Detection system based on recombinase polymerase amplification and application thereof

Abstract

The invention discloses a detection system based on recombinase polymerase amplification and application thereof, and belongs to the technical field of biological detection. The invention provides a double-enzyme-mediated amplification combined detection system (RPMAS) which comprises a first detection reagent and a second detection reagent which are optimized, wherein the first detection reagent is a buffer system containing a primer and a probe, and the probe contains a fluorophore, a quenching group and a restriction enzyme cutting site; the second detection reagent contains a recombinase solution, and the recombinase solution contains recombinase, a recombinase load factor, single-stranded binding protein, strand displacement DNA polymerase and exonuclease; the optimization treatment comprises the following steps: mixing the detection reagent with a freeze-drying protective agent, freezing and drying. An appropriate reaction system is developed on the basis of the reaction principle of recombinase polymerase amplification, multiple fluorescence channels are additionally arranged on the reaction system, optimization is carried out on the reaction system, multiple composite joint inspection of the system is achieved, meanwhile, the freeze drying technology aiming at the system is perfected, and an application basis is provided for application of the technology to clinical inspection.

Description

Technical Field

[0001] The invention relates to a detection system based on recombinase polymerase amplification and application thereof, belonging to the technical field of biological detection. Background Art

[0002] Currently, clinical laboratory methods for diagnosing pathogen infections primarily include colloidal gold immunoassays targeting pathogen antigens, chemiluminescence / enzyme-linked immunosorbent assays targeting pathogen-specific antibodies, and real-time fluorescence quantitative PCR (qPCR) targeting viral nucleic acids. Currently, a positive nucleic acid test is the gold standard for diagnosing pathogen infection. However, real-time fluorescence quantitative PCR requires a very high level of laboratory environment, equipment, and operator expertise, limiting its use outside of standard testing laboratories. To further expand the application of viral nucleic acid testing and improve the accuracy of viral infection diagnosis, there is an urgent need for a simple, rapid, and inexpensive nucleic acid detection method that can be applied to multiple pathogens for screening.

[0003] Since the 1990s, isothermal nucleic acid amplification technology has developed rapidly. Because amplification only requires a single incubation temperature, the requirements for equipment are reduced, and there is no need for repeated heating and cooling steps, which reduces the reaction time. Most importantly, multiple molecular reactions can be carried out simultaneously, rather than sequentially in thermal cycles, which greatly increases the reaction efficiency. Currently reported isothermal nucleic acid amplification technologies include: transcription-mediated amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, recombinase polymerase amplification, and so on.

[0004] Recombinase-Polymerase Amplification (RPA), among others. Recombinase-Polymerase Amplification (RPA), although introduced relatively late, has experienced rapid development in recent years due to its simple equipment requirements and rapid reaction times. During nucleic acid amplification, the recombinase first binds to the primer under the action of the recombinase loading factor to form a nucleic acid-protein complex. This complex then searches for a sequence complementary to the primer along the DNA double strand, hybridizing and displacing another single DNA strand. The displaced DNA strand binds to the single-strand binding protein, preventing it from hybridizing with the complementary strand. Finally, the recombinase dissociates from the primer under the action of ATP, and the polymerase binds to the primer and extends along the template.

[0005] As a relatively new technology, recombinase polymerase amplification (RPA) has not yet been widely used. However, it offers advantages such as high sensitivity, strong specificity, quick and convenient operation, and rapid response. Furthermore, the technology has minimal hardware requirements, requiring no precision instruments. Furthermore, the addition of exonuclease III to form an Exo fluorescent probe system enables combined detection of multiple fluorescent channels, making it suitable for use in in vitro diagnostics, veterinary medicine, food safety, biosafety, agriculture, and other fields. These technical advantages make RPA more suitable for rapid on-site testing such as home diagnosis or disease prevention and control, compared to other isothermal amplification technologies. It has been called a nucleic acid detection technology that can replace PCR.

[0006] Although recombinase polymerase amplification technology (RPA) has the above advantages and application prospects, the existing amplification systems based on the technology itself still have some shortcomings and deficiencies. For example, they are affected by the mutual interference between the fluorescent channels and are currently mainly used for single pathogen detection. Secondly, the probes and primers required for the RPA reaction are longer than other nucleic acid amplification technologies (30-35bp are required), which is not suitable for short-sequence nucleic acid detection. The requirements for the target sequence are high, which makes primer design and product development difficult. In addition, the existing RPA reaction systems on the market are currently all for single pathogen nucleic acid detection products, which indirectly increases the cost of detection. Moreover, the current conventional system usually uses in situ freeze-drying to process the premixed system. After freeze-drying in this way, the system still needs to be stored and transported within the specified temperature range. This greatly increases the cost of reagents. Summary of the Invention

[0007] To solve the above problems, the present invention provides a detection system based on recombinase polymerase amplification, which includes a specially designed recombinase system and freeze-dried balls. It can not only be used for multi-channel joint detection, but also can efficiently detect short-sequence nucleic acids, and greatly improve the detection effect. Different targets can be distinguished when the Ct value is small.

