Microsphere preparation for nucleic acid amplification, amplification method and application in combined detection

Abstract

The present invention relates to the field of molecular diagnostic technology, and in particular to microsphere preparations for nucleic acid amplification, amplification methods, and applications in combined detection. The microsphere preparations for nucleic acid amplification provided by the present invention can be stored for a long time at 2 to 8°C, and when used, the microsphere preparations provided by the present invention do not require additional solvents to be added, but are directly mixed with the sample to be tested, which can significantly increase the template content in the system without changing the concentration of the original system, thereby improving the sensitivity of detection. The microsphere preparations for nucleic acid amplification provided by the present invention are used in RPA, RAA, or in dual or multiple detections composed of a second reaction based on RPA or RAA, which can significantly improve the sensitivity while ensuring the specificity of amplification.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis technology, and in particular to a microsphere preparation for nucleic acid amplification, an amplification method and an application in combined detection. Background Art

[0002] With the development of diagnostic technology, molecular diagnostic technology has gradually become an important means of diagnosing diseases in daily life. With the diversification of detection targets, the application of multiplex detection is becoming more and more extensive. In order to ensure sufficient accuracy, most multiplex detections require amplification of the target fragments to be detected and use the amplified products for detection.

[0003] At present, the amplification reaction system of the target to be detected can only be carried out in a liquid phase environment, and the traditional reagents used for amplification are basically stored and transported in liquid form. The small amount of microsphere preparations used for amplification need to be reconfigured into a solution form with a solvent before amplification, and then used in the amplification reaction. The purpose of using microsphere preparations is only to improve the stability of certain special reagents and reduce their requirements for storage and transportation conditions.

[0004] The detection sensitivity of some emerging detection methods (such as RAA or RPA) is restricted by the amount of template added to the system. Since the original system uses liquid reconstitution, the amount of template that can be added to the system can be adjusted in a small range. Forcibly increasing the amount of template added can easily lead to changes in the concentration of active ingredients in the system. Since the recombinase isothermal amplification technology is a multi-enzyme reaction, changes in the concentration of each component can easily lead to a decrease in the specificity of the reaction. At the same time, in-situ freeze-drying limits the flexibility of the system. Consumables must be fixed during the production stage, which limits the flexibility of reagent use and the adaptability of the machine model.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a microsphere preparation, in which the liquid sample to be tested is directly mixed with the microsphere preparation during the amplification reaction, and the preparation step of the amplification reagent is omitted, so that the primer concentration is always maintained in a stable supersaturated state, thereby improving the sensitivity of the multiple detection reaction, and at the same time will not have a negative impact on the specificity of the amplification reaction.

[0007] In order to solve the above technical problems and achieve the above objectives, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a microsphere preparation for nucleic acid amplification, wherein the microsphere preparation comprises reaction microspheres obtained by freeze-drying a mixed reagent required for an amplification reaction, wherein each gram of the reaction microspheres contains:

[0009] DNA polymerase 131.58~530.5μg, single-stranded binding protein 2.632~13.263mg, recombinase 0.9867~3.316mg, auxiliary protein 0.395~1.33mg, each primer 0.0075~0.02nmol, creatine kinase 657.89~663.13μg, ATP 0.1~0.2μmol, DTT 0.0026~0.015mmol, phosphokinase 0.1~0.5mmol, dNTP each 0.008~0.012μmol, Tris-Ac 0.5~2.5μmol, maltose 0~663.13mg, PEG 131.58~663.13mg.

[0010] In an optional embodiment, the microsphere preparation is used in an RNA amplification system, and each gram of the microsphere preparation further comprises 394.74 to 795.76 μg of reverse transcriptase.

[0011] In an optional embodiment, the microsphere preparation includes reaction microspheres obtained by freeze-drying the mixed reagents required for the amplification reaction, and each gram of reaction microspheres contains:

[0012] DNA polymerase 460.53~464.19μg, each primer 0.013nmol, single-stranded binding protein 10.53~10.61mg, recombinase 1.71~1.72mg, auxiliary protein 1.05~1.06mg, maltose 394.74~397.88mg, PEG 150~151.19mg, ATP 0.15μmol, dNTP 0.01μmol each, Tris-Ac 0.5μmol.

[0013] Preferably, the microsphere preparation comprises 657.89-663.13 μg of reverse transcriptase.

[0014] In an alternative embodiment, the recombinase is selected from T4 UvsX protein, T6 UvsX protein or Rb69 UvsX protein.

[0015] Preferably, the auxiliary protein is selected from T4 UvsY protein, T6 UvsY protein or Rb69 UvsY protein.

