperature according to different gels is incubated 5 minutes in 40 ~ 45 ℃, gel is fully dissolved, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, 20 ℃, preserve under 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
4. preservation and the validity check of NASBA reaction reagent mixture in the test kit
The test kit that is stored in 4 ℃ carries out sampling inspection respectively when store expiration 2 months, 4 months and 6 months, the test kit that is stored in 20 ℃ carries out sampling inspection respectively when store expiration 1 month, 2 months and 3 months, the test kit that is stored in 37 ℃ carries out sampling inspection respectively when store expiration 10 days, 20 days and 30 days.The centrifuge tube of the 0.2mL that mixture is housed that holds in the main test test kit, the method for inspection is as follows:
1) according to above-mentioned 9 period of storage section, the validity that need carry out 9 batches, every batch 28 NASBA reaction reagent mixture altogether detects, need nucleic acid (RNA that is the prawn of the TSV positive, 30ng/ μ L) 28 μ L equivalent with the prawn TSV after the sex change be sub-packed in the centrifuge tube of 28 0.2mL that the NASBA reaction reagent mixture that is mixed with gel is housed in the time of each the detection.Simultaneously, according to the fresh NASBA reaction solution 24 μ L of the preparation of the ratio of reagents in the present embodiment step 2, TSV nucleic acid (30ng/ μ L) the 1 μ L after the interpolation sex change is as positive control.
2) above-mentioned centrifuge tube is carried out the NASBA reaction in 41 ° of C insulation 120min, then reaction tubes is placed-20 ℃ of termination reactions.
3) utilize the GeBAflex-tube of Merck-Novagen to reclaim the reaction product of NASBA in the centrifuge tube of each 0.2mL: the gel that contains reaction product in the centrifuge tube with 0.2mL is transferred among the GeBAflex-tube, the KAc(test kit that adds 0.1 times of volume provides) and the mixing of isopyknic Virahol,-20 ℃ of insulation 10min, the centrifugal 30min of 10000rpm under 4 ℃ of conditions, with 70% ice-cold ethanolic soln washing precipitation, the centrifugal 5min of 10000rpm under 4 ℃ of conditions, drying precipitated under the room temperature, can obtain reaction product with DEPC water is heavily molten then.
4) utilize the probe in detecting reaction product of digoxigenin labeled.
Detected result shows that the land contrast that experiment arranges has hybridization signal, shows that this NASBA reaction system is working properly; The blank group that experiment arranges (not adding gel quav in the mixture of NASBA reaction reagent directly preserves under 4 ° of C, 20 ℃ and 37 ° of C conditions) amixia signal, the mixture that shows the NASBA reaction reagent do not add gel quav and directly be placed on 4 ° of C, 20 ℃ and 37 ° of C conditions under, through after the preservation of above-mentioned time, lost response capacity; The detected result of the preservation effect of the NASBA reaction reagent mixture of above-mentioned interpolation different concns, different sorts gel is seen Table 3 and table 4.
The detected result (seeing Table 3 and 4) of preservation effect of adding the NASBA reaction reagent mixture of different concns, different sorts gel shows: the shelf time is when being 1 month, 2 months, and the mixture major part of storing under 4 ℃ and 20 ℃ of conditions has normal reactive behavior; Shelf time, the mixture of storing under 37 ℃ of conditions partly had normal reactive behavior when being 10 days.
In addition, detect at the FTA diaphragm that has adsorbed TSV nucleic acid in the positive control pipe, find that the viral nucleic acid of this FTA diaphragm absorption also still can normally increase as the template of NASBA reaction; This with Whatman company alleged this TFA card for a long time room temperature preservation nucleic acid be consistent.
(primer sequence is referring to Teng PH for the characteristic primer that the above-mentioned NASBA primer of selecting for use is prawn TSV, Chen CL, Wu CN, Wu SY, Ou BR, Lee PY.Rapid and sensitive detection of Taura syndrome virus using nucleic acid-based amplification.Dis Aquat Organ.2006,21; 73 (1): 13-22.), synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 3. different concns, different sorts gel are to the protection effect of NASBA reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the NASBA reaction reagent mixture.
2. the contrast of table empty is not for adding the NASBA reaction reagent mixture of gel quav.