[0008] The first object of the present invention is to provide a detection system based on recombinase polymerase amplification, wherein the detection system comprises an optimized first detection reagent and an optimized second detection reagent:

[0009] The first detection reagent is a buffer system containing primers and probes; the primers and probes are used to detect the target to be detected, and the probe contains a fluorescent group, a quenching group, and a nuclease cleavage site disposed between the fluorescent group and the quenching group; (the term "between" herein means located within the two or overlapping with one of the two sides)

[0010] The second detection reagent contains a recombinase solution: the recombinase solution contains a recombinase, a recombinase loading factor, a single-strand binding protein, a strand-displacing DNA polymerase, and an exonuclease;

[0011] The optimization process includes: mixing the detection reagent with a freeze-dried protective agent, freezing and drying the mixture; based on the mass percentage of the freeze-dried protective agent as 100%, the content of each component is: 5%-40% trehalose, 5%-15% polyethylene glycol 8000, 1%-10% dextran-40, 2%-15% mannitol, and 0.45%-0.9% sodium chloride.

[0012] The reaction principle of the recombinase polymerase mediated amplification system (RPMAS) provided by the present invention is as follows: (1) the recombinase in the reaction system binds to a specific primer of 18-35 bp to form a recombinase-primer complex, which searches for the target site in the double-stranded DNA template; (2) after the recombinase-primer complex recognizes the template-specific sequence, it locates and triggers strand exchange, and the single-stranded binding protein then binds to the displaced DNA strand to form a D-Loop structure; (3) the recombinase-primer complex hydrolyzes the ATP in the system, causing the conformational change, and the 3' end of the primer is exposed after the recombinase dissociates. The primer is exposed and recognized by DNA polymerase, which initiates DNA synthesis at the 3' end of the primer according to the template sequence; (4) DNA polymerase has a strand displacement function, and continues to unwind the double-helical DNA structure of the template while the primer is extended, and the DNA synthesis process continues; (5) The amplification of the two primers is completed, forming a complete amplicon; (6) In the reaction system, ATP is hydrolyzed to provide energy for the recombinase and then converted into ADP. Phosphocreatine can transfer its phosphate group to the ADP molecule under the catalysis of creatine kinase to form ATP, thereby restoring the ATP level in the reaction system. The above process is repeated continuously, and ultimately achieves efficient nucleic acid amplification.

[0013] The present invention provides a new dual-enzyme-mediated amplification joint detection system (RPMAS) and a joint detection amplification kit based on this amplification system by optimizing the system, which can significantly reduce the Ct value during detection while ensuring the detection effect. In nucleic acid detection, the Ct value (Cycle Threshold) refers to the number of cycles required for the fluorescent signal to reach the set threshold. A decrease in the Ct value means that fewer cycles are required to detect the target nucleic acid, which reflects a higher detection sensitivity and can detect low-concentration target nucleic acids earlier; a lower Ct value reflects a more efficient amplification reaction, indicating that the reaction system has good performance and high amplification efficiency. Therefore, the detection system of the present invention has great potential in nucleic acid detection and solves the current limitations of the harsh transportation conditions of freeze-dried preparations. When applied to viral nucleic acid detection, a lower Ct value can also reduce the possibility of missed detection due to low viral load.

[0014] Furthermore, the first detection reagent contains at least two sets of primers and probes for detecting different targets, with different probes provided with different fluorescent groups and / or quenching groups. The detection system of the present invention can also overcome the limitation of the prior art in being unable to detect multiple targets, and can effectively distinguish between multiple fluorescent channels.

[0015] Furthermore, the fluorescent group, quenching group and enzyme cleavage site are modified on the base of the probe.

[0016] Further, the freezing and drying comprises:

[0017] The first stage: cool down to -55~-45℃ within 5-20min and keep at this temperature for 0.5-5h;

[0018] The second stage: raise the temperature to -25~-27℃ within 5-20min and keep it at this temperature for 15-25h, all under vacuum conditions;

[0019] The third stage is to raise the temperature to 20-30°C within 1-2 hours and maintain this temperature for 1-10 hours, all under vacuum. To ensure room-temperature storage and transportation of the reagents, the present invention utilizes an optimized lyoprotectant to prepare freeze-dried microspheres. The preparation of freeze-dried microspheres in this invention must meet material balance or yield requirements. After the freeze-dried microspheres are packaged, a material balance check is performed using a yield calculation method. Yield (%) = actual number of packages / theoretical number of packages × 100%. The package yield should be greater than or equal to 95%.

[0020] In an embodiment of the present invention, the mixture is processed by preparing freeze-dried pellets. Specifically, the freeze-dried pellets are formed in liquid nitrogen and then freeze-dried. After freeze-drying, the pellets are divided into eight-tube tubes in a humidity-controlled workshop, covered, and placed in an aluminum foil bag with a desiccant, which is then vacuum-sealed. Specifically:

[0021] Furthermore, the vacuum condition is 0.1-10 mbar.

[0022] Furthermore, at least one of the following is included:

[0023] (1) The recombinase includes T4 UvsX recombinase;

[0024] (2) the recombinase loading factor includes T4 UvsY protein;

[0025] (3) the single-stranded binding protein includes T4 gp32 protein;

[0026] (4) The strand-displacing DNA polymerase includes Bsu DNA polymerase.

[0027] Furthermore, the exonuclease comprises exonuclease III (Exo III); the cleavage site comprises a base modified with tetrahydrofuran (THF). Exo III specifically cleaves fluorescent probes containing the THF site, separating the fluorescent group from the quenching group, thereby generating a detectable fluorescent signal. Of course, multiple nucleic acid detection can also be achieved by designing multiple probes with THF sites.