[0016] Preferably, the DNA polymerase is a strand displacement DNA polymerase selected from the large fragment of DNA polymerase I of Staphylococcus aureus, the large fragment of DNA polymerase I of Bacillus subtilis, the large fragment of DNA polymerase I of Escherichia coli or T4 bacteriophage Klewnowexo-polymerase.

[0017] Preferably, the reverse transcriptase comprises M-MLV reverse transcriptase.

[0018] Preferably, the single-stranded binding protein is selected from T4 GP32 protein, T6 GP32 protein or Rb69 GP32 protein.

[0019] In an optional embodiment, the microsphere preparation further comprises a second microsphere, wherein the second microsphere contains a complexing solvent PEG and / or a magnesium salt activator.

[0020] Preferably, each gram of the second microsphere contains 960-980 mg of PEG and / or 265.0-265.5 μmol of magnesium salt.

[0021] In a second aspect, the present invention provides a use of the microsphere preparation described in any one of the aforementioned embodiments in RPA or RAA.

[0022] In a third aspect, the present invention provides a method for preparing a microsphere preparation, wherein the microsphere preparation comprises the microsphere preparation described in any one of the aforementioned embodiments;

[0023] The preparation method comprises mixing the components of the microsphere preparation uniformly, dripping them into liquid nitrogen at time intervals of not less than 25 seconds, storing the microspheres in liquid nitrogen for not less than 1 hour, transferring them to a freeze dryer for freeze drying, and freeze drying them according to a freeze drying procedure to obtain the microsphere preparation;

[0024] The freeze-drying procedure is a gradient temperature rise freeze-drying method, which sequentially includes a pre-freezing step, a main drying step and a final drying step.

[0025] Preferably, the temperature of the pre-freezing step is below -54°C, and the processing time is 0.5 to 1 hour.

[0026] Preferably, the temperature of the main drying step is -27 to -15°C, the treatment time is 2 to 6 hours, and the vacuum degree is 0.01 to 30 Pa; further preferably, the main drying step includes at least two gradient temperature increase treatment processes.

[0027] Preferably, the temperature of the final drying step is 0-20° C., the treatment time is more than 2 hours, and the vacuum degree is 0.01-1 Pa; further preferably, the final drying step includes at least four gradient temperature increase treatment processes.

[0028] In a fourth aspect, the present invention provides a method for nucleic acid amplification using the microsphere preparation described in any of the preceding embodiments, the method comprising adding reaction microspheres to the amplification sample solution at a ratio of 0.263 to 6.58 mL of the sample solution to be amplified per gram of reaction microspheres, and then amplifying according to any one of the following (a) to (c):

[0029] (a) Add liquid resolvent and activator, mix well, and directly amplify at 37-44°C for 20 min;

[0030] (b) adding a second microsphere and amplifying at 37-44° C. for 20 min, wherein the second microsphere contains a complexing solvent PEG and a magnesium salt activator;

[0031] (c) After redissolution, a second microsphere is added and amplified at 37-44°C for 20 min, wherein the second microsphere contains a magnesium salt activator.

[0032] In a fifth aspect, the present invention provides a use of a microsphere preparation in a nucleic acid amplification combined with a second reaction method, wherein the microsphere preparation includes the microsphere preparation described in any one of the aforementioned embodiments;

[0033] The nucleic acid amplification adopts the nucleic acid amplification method described in the above embodiment;

[0034] The preparations used in the second reaction are all microsphere preparations;

[0035] The second reaction includes a fluorescence reaction or a CRISPR reaction.

[0036] Preferably, the fluorescence reaction comprises adding EXO enzyme and probe into the RPA or RAA system before freeze-drying, so that the RPA or RAA fluorescence reaction can be detected in real time.

[0037] Preferably, the EXO enzyme selected from the exonuclease is exonuclease III.

[0038] Preferably, the CRISPR reaction includes a Cas12 CRISPR detection system and a Cas13 CRISPR detection system.

[0039] In an optional embodiment, the second reaction method includes directly adding a microsphere preparation for the second reaction to the amplification product to complete the second reaction.

[0040] In an optional embodiment, the second reaction is a CRISPR reaction, and the method for preparing the microsphere preparation used in the CRISPR reaction is: preparing a CRISPR freeze-drying system, and then using the method described in the above embodiment to prepare the microsphere preparation used in the CRISPR reaction;

[0041] The CRISPR freeze-dried system contains: CRISPR freeze-dried protectant 10uL / test, Buffer 1×, Cas12 protein 40-100nmol / L, Cas13 protein 40-100nmol / L, mRNA enzyme inhibitor 5U, T7 RNA polymerase 14U, rNTP 0.5-0.6mM, CrRNA1 0.1μM, ssDNA 0.8-1.2nmol / L, ssRNA 0.8-1.2nmol / L.

