CN113186349A - Primer probe combination for detecting SARS-CoV-2, kit and application - Google Patents
Primer probe combination for detecting SARS-CoV-2, kit and application Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention discloses a primer-probe combination for detecting SARS-CoV-2, a kit and application thereof. In a first aspect of the application, a primer-probe combination for detecting SARS-CoV-2 is provided, the primer-probe combination comprising a forward primer, a reverse primer and a probe. The primer probe combination according to the embodiment of the application has at least the following beneficial effects: the target sequence targeted by the primer probe combination designed in the embodiment of the application is positioned on a highly conserved Orf1ab region, and the size is 233 bp. The primer probe combination taking the fragment as a target amplification sequence has better recognition effect on SARS-CoV-2 virus, and has higher sensitivity compared with the prior RPA kit.
Description
Technical Field
The application relates to the technical field of nucleic acid detection, in particular to a primer probe combination, a kit and application for detecting SARS-CoV-2.
Background
The nucleic acid detection is the most main mode for diagnosing the novel coronavirus infection at present, a detection person samples a person to be detected on site through a nasopharynx swab and then uniformly extracts nucleic acid, and then the detection is carried out through a fluorescence quantitative PCR instrument, so that the whole process has many steps and is time-consuming, a detection report can be issued generally in 6-8 hours, and the operation of a professional is required. Compared with the traditional fluorescent quantitative PCR detection, the Recombinase Polymerase Amplification (RPA) technology can generally complete detection within 15-20 min, not only the time consumption is shorter, but also the simplicity is greatly improved, and the requirement on equipment is reduced. Therefore, there is a need to develop suitable RPA detection methods for novel coronaviruses.
RPA is a constant temperature nucleic acid amplification technology, and the key is three enzymes and proteins, including recombinase capable of binding primers, single-stranded nucleic acid binding protein (SSB) and strand displacement polymerase, which are active even at normal temperature, and the optimal reaction temperature is around 37 ℃. The specific principle of the RPA is as follows: the recombinase-primer complex is formed by the recombination of the recombinase and the primer under the constant temperature condition, homologous sequence pairing is carried out in the double-stranded DNA, the SSB is combined with the replaced single-stranded DNA and keeps the replaced single-stranded DNA stable, and the strand replacement DNA polymerase mediates the amplification of the complex.
The design of primers and probes required for RPA is more complex than traditional PCR primers. Because too short primers can affect amplification speed and detection sensitivity, the RPA primers are generally longer than the PCR primers, and are usually about 30-38 bases. The reaction process of the RPA does not need to be denatured, and the denaturation temperature considered by the traditional PCR is no longer an important factor influencing the amplification of the RPA. At the same time, RPA is not as mature as traditional PCR and requires the experimenter to individually touch conditions to optimize it. These reasons have led to greater difficulty in primer and probe design for RPA compared to traditional PCR. However, the sensitivity of the RPA detection product for detecting SARS-CoV-2 is low, and it is difficult to ensure the detection accuracy. Therefore, it is necessary to provide a primer-probe combination with higher sensitivity that can be used for detecting SARS-CoV-2.
Disclosure of Invention
The present application is directed to solving at least one of the problems in the prior art. Therefore, the application provides a primer probe combination with higher sensitivity and capable of being used for detecting SARS-CoV-2, a kit and application.
In a first aspect of the present application, there is provided a primer-probe combination for detecting SARS-CoV-2, the primer-probe combination comprising:
a forward primer: TAAGTATGTACAAATACCTACAACTTGTGC, respectively;
reverse primer: CTGTATACGACATCAGTACTAGTGCCTGTG, respectively;
and (3) probe: CTGCGGTATGTGGAAAGGTTATGGCTGTAGTTTGATCAACTCCGCGAAC are provided.
The primer probe combination according to the embodiment of the application has at least the following beneficial effects:
the target sequence targeted by the primer probe combination designed in the embodiment of the application is positioned on a highly conserved Orf1ab region, and the size is 233 bp. The primer probe combination taking the fragment as a target amplification sequence has better recognition effect on SARS-CoV-2 virus, and has higher sensitivity compared with the prior RPA kit.
In addition, the sequence is located in the highly conserved region of SARS-CoV-2, so that not only conventional novel coronavirus but also mutant novel coronavirus strain known at present can be detected. The isothermal amplification system using the primer probe combination can amplify a target gene sequence under the temperature condition of 37 ℃, does not have the steps of denaturation, annealing, extension and other different temperatures in the conventional amplification, and is simpler, more convenient and more time-saving to operate.
In some embodiments of the present application, the probe is inserted with a base analog 30-35 nt away from the 5 'end, and the 3' end of the probe is labeled with a modifying group selected from any one of an amine group, a phosphate group, and C3-spacer. Wherein the modifying group at the 3' -end of the probe can block the progress of extension. And by inserting the base analogues at 30 to 35nt, the probe can be recognized and cleaved by an enzyme having a DNA damage repair activity during an amplification reaction to form 3' -OH, thereby continuing the extension. Non-limiting examples of base analogs include tetrahydrofuran.