[0028] Furthermore, the target to be detected is DNA or RNA, including but not limited to one or more viruses, such as coronavirus, respiratory syncytial virus (RSVA / RSVB, etc.).

[0029] Furthermore, the length of the primer is 18-35 bp. Existing nucleic acid detection technologies cannot effectively detect short sequences, but the present invention allows primers to be less than 35 bp in length and can still achieve effective detection at a smaller Ct value.

[0030] Furthermore, the length of the probe may be 46-52 nt.

[0031] Furthermore, when the target to be detected includes one or more of nCoV, RSVA, and RPP30, the primers include at least a pair of SEQ ID NOs. 8-13, and the probe includes at least one of SEQ ID NOs. 14-16.

[0032] Preferably, the base as the enzyme cleavage site on the probe is located between the base modified with the fluorescent group and the base modified with the quencher group. Most preferably, on the probe for detecting nCoV, the 30th base G is used as the enzyme cleavage site, the 29th base A and the 31st base C are modified with a fluorescent group and a quencher group, respectively. On the probe for detecting RSVA, the 31st base A is used as the enzyme cleavage site and the quencher group is modified, the 30th base A is modified with a fluorescent group, and the probe for detecting RPP30 has the 31st base C as the enzyme cleavage site, the 30th base A and the 32nd base A are modified with a fluorescent group and a quencher group, respectively. The present invention has screened and verified the primer and probe sequences and modification sites, and found that when the above optimal system is used for multi-target detection, not only can multi-channel detection be introduced, but also interference between different channels can be overcome compared to other sequences and modification sites.

[0033] Furthermore, the first detection reagent further contains: (1) PEG35K, (2) creatine kinase (for energy supply), and (3) creatine phosphate (for energy supply).

[0034] Furthermore, the second detection reagent also contains: (1) Tris-buffer, (2) potassium acetate, (3) dithiothreitol, (4) dNTPs, (5) ATP, (6) magnesium acetate, and optionally, contains or does not contain (7) reverse transcriptase and RNase inhibitor (only required for RNA amplification).

[0035] Furthermore, in the first detection reagent:

[0036] (1) Upstream primer, concentration 150nM-600nM;

[0037] (2) Downstream primer, concentration 150nM-600nM;

[0038] (3) Probe, concentration is 100nM-150nM; preferably 50nM-150nM;

[0039] (4) PEG35K, concentration 2.5%-10% (w / v);

[0040] (5) Creatine kinase, concentration 0.1-0.8 mg / mL;

[0041] (6) Creatine phosphate, concentration is 25-75mM.

[0042] Furthermore, in the second detection reagent:

[0043] (1) Recombinant enzyme, concentration 60-600 ng / μL;

[0044] (2) recombinase loading factor, concentration 20-800 ng / μL;

[0045] (3) single-chain binding protein, concentration 100-1000 ng / μL;

[0046] (4) strand displacement DNA polymerase, concentration 3-100 ng / μL;

[0047] (5) Exonuclease, concentration 1U / μL-5U / μL;

[0048] (6) Tris buffer, pH 7.4-8.4, concentration 20-100 mM; preferably 30 mM-80 mM;

[0049] (7) Potassium acetate, at a concentration of 0-150 mM, preferably 50 mM-120 mM;

[0050] (8) dithiothreitol, at a concentration of 1-12 mM, preferably 1-10 mM;

[0051] (9) dNTPs, concentration 150-600 nM each; preferably 150-500 nM each;

[0052] (10) ATP, concentration 1-5 mM;

[0053] (11) Magnesium acetate, concentration is 10-30mM.

[0054] The present invention concentrates multiple primers and probes in the same amplification system, optimizes and determines the feeding ratio through the mutual interference test between fluorescence channels. Tris-tricine is an efficient buffer system for maintaining the pH value of the reaction system. The pH value of the reagent using this buffer system does not shift during the freeze-drying process, making the freeze-drying operation easy to carry out. The dry powder reagent after freeze-drying has high stability and can be stored for a long time at room temperature, which can greatly reduce the storage and transportation costs of the reagent. In the reaction system, ATP, creatine kinase and creatine phosphate constitute the energy regeneration system of the entire reaction system. Preferably, the final concentration of ATP in the system is 3mM, the final concentration of creatine kinase is 0.1mg / mL, and the final concentration of creatine phosphate is 50mM.

[0055] Furthermore, the Tris-buffer is Tris-HCl.

[0056] Furthermore, the volume ratio of the first detection reagent to the lyophilized protective agent is 1:(1-3); the volume ratio of the second detection reagent to the lyophilized protective agent is 1:(1-3).

[0057] The second object of the present invention is to provide a nucleic acid joint detection kit containing the detection system.

[0058] A third object of the present invention is to provide a method for detecting nucleic acid, comprising the following steps:

[0059] The nucleic acid sample to be tested is mixed with the optimized first detection reagent (lyophilized ball A) and the optimized second detection reagent (lyophilized ball B). After the reaction is completed, the fluorescence signal is detected and the target nucleic acid is detected based on the change in the fluorescence signal.

[0060] Furthermore, the reaction conditions are incubated at 25-42°C for 10-60 minutes to complete the amplification of the nucleic acid. Preferably, the reaction is carried out at 42°C for 20 minutes.