[0042] Preferably, the Cas12 protein is selected from LbCas12a, FnCas12a, AsCas12a (cpf1), BbCas12a (cpf1), HkCas12a (cpf1).

[0043] Preferably, the Cas13 protein includes LwaCas13a.

[0044] Preferably, the T7 RNA polymerase is expressed in Escherichia coli.

[0045] In a sixth aspect, the present invention further provides the use of the microsphere preparation described in any of the aforementioned embodiments in an amplified self-chromogenic reaction, wherein each gram of the microsphere preparation further comprises 0.4613 to 12.13 mg of EXO enzyme.

[0046] Preferably, each gram of the microsphere preparation includes 0.6579 to 0.6667 mg of EXO enzyme.

[0047] The microsphere preparation for nucleic acid amplification provided by the present invention can be stored for a long time at 2-8°C. Moreover, when the microsphere preparation provided by the present invention is used, no additional solvent is added, but it is directly mixed with the sample to be tested, which can significantly increase the upper limit of template concentration, thereby improving the sensitivity in multiple detection.

[0048] The microsphere preparation for nucleic acid amplification provided by the present invention is used in RPA, RAA, or in a double or multiple detection consisting of a second reaction combined with RPA or RAA, which can significantly improve the sensitivity while ensuring the specificity of amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 The packaging form of the microsphere preparation provided by the present invention for only RPA or RAA reaction;

[0051] Figure 2 The packaging form of the microsphere preparation provided by the present invention includes reconstituted microspheres or activated microspheres;

[0052] Figure 3 The packaging form of the microsphere preparation for RPA-CRISPR reaction provided by the present invention;

[0053] Figure 4The present invention provides different types of microsphere preparations in independent packaging forms. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0055] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In the description of the present invention, it should be noted that the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0057] In a specific embodiment, in a first aspect, the present invention provides a microsphere preparation for nucleic acid amplification, wherein the microsphere preparation comprises a reaction microsphere obtained by freeze-drying a mixed reagent required for an amplification reaction, wherein each gram of the reaction microsphere contains:

[0058] DNA polymerase 131.58~530.5μg, single-stranded binding protein 2.632~13.263mg, recombinase 0.9867~3.316mg, auxiliary protein 0.395~1.33mg, each primer 0.0075~0.02nmol, creatine kinase 657.89~663.13μg, ATP 0.1~0.2μmol, DTT 0.0026~0.015mmol, phosphokinase 0.1~0.5mmol, dNTP each 0.008~0.012μmol, Tris-Ac 0.5~2.5μmol, maltose 0~663.13mg, PEG 131.58~663.13mg.

[0059] In an optional embodiment, the microsphere preparation is used in an RNA amplification system, and each gram of the microsphere preparation further comprises 394.74 to 795.76 μg of reverse transcriptase.

[0060] In an optional embodiment, the microsphere preparation includes reaction microspheres obtained by freeze-drying the mixed reagents required for the amplification reaction, and each gram of reaction microspheres contains:

[0061] DNA polymerase 460.53~464.19μg, each primer 0.013nmol, single-stranded binding protein 10.53~10.61mg, recombinase 1.71~1.72mg, auxiliary protein 1.05~1.06mg, maltose 394.74~397.88mg, PEG 150~151.19mg, ATP 0.15μmol, dNTP 0.01μmol each, Tris-Ac 0.5μmol.

[0062] Preferably, the microsphere preparation comprises 657.89-663.13 μg of reverse transcriptase.

[0063] In an alternative embodiment, the recombinase is selected from T4 UvsX protein, T6 UvsX protein or Rb69 UvsX protein.

[0064] Preferably, the auxiliary protein is selected from T4 UvsY protein, T6 UvsY protein or Rb69 UvsY protein.

[0065] Preferably, the DNA polymerase is a strand displacement DNA polymerase selected from the large fragment of DNA polymerase I of Staphylococcus aureus, the large fragment of DNA polymerase I of Bacillus subtilis, the large fragment of DNA polymerase I of Escherichia coli or T4 bacteriophage Klewnowexo-polymerase.

[0066] Preferably, the reverse transcriptase comprises M-MLV reverse transcriptase.

[0067] Preferably, the single-stranded binding protein is selected from T4 GP32 protein, T6 GP32 protein or Rb69 GP32 protein.

[0068] In an optional embodiment, the microsphere preparation further comprises a second microsphere, wherein the second microsphere contains a complexing solvent PEG and / or a magnesium salt activator.

[0069] Preferably, each gram of the second microsphere contains 960-980 mg of PEG and / or 265.0-265.5 μmol of magnesium salt.

[0070] In a second aspect, the present invention provides a use of the microsphere preparation described in any one of the aforementioned embodiments in RPA or RAA.