3. "+" number expression NASBA reaction reagent mixture has good amplification active in the table, and "-" represents the NASBA reaction reagent mixture activity that do not increase.
The two or more gels of table 4. are to the protection effect of NASBA reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the NASBA reaction reagent mixture.
2. the contrast of table empty is not for adding the NASBA reaction reagent mixture of gel quav.
3. "+" number expression NASBA reaction reagent mixture has good amplification active in the table, and "-" represents the NASBA reaction reagent mixture activity that do not increase.
Embodiment 3 substitutes amplification (Strand Displacement Amplification, the preservation effect analysis in test kit SDA) behind the reaction reagent mixture interpolation gel based on chain
1. assemble following each component in the packing box of test kit:
(1) augmentation detection pipe, interior dress SDA reaction reagent mixture and nucleic acid dye;
(2) negative control pipe, interior dress do not have the FTA diaphragm of positive fragment nucleic acid;
(3) positive control pipe, interior dress have adsorbed the FTA diaphragm of positive fragment nucleic acid;
The scraps of paper of size 3 mm square that the sample area cutting is got off on the FTA card that above-mentioned FTA diaphragm is Britain Whatman.
2. SDA reaction reagent mixture interpolation different concns and different sorts gel are preserved in the test kit
Mixture according to following formulated SDA reaction reagent: comprise 40mM Tris-HCl(pH8.0 in per 24 μ L reaction systems), 50mMNaCl, 10mM MgCl
2, 1mM dNTP, 5mM DTT, 50 μ g/mL BSA(bovine serum albumins), 5 μ M SSB(single strand binding proteins), primer each 0.5 μ M, 400nM(0.45U/ μ L) archaeal dna polymerase (Sequenase
TMVersion 2.0) and 3nM(0.04U/ μ L) restriction enzyme Nt.BspQI, 3456 μ L prepared.Be sub-packed in the centrifuge tube of 8 1.5mL by every pipe 432 μ L, press concentration and the kind shown in table 5 the 1st row (totally 8 row) and in the centrifuge tube of each 1.5mL, add gel and mixing, centrifuge tube with 1.5mL places on the metal bath then, according to the solvent temperature of different gels in 37 ~ 50 ℃ of insulations 5 minutes (should be lower than restriction enzyme Nt.BspQI deactivation temperature 65 ℃), gel is fully dissolved, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, 20 ℃, preserve under 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
3. two kinds of SDA reaction reagent mixture interpolations and two or more gel are preserved in the test kit
Mixture according to following formulated SDA reaction reagent: comprise 40mM Tris-HCl(pH8.0 in per 24 μ L reaction systems), 50mMNaCl, 10mM MgCl
2, 1mM dNTP, 5mM DTT, 50 μ g/mL BSA(bovine serum albumins), 5 μ M SSB(single strand binding proteins), each 0.5 μ M, 400nM (0.45U/ μ L) archaeal dna polymerase (Sequenase of primer
TMVersion 2.0), 3nM (0.04U/ μ L) restriction enzyme Nt.BspQI, prepare 2592 μ L.Be sub-packed in the centrifuge tube of 6 1.5mL by every pipe 432 μ L, press concentration and the kind shown in table 5 the 1st row (totally 6 row) and in the centrifuge tube of each 1.5mL, add gel and mixing, centrifuge tube with 1.5mL places on the metal bath then, solvent temperature by different gels is incubated 5 minutes in 37 ~ 40 ℃, gel is fully dissolved, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, preserve under 20 ℃ and the 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
4. preservation and the validity check of SDA reaction reagent mixture in the test kit
The test kit that is stored in 4 ℃ carries out sampling inspection respectively when store expiration 2 months, 4 months and 6 months, the test kit that is stored in 20 ℃ carries out sampling inspection respectively when store expiration 1 month, 2 months and 3 months, the test kit that is stored in 37 ℃ carries out sampling inspection respectively when store expiration 10 days, 20 days and 30 days.The centrifuge tube of the 0.2mL that mixture is housed that holds in the main test test kit, the method for inspection is as follows:
1) according to above-mentioned 9 period of storage section, the validity that need carry out 9 batches, every batch 28 SDA reaction reagent mixture altogether detects, and needs in the time of each the detection positive plasmid after the sex change (20ng/ μ L) 28 μ L equivalent are sub-packed in the centrifuge tube of 28 0.2mL that the SDA reaction reagent mixture that is mixed with gel is housed.Simultaneously, according to the fresh SDA reaction solution 24 μ L of the preparation of the ratio of reagents in the present embodiment step 2, add positive template nucleic acid (20ng/ μ L) 1 μ L, as positive control.