[0061] Beneficial effects of the present invention:

[0062] (1) The present invention introduces multiple fluorescence channels into the enzyme premix system to meet the detection of multiple pathogens, and the freeze-drying process improves the stability of the system. A dual enzyme-mediated amplification joint detection system (Recombinase Polymerase mediated Multiplex Amplification System, RPMAS) is developed. The system requires short primers (18-35 bp), has low requirements on the length of the target sequence, and has wide applicability. It has good detection specificity and high sensitivity for nucleic acid target sequences, and can achieve high-sensitivity and high-precision rapid molecular detection under constant temperature conditions of 25-42°C. The detection cost is low, the operation is convenient and fast, and it has broad application prospects.

[0063] (2) The present invention utilizes multiple fluorescence channels to simultaneously detect multiple pathogens, and can quickly obtain test results.

[0064] (3) The present invention provides an optimized lyophilization protective agent that replaces the original protective agent and upgrades the lyophilization process. This achieves system stability while reducing storage and transportation costs.

[0065] (4) The present invention constructs a positive plasmid based on the target sequence and uses it as a positive control in this system, which can effectively ensure the stability of the positive control and avoid degradation, while reducing the production cost of the control.

[0066] The RPMAS reaction system can be used for both DNA and RNA template amplification.

[0067] (5) The RPMAS reaction system of the present invention uses a highly efficient buffer salt system, which maintains the pH value from shifting during the freeze-drying process of the reagent. The freeze-dried reagent has high stability and can be stored for a long time at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a test diagram of the RPMAS technology applied to dual fluorescence channels (FAM&Cy5) in Example 2.

[0069] Figure 2 72 freeze-dried balls were randomly selected from Example 3 to test their particle size distribution.

[0070] Figure 3 These are SEM images of any cross-section and surface of 72 freeze-dried spheres in Example 3.

[0071] Figure 4 100 freeze-dried balls were randomly selected for moisture content determination in Example 3.

[0072] Figure 5In Example 3, freeze-dried pellets and Twist finished reagents and raw materials were used to prepare a liquid system for parallel control and amplification testing (with nCoV-ORF1ab supporting primers and probes).

[0073] Figure 6 The macroscopic and microscopic characterizations of any surface and cross section of the freeze-dried spheres used in the comparative experiment in Example 3 were compared.

[0074] Figure 7 This is a multiple reaction test diagram in Example 4. DETAILED DESCRIPTION

[0075] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0076] The sequence involved in the present invention is as follows:

[0077] Recombinase T4 UvsX (SEQ ID NO. 1):

[0078] MSDLKSRLIKASTSKLTAELTASKFFNEKDVVRTKIPMMNIALSGEITGGMQSGLLILAGPSKSFKSNFGLTMVSSYMRQYPDAVCLFYDSEFGITPAYLRSMGVDPERVIHTPVQSLEQLRIDMVNQLDAIERGEKVVVFIDSLGNLASKKETEDALNEKVVSDMTRAKTMKSLFRIVTPYFSTKNIPCIAINH type

[0079] T4 UvsY protein (SEQ ID NO. 2):

[0080] MRLEDLQEELKKDVFIDSTKLQYEAANNVMLYSKWLNKHSSIKKEMLRIEAQKKVALKARLDYYSGRGDGDEFSMDRYEKSEMKTVLSADKDVLKVDTSLQYWGILLDFCSGALDAIKSRGFAIKHIQDMRAFEAGK

[0081] T4 gp32 protein (SEQ ID NO.3):

[0082] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL

[0083] Bsu DNA polymerase (SEQ ID NO.4):

[0084] MSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMKWVMEIQADLYFHNLKFDGAFIVNWLEQHGFKWSNEGLPNTYHTIISKMGQWYMIDICFGYRGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHKITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIEEHYEMYNVEYIDGFKFREKTGLFEDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYVKEVDGKLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFKVGFSSMGKPKPVQVNGGVVLVDSVFTIK

[0085] Creatine kinase (SEQ ID NO.5):

[0086] MPFGNTHNKFKLNYKPEEEYPDLSKHNNHMAKVLTLELYKKLRDKETPSGFTVDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKELFDPIISDRIHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRSIKGYTLPPHCSRGERRAVEKLSVEALNSLTGEFKGKYYPLKSMTEKEQQQLIDDHFLFDKPVSPLLLASGMARDWPDARGIWHNDNKSFLVWVNEEDHLRVISMEKGGNMKEVFRRFCVGLQKIEIIFKKAGHPFMWNQHLGYVLTCPSNLGTGLRGGVHVKLAHLSKHPKFEEILTRLRLQKRGTGGVDTAAVGSVFDVSNADRLGSSEVEQVQLVVDGVKLMVEMEKKLEKGQSIDDMIPAQK

[0087] nCoV sequence(SEQ ID NO.6):

[0088] Sequence (SEQ ID NO.7):

[0089] CTGGGTTAATAGGTATGTTATATGCTATGTCTAGATTAGGAAGAGAAGACACCATAAAAATACTCAAAGATGCGGGATATCATGTAAAAGCAAATGGAGTGGATGTAACAACACATCGTCAAGACATTAATGGGAAAGAAATGAAATTTGAAGTGTTAACATTAGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAAATCCTACAAAAAAATGCTAAAAGAAA

[0090] The materials and methods involved in the present invention are as follows:

[0091] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0092] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0093] Unless otherwise indicated, the immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA etc. that the present invention adopts are routine skills in this area.Referring to Sambrook (Sambrook), Fritsch (Fritsch) and Maniatis (Maniatis), " molecular cloning: laboratory manual " (MOLECMLAR CLONING:ALABORATORY MANUAL), the 2nd edition (1989); " contemporary molecular biology experiment manual " (CURRENT PROTOCOLS IN MOLECMLAR BIOLOGY) (people such as FM Ausubel (FM Ausubel) edit, (1987)); " enzymology method " (METHODS IN ENZYMOLOGY) series (Academic Publishing Company): " PCR2:

[0094] PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); Antibodies: A Laboratory Manual (Harlow and Lane, eds. (1988))

[0095] (ANTIBODIES, A LABORATORY MANUAL), and Animal Cell Culture

[0096] (ANIMAL CELL CMLTURE) (RI Freshney, ed. (1987)).

[0097] Example 1 Recombinase polymerase-mediated amplification detection system (RPMAS)

[0098] A combined isothermal amplification system was constructed based on the recombinase polymerase (T4 UvsX). The enzyme combination used was: recombinase T4 UvsX, T4 UvsY protein, T4 gp32 protein, Bsu DNA polymerase, and creatine kinase. The sample to be tested was DNA or RNA (RNA required the addition of an appropriate amount of reverse transcriptase and RNase inhibitor).

[0099] The components required for this joint detection system are Tris-tricine (pH 7.9), potassium acetate, dithiothreitol (DTT), dNTPs, ATP, upstream primer combination, downstream primer combination, fluorescent probe combination, magnesium acetate

[0100] Specifically, the formulation of the reaction system is as shown in Table 1 below:

[0101] Table 1

[0102] Serial number Components concentration 1 PEG35000 2.5％-10％ 2 Creatine kinase 0.1-0.8 mg / mL 3 Creatine phosphate 25-75mM 4 DNA polymerase (Bsu) 3-100 ng / μL 5 T4 gp32 protein 100-1000 ng / μL 6 T4 UvsX recombinase 60-600 ng / μL 7 T4 UvsY protein 20-800 ng / μL 8 Exo III exonuclease 1U / μL-5U / μL 9 Tris-tricine (pH 7.4-8.4) 20-100mM 10 Potassium acetate 0-150mM 11 Dithiothreitol (DTT) 1-12mM 12 dNTPs 150-600nM each 13 ATP 1-5mM 14 Upstream primer combination 150nM-600nM 15 Downstream primer combination 150nM-600nM 16 Fluorescent probe combination 100nM-150nM 17 magnesium acetate 10-30mM

[0103] The optimal ratio of the reaction system is as shown in Table 2 below:

[0104] Table 2

[0105] Serial number Components concentration 1 PEG35000 5％ 2 Creatine kinase 0.1mg / mL 3 Creatine phosphate 50mM 4 DNA polymerase (Bsu) 50 ng / μL 5 T4 gp32 protein 600ng / μL 6 T4 UvsX recombinase 120 ng / μL 7 T4 UvsY protein 30 ng / μL 8 Exo III exonuclease 1U / μL 9 Tris-tricine (pH 7.9) 50mM 10 Potassium acetate 100mM 11 Dithiothreitol (DTT) 2mM 12 dNTPs 200nM each 13 ATP 3mM 14 Upstream primer combination 420nM 15 Downstream primer combination 420nM 16 Fluorescent probe combination 120nM 17 magnesium acetate 14mM

[0106] The reaction conditions of the reaction system are: reacting for 10 - 60 min at 25 - 45 °C.

[0107] The optimal reaction conditions are: reacting for 20 min at 42 °C.

[0108] Example 2 Conducted a dual-channel primer test according to the RPMAS system

[0109] Designed upstream and downstream primers and corresponding probes for the target sequence (nCoV). The design guidelines are: a. The length is between 30 - 36; b. 40% < GC content < 60%; c. 50 < Tm value < 100; d. The maximum allowable single nucleotide repeat length is 5. The specific design results are shown in Table 3:

[0110] Table 3 [[ID=二十九]]

[0111] [[ID=三十]] [[ID=三十一]] [[ID=三十二]]

[0112] [[ID=三十三]]Designed and prepared the target positive plasmid, and completed extraction and concentration calculation. The results are shown in Table 4[[ID=三十四]] [[ID=三十五]]

[0113] [[ID=三十六]]Table 4[[ID=三十七]] [[ID=三十八]]

[0114] [[ID=三十九]] [[ID=四十]] [[ID=四十一]]

[0115] [[ID=四十二]]Set the constant temperature amplification program (42 °C, 20 minutes, detecting the fluorescence intensity every 30 s) on the PCR device (SLAN96), prepared the amplification system according to the formula, set two replicates, and the concentrations of the positive plasmid and the internal reference plasmid were both 10:[[ID=四十三]] 3 [[ID=四十四]]copies / μL. The test results are as[[ID=四十五]] Figure 1 [[ID=四十六]](Ct is the amplification cycle number, and Rn is the relative fluorescence intensity). As can be seen from the figure, the curve is complete and the Ct value ≤ 2 / 3 of the total cycle number. The fluorescence intensities of the FAM channel and the Cy5 channel are basically the same, and the two replicates perform consistently. This test result meets the reagent design requirements.[[ID=四十七]] [[ID=四十八]]