[0071] In a third aspect, the present invention provides a method for preparing a microsphere preparation, wherein the microsphere preparation comprises the microsphere preparation described in any one of the aforementioned embodiments;

[0072] The preparation method comprises mixing the components of the microsphere preparation uniformly, dripping them into liquid nitrogen at time intervals of not less than 25 seconds, storing the microspheres in liquid nitrogen for not less than 1 hour, transferring them to a freeze dryer for freeze drying, and freeze drying them according to a freeze drying procedure to obtain the microsphere preparation;

[0073] The freeze-drying procedure is a gradient temperature rise freeze-drying method, which sequentially includes a pre-freezing step, a main drying step and a final drying step.

[0074] Preferably, the temperature of the pre-freezing step is below -54°C, and the processing time is 0.5 to 1 hour.

[0075] Preferably, the temperature of the main drying step is -27 to -15°C, the treatment time is 2 to 6 hours, and the vacuum degree is 0.01 to 30 Pa; further preferably, the main drying step includes at least two gradient temperature increase treatment processes.

[0076] Preferably, the temperature of the final drying step is 0-20° C., the treatment time is more than 2 hours, and the vacuum degree is 0.01-1 Pa; further preferably, the final drying step includes at least four gradient temperature increase treatment processes.

[0077] In a fourth aspect, the present invention provides a method for nucleic acid amplification using the microsphere preparation described in any of the preceding embodiments, the method comprising adding reaction microspheres to the amplification sample solution at a ratio of 0.263 to 6.58 mL of the sample solution to be amplified per gram of reaction microspheres, and then amplifying according to any one of the following (a) to (c):

[0078] (a) Add liquid resolvent and activator, mix well, and directly amplify at 37-44°C for 20 min;

[0079] (b) adding a second microsphere and amplifying at 37-44° C. for 20 min, wherein the second microsphere contains a complexing solvent PEG and a magnesium salt activator;

[0080] (c) After redissolution, a second microsphere is added and amplified at 37-44°C for 20 min, wherein the second microsphere contains a magnesium salt activator.

[0081] In a fifth aspect, the present invention provides a use of a microsphere preparation in a nucleic acid amplification combined with a second reaction method, wherein the microsphere preparation includes the microsphere preparation described in any one of the aforementioned embodiments;

[0082] The nucleic acid amplification adopts the nucleic acid amplification method described in the above embodiment;

[0083] The preparations used in the second reaction are all microsphere preparations;

[0084] The second reaction includes a fluorescence reaction or a CRISPR reaction.

[0085] Preferably, the fluorescence reaction comprises adding EXO enzyme and probe into the RPA or RAA system before freeze-drying, so that the RPA or RAA fluorescence reaction can be detected in real time.

[0086] Preferably, the EXO enzyme selected from the exonuclease is exonuclease III.

[0087] Preferably, the CRISPR reaction includes a Cas12 CRISPR detection system and a Cas13 CRISPR detection system.

[0088] In an optional embodiment, the second reaction method includes directly adding a microsphere preparation for the second reaction to the amplification product to complete the second reaction.

[0089] In an optional embodiment, the second reaction is a CRISPR reaction, and the method for preparing the microsphere preparation used in the CRISPR reaction is: preparing a CRISPR freeze-drying system, and then using the method described in the above embodiment to prepare the microsphere preparation used in the CRISPR reaction;

[0090] The CRISPR freeze-dried system contains: CRISPR freeze-dried protectant 10uL / test, Buffer 1×, Cas12 protein 40-100nmol / L, Cas13 protein 40-100nmol / L, mRNA enzyme inhibitor 5U, T7 RNA polymerase 14U, rNTP 0.5-0.6mM, CrRNA1 0.1μM, ssDNA 0.8-1.2nmol / L, ssRNA 0.8-1.2nmol / L.

[0091] Preferably, the Cas12 protein is selected from LbCas12a, FnCas12a, AsCas12a (cpf1), BbCas12a (cpf1), HkCas12a (cpf1).

[0092] Preferably, the Cas13 protein includes LwaCas13a.

[0093] Preferably, the T7 RNA polymerase is expressed in Escherichia coli.

[0094] In a sixth aspect, the present invention further provides the use of the microsphere preparation described in any of the aforementioned embodiments in an amplified self-chromogenic reaction, wherein each gram of the microsphere preparation further comprises 0.4613 to 12.13 mg of EXO enzyme.

[0095] Preferably, each gram of the microsphere preparation includes 0.6579 to 0.6667 mg of EXO enzyme.