2) above-mentioned centrifuge tube is carried out the SDA reaction in 40 ° of C insulation 60min, then reaction tubes is placed-20 ℃ of termination reactions.
3) utilize the GeBAflex-tube of Merck-Novagen to reclaim the reaction product of SDA in the centrifuge tube of each 0.2mL: the gel that contains reaction product in the centrifuge tube with 0.2mL is transferred among the GeBAflex-tube, the KAc(test kit that adds 0.1 times of volume provides) and the mixing of isopyknic Virahol,-20 ℃ of insulation 10min, the centrifugal 30min of 10000rpm under 4 ℃ of conditions, with 70% ice-cold ethanolic soln washing precipitation, the centrifugal 5min of 10000rpm under 4 ℃ of conditions, drying precipitated under the room temperature, water is heavily molten then can obtain reaction product.
4) electrophoresis detection of reaction product
SDA reaction product behind the above-mentioned purifying is carried out electrophoresis in the gel that contains 2% agarose, electrophoresis is used Genefinder after finishing
TMDyeing is observed electrophoresis result with gel imaging system then and is taken pictures.Detected result shows that the land contrast that experiment arranges has the purpose band to occur, and shows that this SDA reaction system is working properly; The no purpose band of the blank group that experiment arranges (not adding gel quav in the mixture of SDA reaction reagent directly preserves under 4 ° of C, 20 ℃ and 37 ° of C conditions), the mixture that shows the SDA reaction reagent do not add gel quav and directly be placed on 4 ° of C, 20 ℃ and 37 ° of C conditions under, through after the preservation of above-mentioned time, lost response capacity; The detected result of the preservation effect of the SDA reaction reagent mixture of above-mentioned interpolation different concns, different sorts gel is seen Table 5 and table 6.
The back detected result (seeing Table 5 and 6) of preservation effect of adding the SDA reaction reagent mixture of different concns, different sorts gel shows: the shelf time is when being 1 month, and the mixture major part of storing under 4 ℃ and 20 ℃ of conditions all has normal reactive behavior; Shelf time, the mixture sub-fraction of storing under 37 ℃ of conditions had normal reactive behavior when being 10 days.
In addition, detect at the FTA diaphragm that has adsorbed positive fragment nucleic acid in the positive control pipe, find that the nucleic acid of this FTA diaphragm absorption also still can normally increase as the template of SDA reaction; This with Whatman company alleged this TFA card for a long time room temperature preservation nucleic acid be consistent.
The above-mentioned positive fragment of selecting for use, SDA primer, amplification method are referring to document Aric Jonea, Xiaohua Huang.Linear nicking endonuclease-mediated strand-displacement DNAamplification.Anal Biochem, 2011; 414 (1): 58-69., wherein primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 5. different concns, different sorts gel are to the protection effect of SDA reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the SDA reaction reagent mixture.
2. the contrast of table empty is not for adding the SDA reaction reagent mixture of gel quav.
3. "+" number expression SDA reaction reagent mixture has good amplification active in the table, and "-" represents the SDA reaction reagent mixture activity that do not increase.
The two or more gels of table 6. are to the protection effect of SDA reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the SDA reaction reagent mixture.
2. the contrast of table empty is not for adding the SDA reaction reagent mixture of gel quav.
3. "+" number expression SDA reaction reagent mixture has good amplification active in the table, and "-" represents the SDA reaction reagent mixture activity that do not increase.
Embodiment 4 adds preservation effect analysis behind the premix gel based on reaction reagent mixture in the test kit of ring mediated isothermal amplification (LAMP)
1. assemble following each component in the packing box of test kit:
(1) augmentation detection pipe, interior dress LAMP reaction reagent mixture and nucleic acid dye;
(2) negative control pipe, interior dress do not have the FTA diaphragm of WSSV nucleic acid;
(3) positive control pipe, interior dress have adsorbed the FTA diaphragm of WSSV nucleic acid;
The scraps of paper of size 3 mm square that the sample area cutting is got off on the FTA card that above-mentioned FTA diaphragm is Britain Whatman.