[0116] [[ID=四十九]]Example 3 Freeze-dried the amplification system to prepare freeze-dried microspheres[[ID=五十]] [[]][[ID=五十一]]

[0117] [[ID=五十二]](1) Preparation of the enzyme reaction solution: Strictly follow the production process formula (components 1 - 8 in Table 2), and the water used for preparation is sterilized ultrapure water;[[ID=五十三]] [[ID=五十四]]

[0118] It should be noted that in the provided text, there are some incorrect line numbers in the Chinese part (such as "二十九", "三十", etc.). I have translated them as they are while keeping the incorrect line numbers for the sake of following the format requirements. If this is an error in the original text, please correct it for a more accurate translation. Also, the " 3 " and some other tags are left unchanged as per the instructions.Preparation of reaction lyophilized beads A: Mix the enzyme reaction solution and lyophilization protectant in a volume ratio of 1:1, and clean the instrument according to the operating procedures of the liquid nitrogen lyophilization bead system. The details are as follows:

[0119] Add the prepared enzyme reaction solution and lyoprotectant A mixture to the syringe pump of the liquid nitrogen lyophilization bead dot system and set the dispensing volume to 20-50 μL. Prepare the reaction system pre-frozen pellets according to the liquid nitrogen lyophilization bead dot system operating procedures. Transfer the prepared reaction system pre-frozen pellets to a thermos that has been pre-filled with liquid nitrogen. The lyoprotectant A is as follows:

[0120] Serial number Components concentration 1 Trehalose 35％ 2 Polyethylene glycol 8000 10％ 3 Glucan-40 5％ 4 Mannitol 5％ 5 NaCl 0.9％

[0121] After the packaging is completed, clean the instrument according to the operating procedures of the liquid nitrogen freeze-drying bead liquid system.

[0122] Operate the pre-freeze dryer according to the vacuum freeze dryer operating procedures. After the plate temperature drops to -45°C to -50°C, transfer the pre-frozen pellets to a vial containing liquid nitrogen. Press the stopper (note: press the stopper halfway), then transfer the vial to the pre-freeze dryer. Set the freeze-drying curve for freeze-drying. The freeze-drying conditions are shown in Table 5. After freeze-drying is complete, click the plate raise button. After tightening the stopper, open the vial, remove the freeze-dried pellets, and transfer them to the drying room.

[0123] Table 5

[0124]

[0125] For sampling inspection, place the freeze-dried pellets in an aluminum foil bag containing desiccant and vacuum seal it. After passing the inspection, take out the freeze-dried pellets from the vial and divide the pellets into eight-tube tubes. After capping, place them in an aluminum foil bag together with the desiccant and store them at 2℃-25℃ until use.

[0126] (2) Preparation of reaction freeze-dried ball B:

[0127] Take the system buffer (components 9-17 in Table 2) and mix with the lyophilization protectant in a volume ratio of 1:1.

[0128] Clean the instrument according to the operating procedures of the liquid nitrogen lyophilization bead liquid system.

[0129] Add the prepared mixture of system buffer and lyoprotectant B to the syringe pump of the liquid nitrogen lyophilization bead dot system and set the dispensing volume to 20-50 μL. Prepare the reaction system pre-frozen pellets according to the liquid nitrogen lyophilization bead dot system operating procedures. Transfer the prepared reaction system pre-frozen pellets to a thermos that has been pre-filled with liquid nitrogen.

[0130] The components of lyoprotectant B are as follows:

[0131]

[0132]

[0133] After the packaging is completed, clean the instrument according to the operating procedures of the liquid nitrogen freeze-drying bead liquid system.

[0134] Operate the pre-freeze dryer according to the vacuum freeze dryer operating procedures. After the plate temperature drops to -45°C to -50°C, transfer the pre-frozen pellets to a vial containing liquid nitrogen. Press the stopper (note: press the stopper halfway), then transfer the vial to the pre-freeze dryer. Set the freeze-drying curve for freeze-drying. The freeze-drying conditions are shown in Table 5. After freeze-drying is complete, click the plate raise button. After tightening the stopper, open the vial, remove the freeze-dried pellets, and transfer them to the drying room.

[0135] For sampling inspection, place the freeze-dried pellets in an aluminum foil bag containing desiccant and vacuum seal it. After passing the inspection, take out the freeze-dried pellets from the vial, divide the pellets into eight-tube tubes, cover them, place them in an aluminum foil bag together with the desiccant, vacuum seal it and store it at 2℃-25℃.

[0136] (3) Preparation of negative control lyophilized powder:

[0137] The internal reference plasmid was diluted to 1000 copies / mL with 0.9% NaCl solution, and mixed with lyophilized protective agent B at a volume ratio of 1:1 and shaken to mix.

[0138] Mix the negative control thoroughly and dispense 180 μL / vial into 2 mL brown glass bottles. Transfer the sample to a freeze dryer and set the freeze drying curve according to the freeze dryer operating procedures.

[0139] After freeze-drying is completed, take out the brown glass bottle, tighten the tube cap, place it together with the desiccant in an aluminum foil bag, vacuum it and store it at 2℃-25℃.

[0140] (4) Preparation of positive control lyophilized powder

[0141] Dilute the internal reference plasmid and RV-A (syncytial A & novel coronavirus plasmid) to 1000 copies / mL with 0.9% NaCl solution, mix with lyophilized protective agent B at a ratio of 1:1, and shake to mix.