[0096] 1. The specific embodiments of the present invention involve different specific reaction methods as follows:

[0097] 1. Lyophilization preparation method of microsphere preparation

[0098] In some specific embodiments below, the freeze-drying method used to prepare the microsphere preparation is: after the components of the microsphere preparation are evenly mixed, they are dripped into liquid nitrogen at a time interval of not less than 25 seconds. After the microspheres are stored in liquid nitrogen for not less than 1 hour, they are transferred to a freeze dryer for freeze drying, and freeze-dried according to the freeze-drying procedure to obtain a microsphere preparation. The freeze-drying procedure is a gradient temperature rise freeze-drying method, which includes a pre-freezing step, a main drying step, and a final drying step in sequence. The temperature of the pre-freezing step is below -54°C, and the processing time is 0.5 to 1h; the temperature of the main drying step is -27 to -15°C, the processing time is 2 to 6h, and the vacuum degree is 0.01 to 30Pa; the main drying step includes at least two gradient temperature rise treatment processes; the temperature of the final drying step is 0 to 20°C, the processing time is more than 2h, and the vacuum degree is 0.01 to 1Pa; the final drying step includes at least four gradient temperature rise treatment processes.

[0099] 2. RAA, RPA (basal and fluorescence) reaction parameters

[0100] The composition of the microspheres used in the RAA and RPA (basic and fluorescent) reactions is different as described above. However, the specific reaction method can adopt the same steps, including but not limited to mixing the reaction system, keeping the temperature at 37°C to 44°C for 20 minutes, and if it is a fluorescent reaction, the fluorescence collection rule is 30s, collecting fluorescence once, and a total of 40 cycles.

[0101] 3. CRISPR reaction

[0102] In some specific embodiments below, the CRISPR reaction includes, but is not limited to, taking 10 μL of the inactivated amplified product, mixing it with 25 μL of water, and adding it to the CRISPR microspheres. The reaction was carried out at 45°C to 65°C with ABI7500, and fluorescence was collected every 30 seconds for a total of 30 times.

[0103] 2. The specific packaging forms of the microsphere preparations for different purposes provided by the present invention are as follows:

[0104] 1. Microsphere preparations for RPA or RAA reactions only, such as Figure 1 As shown, the amplification reagents including the required primers are prepared by the freeze-drying method of the above-mentioned microsphere preparation to obtain independent microspheres, each of which is packaged separately. When performing RPA or RAA reaction, the sample to be tested, the liquid resolvent and the activator can be directly added.

[0105] 2. In some reactions, it is necessary to add reconstituted microspheres. In this case, Figure 2 In the packaging form shown, each packaging tube contains a reaction microsphere and a reconstituted microsphere, and the sample to be tested and the liquid activator can be directly added during the reaction. It is understandable that the liquid magnesium salt activator can also be mixed with the reconstituted solvent and frozen into a reconstituted / activated mixed microsphere, and the sample to be tested can be directly added during the reaction.

[0106] 3. When the RPA or RAA reaction is completed, a second reaction is also combined, such as a CRISPR reaction. The corresponding packaging format is as follows Figure 3 As shown, the two tubes on the left are packaging cans for completing RPA or RAA reactions, and the two tubes on the right contain reaction microspheres for CRISPR reactions. The products after amplification on the left are directly transferred into the CRISPR reaction microsphere packaging tube on the right to carry out the CRISPR reaction.

[0107] It should be noted that the packaging tubes used in the above three packaging forms can be adjusted and replaced according to actual needs, and are not limited to the specific structure and shape of the packaging tubes in the drawings.

[0108] 4. For bulk storage, transportation and sales, when there is no immediate need for immediate use, different types of microsphere preparations can also be packaged separately, such as Figure 4 shown.

[0109] Some embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0110] Example 1

[0111] This example provides RPA freeze-dried reaction microspheres for respiratory syncytial typing detection. The components contained in each gram of reaction microspheres are shown in the following table:

[0112]

[0113]

[0114]

[0115] The combination of RSV-F, RSV-R and RSV-P can specifically detect respiratory syncytial type A; RSV-F, RSV-2R and RSV-P1 can specifically detect respiratory syncytial type B. The 35th and 38th base Ts of the RSV-P nucleotide sequence were coupled to FAM and BHQ1 fluorescent groups, respectively, and the 36th base was replaced by THF. The 27th and 29th base Ts of the RSV-P1 nucleotide sequence were coupled to CY5 and BHQ2 fluorescent groups, respectively, and the 28th base was replaced by THF.

[0116] It should be noted that the microsphere preparation provided by the present invention does not have selectivity for primers and probes. Those skilled in the art can independently design corresponding primers and probes according to the actual amplification target. The microsphere preparation provided by the present invention has good compatibility with different primers and probes.

[0117] The white microsphere preparation was prepared according to the above freeze-dried microsphere preparation method, and the specific freeze-drying program parameters are as follows:

[0118]

[0119] The obtained reaction microspheres weigh about 7.54-7.60 mg, have a diameter of 3.8-4.5 mm, and are white spherical.