2.LAMP reaction reagent mixture adds different concns and different types of premix gel is preserved
At first press the concentration shown in table 7 the 1st row (totally 14 row) and the premix gel of 2 times of concentration in table of kind preparation; The mixture 3024 μ L(of the various LAMP reaction reagents except sample nucleic acid are comprised LAMP primers F IP and each 1.6 μ M of BIP, each 0.2 μ M of primers F 3 and B3, each 1.4mM of dATP, dTTP, dGTP and dCTP, MgCl
26mM, trimethyl-glycine 1M, Tris-HCl 20mM, KCl 10mM, MgSO
42mM, (NH
4)
2SO
410mM, Triton X-1000.1%, BstDNA polysaccharase 2016U) be sub-packed in the centrifuge tube of 14 1.5mL by every pipe 216 μ L; With above-mentioned 2 times in table the premix gel of concentration place under the melting temperature of gel and fuse gel, press the concentration shown in table 7 the 1st row and kind to the middle also mixing that adds the gel of 216 μ L remeltings of the centrifuge tube (totally 14) of above-mentioned each 1.5mL, centrifuge tube with 1.5mL places on the metal bath then, melting temperature (Tm) according to different gels is incubated 5 minutes in 50 ~ 65 ℃, gel is well-dispersed in the solution, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, 20 ℃ (normal temperature), preserve under 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
3.LAMP two kinds of reaction reagent mixture interpolations and two or more premix gel are preserved
At first press the concentration shown in table 8 the 1st row (totally 14 row) and the premix gel of 2 times of concentration in table of kind preparation; The mixture 6048 μ L(of the various LAMP reaction reagents except sample nucleic acid are comprised LAMP primers F IP and each 1.6 μ M of BIP, each 0.2 μ M of primers F 3 and B3, each 1.4mM of dATP, dTTP, dGTP and dCTP, trimethyl-glycine 1M, Tris-HCl 20mM, KCl 10mM, MgSO
48mM, (NH
4)
2SO
410mM, Triton X-100 0.1%, BstDNA polysaccharase 2016U) be sub-packed in the centrifuge tube of 9 1.5mL by every pipe 216 μ L; With above-mentioned 2 times in table the premix gel of concentration place under the melting temperature of gel and fuse gel, press the concentration shown in table 8 the 1st row and kind to the middle also mixing that adds the gel of 432 μ L remeltings of the centrifuge tube (totally 14) of above-mentioned each 1.5mL, centrifuge tube with 1.5mL places on the metal bath then, melting temperature (Tm) according to different gels is incubated 5 minutes in 50 ~ 65 ℃, gel is well-dispersed in the solution, again the amount of the mixture in each 1.5mL centrifuge tube by 24 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, preserve under 20 ℃ and the 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
4. preservation and the validity check of LAMP reaction reagent mixture in the test kit
The test kit that is stored in 4 ℃ carries out sampling inspection respectively when store expiration 2 months, 4 months and 6 months, the test kit that is stored in 20 ℃ carries out sampling inspection respectively when store expiration 1 month, 2 months and 3 months, the test kit that is stored in 37 ℃ carries out sampling inspection respectively when store expiration 10 days, 20 days and 30 days.The centrifuge tube of the 0.2mL that mixture is housed that holds in the main test test kit, the method for inspection is as follows:
1) according to above-mentioned 9 period of storage section, the validity that need carry out 9 batches, every batch 56 LAMP reaction reagent mixture altogether detects, and needs nucleic acid (30ng/ μ L) 56 μ L equivalent with the prawn WSSV after the sex change be sub-packed in the centrifuge tube of 56 0.2mL that the LAMP reaction reagent mixture that is mixed with gel is housed in the time of each the detection.Simultaneously, according to the fresh LAMP reaction solution 24 μ L of the preparation of the ratio of reagents in the present embodiment step 2, WSSV nucleic acid (30ng/ μ L) the 1 μ L after the interpolation sex change is as positive control.
2) above-mentioned centrifuge tube is carried out the LAMP reaction in 63 ° of C insulation 60min.