[0142] Mix the positive control thoroughly and dispense 180 μL / vial into 2 mL brown glass bottles. Transfer the sample to a freeze dryer and set the freeze drying curve according to the freeze dryer operating procedures.

[0143] After freeze-drying is completed, take out the brown glass bottle, tighten the tube cap, place it together with the desiccant in an aluminum foil bag, vacuum it and store it at 2℃-25℃.

[0144] The quality control diluent A is 0.9% NaCl solution (normal saline), which is dispensed into 2 mL screw-cap tubes at a volume of 1.9 mL / tube.

[0145] (5) Characterization of freeze-dried pellets:

[0146] Characterize the particle size and appearance of the freeze-dried pellets. Figure 2 The particle size of randomly selected freeze-dried balls was measured using a vernier caliper (72 cases in total, with smooth surface, no collapse or cracking, and complete dissolution without precipitation in 50 μL buffer system). Figure 2 It can be seen that the particle size distribution of the freeze-dried balls is uniform (centered at 2.99±0.03mm), and they do not stick to each other, meeting the conditions for packaging and use. SEM characterizes the morphological characteristics of the freeze-dried balls (any cross section and surface). The freeze-dried microsphere sample was adhered to the conductive glue and gold was sprayed for 45s using a Quorum SC7620 sputtering coater with a gold spraying current of 10mA; then the sample morphology was photographed using a ZEISS GeminiSEM 300 scanning electron microscope with an acceleration voltage of 3kV. Figure 3 As shown in the figure, as excipients, the matrix structure composed of trehalose, polyethylene glycol 8000 and dextran-40 is dense, the skeleton is complete, and there is no sign of cracking or collapse; the use of NaCl as an excipient can change the crystallization trend and keep mannose in an amorphous state, thereby protecting the various enzymes in the system components. Because they cannot be completely dissolved in the excipients, the protective agent-enzyme composite blocks are basically distributed around the holes and grooves produced by the sublimation of ice crystals during freeze-drying, so as to have a protective effect on them as much as possible.

[0147] 100 freeze-dried pellets were randomly selected for moisture content determination (using a Karl Fischer moisture analyzer). The test results are as follows: Figure 4 The moisture content of all samples was less than 10%, meeting the requirements of freeze-dried finished products.

[0148] The freeze-dried pellets were amplified using nCoV-ORF1ab primers and probes, and the Twist finished reagents were compared with the raw material liquid system. The test results were as follows: Figure 5 As can be seen from the figure, this freeze-drying process can meet the detection performance requirements, and the advantage in Ct value is more obvious, which is in line with the product itself and process expectations.

[0149] Compared with other ratio protective agents and freeze-drying processes:

[0150] (1) Compared with other protective agents (replacing the above-mentioned freeze-dried protective agent A with the protective agents in the table below), the solution of the present invention achieves optimal effects in terms of morphology and many aspects. Figure 6 A in the middle is a photo of freeze-dried microspheres prepared using the following surface protective agent. The appearance of the obtained freeze-dried microspheres is incomplete and there are adhesions between them;

[0151] Serial number Components concentration 1 Trehalose 35％ 2 polyethylene glycol 4000 20％ 3 Glucan-40 15％ 4 Mannitol 8％ 5 NaCl 0.9％

[0152] (2) If other lyophilization processes are used under the condition of the same lyoprotectant (taking the conditions in Table 6 below as an example), it can be known from SEM observation that the internal structure is uneven, and the local pores are too large, resulting in loose texture, which does not meet the production and use standards of lyophilized reagents ( Figure 6 in B and C).

[0153] Table 6

[0154]

[0155] Example 4 A triple detection amplification kit relying on an amplification system

[0156] A constant-temperature amplification joint detection system was constructed based on recombinase polymerase (T4 UvsX), and the enzyme combination used was: recombinase T4 UvsX, T4 UvsY protein, T4 gp32 protein, Bsu DNA polymerase, creatine kinase. Such as the DNA or RNA of the test sample (RNA needs to add an appropriate amount of reverse transcriptase and RNase inhibitor).

[0157] The components required for this joint detection system relying on the buffer are: Tris-tricine (pH7.9), potassium acetate, dithiothreitol (DTT), dNTPs, ATP, upstream primer combination, downstream primer combination, fluorescence probe combination, magnesium acetate.

[0158] Specifically, the ratio of the reaction system is as shown in Table 7 below:

[0159] Table 7

[0160]

[0161]

[0162] The reaction conditions of the reaction system are: react at 42°C for 20 min.

[0163] Upstream and downstream primers and corresponding probes were designed for the target sequences (nCoV, RSVA), and the design guiding principles were: a. The length is between 30 and 36; b. 40% < GC content < 60%; c. 50 < Tm value < 100; d. The maximum allowable single nucleotide repeat length is 5. The specific design results are shown in Table 8:

[0164] Table 8

[0165]

[0166] The target positive plasmid was designed, prepared, extracted and its concentration was calculated. The results are shown in Table 9

[0167] Table 9

[0168]

[0169]

[0170] The constant temperature amplification program (42°C, 20 minutes, fluorescence intensity detection every 30 seconds) was set on the PCR equipment (SLAN96). The amplification system was prepared according to the formula and freeze-dried microspheres were prepared (prepared according to the optimal system in Example 3). The concentrations of positive plasmid and internal reference plasmid were both 10 3 copies / μL, test results such as Figure 7 As shown in the figure, the curve remains intact at low nucleic acid concentrations, with Ct values ≤ 2 / 3 of the total cycle number. The fluorescence intensities of the FAM, HEX, and Cy5 channels meet the detection criteria. This test result meets the reagent design requirements. However, the crosstalk between different channels and the development and debugging costs are key considerations for developers, and the system of the present invention also addresses this issue.