[0120] Oropharyngeal swab samples of respiratory syncytial type a / b quantified by digital PCR (sample source: provided by Shanghai Bojian Medical Laboratory) were diluted to 1×10 4 copies / mL, 5×10 3 copies / mL, 5×10 2 copies / mL, 2.5×10 2 copies / mL and 1.5×10 2 The samples were prepared as samples for future testing. Each sample was tested 10 times using the reaction microspheres prepared above, and the sample with a detection rate of 90% was the minimum detection limit of the reagent.

[0121] Testing process:

[0122] (1) Pre-pack the reaction microspheres into reaction tubes, with one RPA reaction microsphere per well in the RPA reaction tube;

[0123] (2) Add 15 μL of sample to the RPA reaction microspheres, add 30 μL of resolvent and 5 μL of activator; the resurrection agent is a PEG (35K) aqueous solution with a mass volume ratio of 7.35%, and the activator is a magnesium acetate aqueous solution with a magnesium acetate concentration of 140 mM;

[0124] (3) After the reaction system was fully mixed, the ABI7500 reaction was carried out at 44°C, and the fluorescence was collected every 30 seconds for a total of 30 times, and the corresponding detection rate was calculated.

[0125] Results Statistics:

[0126]

[0127]

[0128] Conclusion: The dual system microspheres can detect different types of respiratory syncytial samples, with a detection sensitivity as low as 2.5×10 2 copies / mL.

[0129] Example 2

[0130] The only difference between this example and Example 1 is that this example also uses a freeze-drying preparation method to prepare reconstituted microspheres, and the component of the reconstituted microspheres is PEG (35K) (976 mg / g).

[0131] Respiratory syncytial type a / b oropharyngeal swab samples quantified by digital PCR (sample source: provided by Shanghai Bojian Medical Laboratory) were diluted to 5×10 3 copies / mL, 5×10 2 copies / mL, 2.5×10 2 copies / mL, 1.5×10 2 copies / mL and 1.0×10 2 The remaining tests were the same as in Example 1, and the results were as follows:

[0132]

[0133] Conclusion: The dual system microspheres can detect different types of respiratory syncytial samples, with a detection sensitivity as low as 1.5×10 2 copies / mL.

[0134] Example 3

[0135] This embodiment provides RPA-CRISPR freeze-dried microspheres for influenza A virus and influenza B virus detection, including RPA reaction microspheres, reconstituted microspheres and CRISPR microspheres. The reaction microspheres weigh about 7.54-7.60 mg, have a diameter of 3.8-4.5 mm, and are white spherical; the reconstituted microspheres weigh about 2.52-2.58 mg, have a diameter of 3.2-3.5 mm, and are white spherical; the CRISPR microspheres weigh about 2.2-2.32 mg, have a diameter of 3.2-3.8 mm, and are light pink spherical. The components contained in each gram of RPA reaction microspheres are shown in the following table:

[0136] Component name content Single-chain binding protein 10.53~10.61mg Recombinase 1.71~1.72mg Accessory proteins 1.05~1.06mg DNA polymerase 460.53~464.19μg Reverse transcriptase 657.89~663.13μg maltose 394.74~397.88mg PEG(20K-36K) 150~151.19mg Creatine kinase 657.89~663.13μg ATP 0.15 μmol DTT 0.013mmol Phosphokinase 0.331mmol dNTP 0.01 μmol each Tris-Ac 0.5 μmol Primers 0.015nmol each

[0137] The primers are:

[0138]

[0139]

[0140] The combination of IFA-F and IFA-R can specifically amplify the target sequence of influenza A virus, and the combination of IFB-F and IFB-R can specifically amplify the target sequence of influenza B virus.

[0141] The components of the reconstituted microspheres are PEG (35K): 976 mg / g.

[0142] The CRISPR microsphere freeze-drying system is as follows:

[0143] Component name Addition amount CRISPR Lyoprotectant 10uL / test Buffer 3.1 1× LbCas12a(cpf1) 100nmol / L LwaCas13a 100nmol / L Murine RNase inhibitor(40U / uL) 0.125uL T7 RNA polymerase 0.05μL rNTP 0.8μL CrRNA1 20ng / μL CrRNA2 20ng / μL ssDNA 2μmol / L ssRNA 2μmol / L

[0144] The nucleotide sequences of CrRNA1, CrRNA2, ssDNA and ssRNA are as follows:

[0145]

[0146] Among them, CrRNA1 can specifically bind to the amplification products of IFA-F and IFA-R under the CRISPR system, activate the trans-cutting activity of Cas13 protein, cut ssRNA, and emit fluorescence; CrRNA2 can specifically bind to the amplification products of IFB-F and IFB-R under the CRISPR system, activate the trans-cutting activity of Cas12 protein, cut ssDNA, and emit fluorescence.