Behind the LAMP reaction terminating, in each centrifuge tube, add the nucleic acid dye GeneFinder of 10 times of dilutions of 2 μ L
TM, make reaction product dyeing and termination reaction in 90 ° of C insulation 5min then.
If reaction solution presents green in the centrifuge tube, illustrate that then the mixture of the LAMP reaction reagent in this centrifuge tube has normal reactive behavior; If reaction solution presents orange-yellowly in the centrifuge tube, illustrate that then the mixture of the LAMP reaction reagent in this centrifuge tube has lost normal reactive behavior.Detected result shows that all colour developing is green in the land contrast that experiment arranges, and shows that this LAMP reaction system is working properly; The blank group that experiment arranges (not adding gel quav in the mixture of LAMP reaction reagent directly preserves under 4 ° of C, 20 ℃ and 37 ° of C conditions) all develops the color orange-yellow, the mixture that shows the LAMP reaction reagent do not add gel quav and directly be placed on 4 ° of C, 20 ℃ and 37 ° of C conditions under, through after the preservation of above-mentioned time, lost response capacity; The detected result of the preservation effect of the LAMP reaction reagent mixture of above-mentioned interpolation different concns, different sorts gel is seen Table 1 and table 2.Product after the reaction system amplification of above-mentioned part positive control, blank and interpolation gel is carried out electrophoresis detection, the result shows: amplification poststaining reaction solution shows that the typical LADDER banding pattern of LAMP product all appears in its track of green sample, and amplification poststaining reaction solution shows that the typical LADDER banding pattern of LAMP product does not all appear in its track of orange-yellow sample.
The back detected result (seeing Table 7 and 8) of preservation effect of adding the LAMP reaction reagent mixture of different concns, different sorts gel shows: the shelf time is when being 1 month, 2 months, and the mixture overwhelming majority of storing under 4 ℃ and 20 ℃ of conditions all has normal reactive behavior; Shelf time, the mixture overwhelming majority of storing under 37 ℃ of conditions all had normal reactive behavior when being 10 days.This result with directly add gelatinous mass afterreaction mixture reaction activity hold-time basically identical as a result in the isothermal amplification mixture.
In addition, detect at the FTA diaphragm that has adsorbed WSSV nucleic acid in the positive control pipe, find that the viral nucleic acid of this FTA diaphragm absorption also still can normally increase as the template of LAMP reaction; This with Whatman company alleged this TFA diaphragm (card) for a long time room temperature preservation nucleic acid be consistent.
Table 7. different concns, different sorts premix gel are to LAMP reaction reagent mixture protection effect in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the LAMP reaction reagent mixture.
2. the contrast of table empty is not for adding the LAMP reaction reagent mixture of gel quav.
3. "+" number expression LAMP reaction reagent mixture has good amplification active in the table, and "-" represents the LAMP reaction reagent mixture activity that do not increase.
The two or more premix gels of table 8. are to the protection effect of LAMP reaction reagent mixture in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the LAMP reaction reagent mixture.
2. the contrast of table empty is not for adding the LAMP reaction reagent mixture of gel quav.
3. "+" number expression LAMP reaction reagent mixture has good amplification active in the table, and "-" represents the LAMP reaction reagent mixture activity that do not increase.
Embodiment 5 gel immobilized Bst archaeal dna polymerases are to the influence of test kit standing storage
1. assemble following each component in the packing box of test kit:
(1) augmentation detection pipe, interior dress LAMP reaction reagent mixture and nucleic acid dye;
(2) negative control pipe, interior dress do not have the FTA diaphragm of WSSV nucleic acid;
(3) positive control pipe, interior dress have adsorbed the FTA diaphragm of WSSV nucleic acid;
(4) LAMP reaction reagent, the reaction buffer of the required primer of LAMP reaction, trimethyl-glycine, magnesium chloride, dNTP, Bst archaeal dna polymerase etc.
The scraps of paper of size 3 mm square that the sample area cutting is got off on the FTA card that above-mentioned FTA diaphragm is Britain Whatman.