[0171] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A detection system based on recombinase polymerase amplification, characterized in that: The detection system includes an optimized first detection reagent and an optimized second detection reagent: The first detection reagent is a buffer system containing primers and probes; the probe contains a fluorescent group, a quenching group, and an exonuclease cleavage site disposed between the fluorescent group and the quenching group; the buffer system contains PEG35K, creatine kinase, and creatine phosphate; The second detection reagent contains a recombinase solution: the recombinase solution contains recombinase, recombinase loading factor, single-strand binding protein, strand-displacing DNA polymerase and exonuclease, as well as Tris-buffer, potassium acetate, dithiothreitol, dNTPs, ATP and magnesium acetate, and optionally contains or does not contain reverse transcriptase and RNase inhibitor; The optimization process includes: mixing the detection reagent with a freeze-dried protective agent, freezing and drying the mixture; based on the mass percentage of the freeze-dried protective agent as 100%, the content of each component is: 5%-40% trehalose, 5%-15% polyethylene glycol 8000, 1%-10% dextran-40, 2%-15% mannitol, and 0.45%-0.9% sodium chloride.

2. The detection system according to claim 1, characterized in that Include at least one of the following characteristics: (1) In the first detection reagent: The upstream primer concentration was 150nM-600nM; The concentration of downstream primers was 150nM-600nM; The probe concentration is 100nM-150nM; PEG35K concentration is 2.5%-10% (w / v); Creatine kinase concentration is 0.1-0.8 mg / mL; Creatine phosphate concentration is 25-75mM; (2) In the second detection reagent: The recombinant enzyme concentration was 60-600 ng / μL; The recombinase loading factor concentration was 20-800 ng / μL; The concentration of single-chain binding protein was 100-1000 ng / μL; The concentration of strand-displacing DNA polymerase was 3-100 ng / μL; The exonuclease concentration is 1U / μL-5U / μL; Tris-buffer pH 7.4-8.4, concentration 20-100 mM; Potassium acetate concentration was 0-150 mM; Dithiothreitol concentration was 1-12 mM; dNTPs, concentration 150-600 nM each; ATP, concentration 1-5 mM; Magnesium acetate, concentration 10-30 mM; (3) The Tris-buffer comprises Tris-HCl.

3. The detection system according to claim 1, characterized in that Include at least one of the following characteristics: (1) The freezing and drying comprises: The first stage: cool down to -55~-45℃ within 5-20min and keep at this temperature for 0.5-5h; The second stage: raise the temperature to -25~-27℃ within 5-20min and keep it at this temperature for 15-25h, all under vacuum conditions; The third stage: heating to 20-30℃ within 1-2 hours and keeping at this temperature for 1-10 hours, all under vacuum conditions; (2) The recombinase includes T4 UvsX recombinase; (3) The recombinase loading factor includes T4 UvsY protein; (4) the single-stranded binding protein includes T4 gp32 protein; (5) the strand-displacing DNA polymerase comprises Bsu DNA polymerase; (6) The exonuclease comprises exonuclease III; (7) The enzyme cleavage site includes tetrahydrofuran.

4. The detection system according to claim 1, characterized in that Include at least one of the following characteristics: (1) The length of the primer is 18 bp or more; (2) The probe is 46-52 nt in length; (3) The first detection reagent contains at least two sets of primers and probes for detecting different targets, and different probes are provided with different fluorescent groups and / or quenching groups; (4) The fluorescent group, quenching group and enzyme cleavage site are modified on the base of the probe; (5) The target to be detected is DNA or RNA; (6) When the target to be detected includes one or more of nCoV, RSVA, and RPP30, the primers include at least one pair of SEQ ID NOs. 8-13, and the probe includes at least one of SEQ ID NOs. 14-16.

5. The detection system according to claim 1, characterized in that Include at least one of the following characteristics: (1) The volume ratio of the first detection reagent to the lyophilized protective agent is 1:(1-3); (2) The volume ratio of the second detection reagent to the lyophilized protective agent is 1:(1-3).

6. A nucleic acid detection kit, characterized in that: A detection system comprising any one of claims 1 to 5.

7. The nucleic acid detection kit according to claim 6, characterized in that The nucleic acid detection kit is a kit for the joint detection of multiple nucleic acids.

8. Use of the detection system according to any one of claims 1 to 5 or the nucleic acid detection kit according to claim 6 or 7 in nucleic acid detection.

9. A nucleic acid detection method, characterized in that: The following steps are involved: The nucleic acid sample to be tested is mixed with the detection system according to any one of claims 1 to 5. After the reaction is completed, the fluorescent signal is detected and the target nucleic acid is detected according to the change of the fluorescent signal.

10. The nucleic acid detection method according to claim 9, characterized in that: Include at least one of the following characteristics: (1) The reaction temperature is 25-42°C; (2) The reaction time is 10-60 min.

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