[0147] Oropharyngeal swab samples of influenza A virus and influenza B virus quantified by digital PCR (sample source: provided by Shanghai Bojian Medical Laboratory) were diluted to 1×10 4 copies / mL, 5×10 3 copies / mL, 5×10 2 copies / mL, 2.5×10 2 copies / mL, 2.0×10 2 copies / mL and 1.0×10 2 copies / mL are used as samples to be tested.

[0148] The testing process is as follows:

[0149] (1) Pre-pack the reaction microspheres into reaction tubes. In each well of the RPA reaction tube, there is one RPA reaction microsphere and one resolvent microsphere; in each well of the CRISPR reaction tube, there is one CRISPR microsphere.

[0150] (2) Add 45 μL of sample to the RPA reaction microspheres and add 5 μL of activator;

[0151] (3) After the reaction system is fully mixed, incubate in a metal bath at 42°C for 20 min, and inactivate at 95°C for 2 min;

[0152] (4) Take 10 μL of the inactivated RPA product, mix it with 25 μL of water, and add it to the CRISPR microspheres.

[0153] (5) The ABI7500 reaction was performed at 45°C, and fluorescence was collected every 30 seconds for a total of 30 times, and the corresponding detection rate was calculated.

[0154] The statistical results are as follows:

[0155]

[0156]

[0157] Conclusion: The minimum detection limit of dual alpha and beta flow RPA-CRISPR freeze-dried microspheres was 2.0×10 2 copies / mL.

[0158] Example 4

[0159] The difference between this embodiment and embodiment 3 is that the re-dissolving microspheres is omitted, and 30 μL of re-dissolving agent is added in step (2) of the detection process. The dilution concentration of the sample to be tested is 1×10 4 copies / mL, 5×10 3 copies / mL, 5×10 2 copies / mL, 4×10 2 copies / mL, 2.0×10 2 copies / mL and 1.0×10 2 copies / mL.

[0160] The test results are as follows:

[0161] Template concentration Influenza A positive detection rate Positive detection rate of influenza B Negative control negative detection rate <![CDATA[1×10 4 copies / mL]]> 10 / 10 10 / 10 3 / 3 <![CDATA[5×10 3 copies / mL]]> 10 / 10 10 / 10 3 / 3 <![CDATA[5×10 2 copies / mL]]> 10 / 10 10 / 10 3 / 3 <![CDATA[4.0×10 2 copies / mL]]> 10 / 10 10 / 10 3 / 3 <![CDATA[2.0×10 2 copies / mL]]> 8 / 10 9 / 10 3 / 3 <![CDATA[1.0×10 2 copies / mL]]> 6 / 10 8 / 10 3 / 3

[0162] Conclusion: The minimum detection limit of dual influenza A virus and influenza B virus RPA-CRISPR freeze-dried microspheres was 4.0×10 2 copies / mL.

[0163] Example 5

[0164] The RPA-CRISPR freeze-dried microspheres for influenza A virus and influenza B virus detection provided in Example 3 were stored at room temperature for 15 days, 30 days, 90 days, 180 days, 270 days and 360 days, and then stability tests were performed.

[0165] Sample preparation: The influenza A and B virus samples quantified by digital PCR were diluted to a minimum detection limit of 200 copies / mL and used as samples to be tested.

[0166] The RPA-CRISPR freeze-dried microspheres with different storage times were used to detect the above-mentioned samples according to the detection method of Example 3. The results are as follows:

[0167]

[0168] Conclusion: The detection performance of freeze-dried microspheres was not affected after being stored at room temperature for 360 days.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Microsphere preparations for nucleic acid amplification, It is characterized in that The microsphere preparation comprises reaction microspheres obtained by freeze-drying the mixed reagents required for the amplification reaction, and each gram of the reaction microspheres contains: T4 bacteriophage Klewnowexo-polymerase 131.58~530.5 μg, T4 GP32 protein 2.632~13.263 mg, T4UvsX protein 0.9867~3.316 mg, T4 UvsY protein 0.395~1.33 mg, M-MLV reverse transcriptase 394.74~795.76μg, each primer 0.0075~0.02 nmol, creatine kinase 657.89~663.13 μg, ATP 0.1~0.2 μmol, DTT 0.0026~0.015 mmol, phosphokinase 0.1~0.5 mmol, dNTP 0.008~0.012 μmol each, Tris-Ac 0.5~2.5μmol, maltose 394.74~663.13 mg, PEG 131.58~663.13 mg; After the components of the microsphere preparation are evenly mixed, freeze-drying is performed, and freeze-drying is performed according to the freeze-drying procedure to obtain the microsphere preparation.