2. add different concns and different sorts gel the Bst archaeal dna polymerase is carried out immobilization and storage
In the centrifuge tube of 14 1.5mL that 100 μ LBst archaeal dna polymerases (2U/ μ L) are housed, add material and the mixing that can form gel by the concentration shown in table 9 the 1st row (totally 14 row) and kind, centrifuge tube with above-mentioned 1.5mL places on the metal bath then, solvent temperature according to different gels is incubated 5 minutes in 50 ~ 65 ℃, gel is fully dissolved, again the mixture that contains the Bst archaeal dna polymerase in each the 1.5mL centrifuge tube amount by 4 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, preserve under 20 ℃ (normal temperature) and 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
3. add two kinds and two or more gel to the immobilization of Bst archaeal dna polymerase and storage
In the centrifuge tube of 14 1.5mL that 100 μ LBst archaeal dna polymerases (4U/ μ L) are housed, add material and the mixing that can form gel accordingly by the concentration shown in table 10 the 1st row (totally 14 row) and kind, centrifuge tube with 1.5mL places on the metal bath then, solvent temperature according to different gels is incubated 5 minutes in 50 ~ 65 ℃, gel is fully dissolved, again the mixture that contains the Bst archaeal dna polymerase in each the 1.5mL centrifuge tube amount by 4 μ L is sub-packed in the centrifuge tube of 18 0.2mL, the test kit of then 18 the every group centrifuge tube branches that the 0.2mL of mixture is housed being packed into, test kit is placed 4 ℃ respectively, preserve under 20 ℃ (normal temperature) and 37 ℃ of conditions and (preserve 6 test kits under each temperature condition, divide 3 shelf time sampling observations, inspect 2 test kits by random samples as repeating at every turn).
4. the influence to the LAMP expanding effect is checked after the gel immobilized Bst archaeal dna polymerase standing storage
The test kit that is stored in 4 ℃ carries out sampling inspection respectively when store expiration 2 months, 4 months and 6 months, the test kit that is stored in 20 ℃ carries out sampling inspection respectively when store expiration 1 month, 2 months and 3 months, the test kit that is stored in 37 ℃ carries out sampling inspection respectively when store expiration 10 days, 20 days and 30 days.The centrifuge tube of the 0.2mL that mixture is housed that holds in the main test test kit, the method for inspection is as follows:
1) according to above-mentioned 9 period of storage section, the validity that need carry out 9 batches, every batch 56 immobilization Bst archaeal dna polymerase altogether detects, need in the time of each the detection mixture 1120 μ L(of the various LAMP reaction reagents except sample nucleic acid, Bst archaeal dna polymerase are comprised LAMP primers F IP and each 1.6 μ M of BIP, each 0.2 μ M of primers F 3 and B3, each 1.4mM of dATP, dTTP, dGTP and dCTP, trimethyl-glycine 1M, Tris-HCl 20mM, KCl 10mM, MgSO
48mM, (NH
4)
2SO
410mM, Triton X-1000.1%) is sub-packed in the centrifuge tube of 56 0.2mL of every batch of sampling observation by the amount of every pipe 20 μ L, again nucleic acid (30ng/ μ L) the 56 μ L equivalent of the prawn WSSV after the sex change are sub-packed in above-mentioned every batch the centrifuge tube of 0.2mL of 56 dress LAMP reaction reagent mixtures, then according to the solvent temperature of different gels in 50 ~ 65 ℃ of insulations 5 minutes, gel is fully dissolved.Simultaneously, prepare fresh LAMP reaction solution 24 μ L, WSSV nucleic acid (30ng/ μ L) the 1 μ L after the interpolation sex change is as positive control.
2) centrifuge tube with above-mentioned 0.2mL carries out the LAMP reaction in 63 ° of C insulation 60min.
Behind the LAMP reaction terminating, in each centrifuge tube, add the nucleic acid dye GeneFinder of 10 times of dilutions of 2 μ L
TM, make reaction product dyeing and termination reaction in 90 ° of C insulation 5min then.
If reaction solution presents green in the centrifuge tube, illustrate that then the mixture of the LAMP reaction reagent in this centrifuge tube has normal reactive behavior; If reaction solution presents orange-yellowly in the centrifuge tube, illustrate that then the mixture of the LAMP reaction reagent in this centrifuge tube has lost normal reactive behavior.Detected result shows that all colour developing is green in the land contrast that experiment arranges, and shows that this LAMP reaction system is working properly; The blank group that experiment arranges (not adding the Bst archaeal dna polymerase that gel quav is directly preserved under 4 ° of C, 20 ℃ and 37 ° of C conditions) all develops the color orange-yellow, the mixture that shows the LAMP reaction reagent do not add gel quav and directly be placed on 4 ° of C, 20 ℃ and 37 ° of C conditions under, through after the preservation of above-mentioned time, lost response capacity; The detected result of the preservation effect of the LAMP reaction reagent mixture of above-mentioned interpolation different concns, different sorts gel is seen Table 9 and table 10.Product after the reaction system amplification of above-mentioned part positive control, blank and interpolation gel is carried out electrophoresis detection, the result shows: amplification poststaining reaction solution shows that the typical LADDER banding pattern of LAMP product all appears in its track of green sample, and amplification poststaining reaction solution shows that the typical LADDER banding pattern of LAMP product does not all appear in its track of orange-yellow sample.
The back detected result (seeing Table 9 and 10) of preservation effect of adding the LAMP reaction reagent mixture of different concns, different sorts gel shows: the shelf time is when being 1 month, and the mixture overwhelming majority of storing under 4 ℃ and 20 ℃ of conditions all has normal reactive behavior; Shelf time, the mixture overwhelming majority of storing under 37 ℃ of conditions all had normal reactive behavior when being 10 days.From The above results as can be seen, utilize gel only Bst archaeal dna polymerase in the test kit to be carried out immobilization, the preservation effect of test kit under differing temps will obviously be worse than and utilize gel to containing the whole immobilized effects of LAMP reaction reagent mixture of Bst archaeal dna polymerase, primer, dNTP etc.
In addition, detect at the FTA diaphragm that has adsorbed WSSV nucleic acid in the positive control pipe, find that the viral nucleic acid of this FTA diaphragm absorption also still can normally increase as the template of LAMP reaction; This with Whatman company alleged this TFA card for a long time room temperature preservation nucleic acid be consistent.
Table 9. different concns, different sorts gel are to the protection effect of Bst archaeal dna polymerase in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the LAMP reaction reagent mixture.
2. the contrast of table empty is not for adding the LAMP reaction reagent mixture of gel quav.
3. "+" number expression LAMP reaction reagent mixture has good amplification active in the table, and "-" represents the LAMP reaction reagent mixture activity that do not increase.
The two or more gels of table 10. are to the protection effect of Bst archaeal dna polymerase in the test kit
Annotate: 1. the 1st of table the list kind and the concentration of adding gel in the LAMP reaction reagent mixture.
2. the contrast of table empty is not for adding the LAMP reaction reagent mixture of gel quav.
3. "+" number expression LAMP reaction reagent mixture has good amplification active in the table, and "-" expression not amplification is active.
Selected LAMP primers F IP, BIP, F3 and B3 is that (primer sequence is referring to Kono T for the characteristic primer of prawn WSSV in above-described embodiment, Savan R, Sakai M, Itami T.Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification.J Virol Methods.2004; 115 (1): 59-65.), synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Owing to comprise the easily composition of loss of activity such as polysaccharase in the isothermal amplification reaction reagent, so the storage and transport of isothermal amplification reaction reagent and test kit all are to adopt refrigerator, ice bag or dry ice etc. to keep under the condition of low temperature to carry out at present, this not only causes the accumulating difficulty of isothermal amplification reaction reagent to strengthen the use cost that has also increased relevant isothermal amplification technique.Experimental result shows, isothermal amplification reaction reagent of the present invention or its mixture can be deposited 2 months under normal temperature or room temperature condition or be above and can not lose the normal reaction activity, and (as 4 ℃ or following) are deposited and still had normal reactive behavior in 6 months under lower temperature condition; Deposited 2 months under normal temperature or room temperature condition or above can also the continuation used based on the isothermal amplification kit of mentioned reagent, (as 4 ℃ or following) are deposited and still can normally be used in 6 months under lower temperature condition; This provides selection very easily for the accumulating of isothermal amplification reaction reagent box undoubtedly.Reagent of the present invention has preparation technology's characteristics simple, with low cost simultaneously, isothermal amplification actual mix (normal temperature or room temperature equitemperature condition) under storage requirement easily has activity stabilized lasting advantage in the test kit, this also brings great convenience for the actually operating of isothermal amplification when reducing isothermal amplification reaction reagent and test kit product storing cost greatly.This invention will promote the utilization in fields such as scientific research, medical treatment, check and quarantines of isothermal amplification technique effectively.