2. The microsphere preparation according to claim 1, It is characterized in that The microsphere preparation includes 657.89-663.13 μg of M-MLV reverse transcriptase.

3. The microsphere preparation according to claim 1, It is characterized in that The microsphere preparation comprises reaction microspheres obtained by freeze-drying the mixed reagents required for the amplification reaction, and each gram of the reaction microspheres contains: T4 bacteriophage Klewnowexo-polymerase 460.53~464.19 μg, each primer 0.013 nmol, T4 GP32 protein 10.53~10.61 mg, T4 UvsX protein 1.71~1.72 mg, T4 UvsY protein 1.05~1.06 mg, maltose 394.74~397.88 mg, PEG 150~151.19 mg, ATP 0.15 μmol, dNTP 0.01 μmol each, Tris-Ac 0.5 μmol, M-MLV reverse transcriptase 657.89~663.13 μg, creatine kinase 657.89~663.13 μg, DTT 0.013 mmol and phosphokinase 0.331 mmol.

4. The microsphere preparation according to any one of claims 1 to 3, It is characterized in that The microsphere preparation further includes a second microsphere containing a complexing solvent PEG.

5. The microsphere preparation according to claim 4, It is characterized in that Each gram of the second microsphere contains 960-980 mg of PEG.

6. Use of the microsphere preparation according to any one of claims 1 to 5 in RPA or RAA.

7. Preparation method of microsphere preparation, It is characterized in that The microsphere preparation includes the microsphere preparation according to any one of claims 1 to 5; The preparation method comprises mixing the components of the microsphere preparation uniformly, dripping them into liquid nitrogen at time intervals of not less than 25 seconds, storing the microspheres in liquid nitrogen for not less than 1 hour, transferring them to a freeze dryer for freeze drying, and freeze drying them according to a freeze drying procedure to obtain the microsphere preparation; The freeze-drying procedure is a gradient temperature rise freeze-drying method, which sequentially includes a pre-freezing step, a main drying step and a final drying step.

8. The preparation method according to claim 7, It is characterized in that The temperature of the pre-freezing step is below -54°C, and the processing time is 0.5 to 1 h.

9. The preparation method according to claim 7, It is characterized in that The temperature of the main drying step is -27~-15°C, the processing time is 2~6h, and the vacuum degree is 0.01~30 Pa.

10. The preparation method according to claim 9, It is characterized in that The main drying step includes at least two gradient temperature rising processes.

11. The preparation method according to claim 10, It is characterized in that The temperature of the final drying step is 0-20° C., the processing time is more than 2 hours, and the vacuum degree is 0.01-1 Pa.

12. The preparation method according to claim 11, It is characterized in that The final drying step includes at least four gradient temperature increase treatment processes.

13. A method for nucleic acid amplification using the microsphere preparation according to any one of claims 1 to 5, It is characterized in that The method comprises adding reaction microspheres to the amplified sample solution in a ratio of 0.263 to 6.58 mL of the sample solution to be amplified per gram of reaction microspheres, and then amplifying according to any one of the following (a) to (b): (a) Add liquid resolvent and activator, mix well, and directly amplify at 37-44°C for 20 min. (b) Adding a second microsphere and an activator, and amplifying at 37-44°C for 20 min, wherein the second microsphere contains a complexing solvent PEG.

14. Application of microsphere preparation in nucleic acid amplification combined with second reaction method, It is characterized in that The microsphere preparation includes the microsphere preparation according to any one of claims 1 to 5; The nucleic acid amplification adopts the method of claim 13; The preparations used in the second reaction are all microsphere preparations; The second reaction includes a fluorescence reaction or a CRISPR reaction.

15. The use according to claim 14, It is characterized in that The fluorescence reaction includes adding EXO enzyme and probe into the RPA or RAA system before freeze-drying, so as to detect the RPA or RAA fluorescence reaction in real time.

16. The use according to claim 15, It is characterized in that The EXO enzyme selected from the exonuclease is exonuclease III.

17. The use according to claim 14, It is characterized in that The CRISPR reaction includes a Cas12 CRISPR detection system and a Cas13 CRISPR detection system.

18. The use according to claim 14, It is characterized in that The second reaction method includes directly adding a microsphere preparation for the second reaction to the amplification product to complete the second reaction.

19. Use of the microsphere preparation according to any one of claims 1 to 5 in amplifying a fluorescent reaction, It is characterized in that Each gram of microsphere preparation also includes 0.4613~12.13 mg of EXO enzyme.

20. The use according to claim 19, It is characterized in that Each gram of microsphere preparation contains 0.6579~0.6667mg of EXO enzyme.

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