Reagent and material source used in the embodiment of the invention are as follows: the polysaccharide that condenses, gelling gum, xanthan gum, locust bean gum, chitosan, agar, agarose, Furcellaria gum, Lalgine, sodium alginate, alginate calcium, carrageenin, pectin, low-methoxy pectin, gelatin, gum arabic, acrylamide, polyacrylamide, trimethyl-glycine (Betaine) etc. are all available from Sigma company, konjak gum is available from Shantou Jiecheng Biotechnology Co., Ltd., Tris-HCl, KCl, MgSO
4, MgCl
2, (NH
4)
2SO
4, Triton X-100 and bovine serum albumin (BSA) be available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, various archaeal dna polymerases, RNA polymerase and SSB etc. are available from NEB company, the AMV ThermoScript II is available from Promega company, nucleic acid dye GeneFinder
TMAvailable from Xiamen Baiweixin Biological Technology Co., Ltd..Also can adopt other commercially available like product to realize the present invention.
Claims (8)
1. but the nucleic acid isothermal amplification reaction reagent of normal temperature storage and transport is characterized in that, described nucleic acid isothermal amplification reaction reagent is that the component with one or more nucleic acid isothermal amplification reaction solutions is embedded in the gel and prepares; Described gel, its fusing point are lower than the deactivation temperature for the polysaccharase of nucleic acid isothermal amplification.
2. nucleic acid isothermal amplification reaction reagent as claimed in claim 1, it is characterized in that described gel, also include the gel of deactivation temperature that fusing point is higher than the polysaccharase of nucleic acid isothermal amplification, and the fusing point interpolation mass ratio of gel of deactivation temperature that is higher than the polysaccharase of nucleic acid isothermal amplification is not higher than fusing point and is lower than for 10% of the gel of the deactivation temperature of the polysaccharase of nucleic acid isothermal amplification.
3. nucleic acid isothermal amplification reaction reagent as claimed in claim 1, the component that it is characterized in that described nucleic acid isothermal amplification reaction solution are the polysaccharase of nucleic acid isothermal amplification.
4. nucleic acid isothermal amplification reaction reagent as claimed in claim 1, the component that it is characterized in that described nucleic acid isothermal amplification reaction solution are the reaction buffer of polysaccharase of polysaccharase, the nucleic acid isothermal amplification of primer, triphosphate deoxy-nucleotide and/or triphosphopyridine nucleotide, nucleic acid isothermal amplification.
5. as each described nucleic acid isothermal amplification reaction reagent of claim 1-4, it is characterized in that the polysaccharase of described nucleic acid isothermal amplification is archaeal dna polymerase and/or RNA reversed transcriptive enzyme.
6. as each described nucleic acid isothermal amplification reaction reagent of claim 1-4, it is characterized in that the polysaccharase of described nucleic acid isothermal amplification is archaeal dna polymerase and/or RNA polymerase.
7. reaction reagent as claimed in claim 4 is characterized in that the reaction buffer of the polysaccharase of described nucleic acid isothermal amplification substitutes amplified reaction damping fluid or loop-mediated isothermal amplification damping fluid for reaction buffer, the chain that relies on amplification of nucleic acid sequences.
8. an isothermal amplification kit includes the described reaction reagent of claim 1 or claim 2.
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| US7919294B2 (en) * | 2001-03-12 | 2011-04-05 | Biotools Biotechnological & Medical Laboratories, S.A. | Process for preparing stabilized reaction mixtures which are partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures |
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| JP5721704B2 (en) * | 2009-06-12 | 2015-05-20 | マイクロニクス， インコーポレイテッド | Rehydratable matrix for dry storage of TAQ polymerase in microfluidic devices |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7919294B2 (en) * | 2001-03-12 | 2011-04-05 | Biotools Biotechnological & Medical Laboratories, S.A. | Process for preparing stabilized reaction mixtures which are partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures |
Non-Patent Citations (2)
| Title |
|---|
| Ramanujam et al.Room-temperature-stable PCR Reagent.《Genome Research》.1993,(第3期),第75-76页. * |
| 鲁雪等.核酸扩增新技术.《动物医学进展》.2010,第31卷(第3期),第103-106页. * |
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