In some embodiments of the present application, the probe is inserted with a base analog 32nt from the 5' end.
In some embodiments of the present application, the probe is labeled with C3-spacer at the 3' end.
In some embodiments of the present application, the reverse primer is labeled with a first binding label at the 5 'end and the probe is labeled with a second binding label at the 5' end. By labeling the reverse primer and the probe with corresponding binding labels, the amplification product simultaneously carries the first binding label and the second binding label, so that the binding labels can be used for detecting and judging the amplification result through a chromatographic reaction by virtue of the actions of antigen-antibody binding and the like. Wherein the first binding label and the second binding label are different binding labels, and have different binding properties, and can bind to different substances, so that the different substances are labeled at different positions by a chromatography reaction, and different binding reactions occur according to different amplification results, thereby performing judgment.
In addition, it should be noted that, in addition to using a chromatography reaction, the amplification result can also be directly detected and determined by means of a fluorescence reaction (for example, a fluorescence reporter group and a fluorescence quencher group are respectively labeled about 2nt upstream and downstream of the base analog position of the probe), gel electrophoresis, and the like, and the difference is that the detection and determination can be directly carried out by naked eyes by using a chromatography reaction, which is simpler and faster, and does not need any complicated instrument and equipment.
In some embodiments of the present application, the first binding tag is biotin and the second binding tag is fluorescein.
In a second aspect of the present application, a kit is provided, which comprises the primer probe combination described above.
In some embodiments of the present application, a test strip is also included.
In some embodiments of the present application, the test strip is a colloidal gold chromatography test strip.
In some embodiments of the present application, the colloidal gold chromatography test paper includes a bonding pad and a detection film, which are attached in sequence, the bonding pad is coated with colloidal gold, and the detection film is provided with a detection line and a quality control line.
In some embodiments of the present application, the colloidal gold is labeled with a third binding label, the detection line is coated with a fourth binding label, and the quality control line is coated with a fifth binding label. The third binding label is capable of specifically binding to the first (or second) binding label, thereby allowing the colloidal gold to bind to the amplification product; the fourth binding label can be specifically combined with the second (or the first) binding label, so that the amplification product combined with the colloidal gold is fixed on the detection line; the fifth binding label can be specifically combined with the third binding label, so that the colloidal gold or the amplification product combined with the colloidal gold is fixed on the quality control line.
In some embodiments of the present application, the third binding label is capable of specifically binding to the second binding label, thereby allowing the colloidal gold to bind to the amplification product; the fourth binding label can be specifically bound with the first binding label, so that the amplification product bound with the colloidal gold is fixed on the detection line; the fifth binding label can be specifically combined with the third binding label, so that the colloidal gold or the amplification product combined with the colloidal gold is fixed on the quality control line.
In some embodiments of the present application, the third binding label, the fourth binding label, and the fifth binding label are bound by antigen-antibody binding, and the detection is performed by a double antibody sandwich method.
In some embodiments of the present application, the third binding label is an antibody to the second binding label, such as a fluorescein antibody.
In some embodiments of the present application, the fourth binding label is an antibody, such as a biotin antibody, to the first binding label. It is obvious that the fourth binding tag can equally be avidin, streptavidin or the like capable of producing a biotin-avidin binding effect with biotin.
In some embodiments of the present application, the fifth binding label is an antibody to the third binding label, such as a fluorescein secondary antibody.
In some embodiments of the present application, the kit further comprises a recombinase required for the recombinase polymerase amplification reaction, a single-stranded DNA binding protein, a strand-displacing DNA polymerase, a buffer, and nfo enzyme, reverse transcriptase, and the like.
In a third aspect of the present application, there is provided the use of the primer-probe combination described above in the preparation of a kit for detecting SARS-CoV-2.
In a fourth aspect of the present application, there is provided a method for detecting SARS-CoV-2, the method comprising the steps of:
providing a nucleic acid sample;
mixing a nucleic acid sample with the primer probe combination to perform recombinase polymerase amplification reaction;
analyzing the result of the polymerase amplification reaction of the recombinant enzyme;
the detection method is used for non-disease diagnostic purposes.
Non-disease diagnostic purposes are defined herein as not directly targeting a living human or animal body and/or not identifying, identifying or eliminating a lesion. For example, the spread of SARS-CoV-2 virus in an environment such as wastewater is examined and monitored.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
FIG. 1 shows the results of the specificity test of the colloidal gold chromatography test paper of example 1 of the present application.
FIG. 2 shows the results of the sensitivity test of the colloidal gold chromatography test paper of example 1 of the present application.
Detailed Description
The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.
The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.
In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, unless otherwise expressly limited, terms such as set, mounted, connected and the like should be construed broadly, and those skilled in the art can reasonably determine the specific meaning of the terms in the present application by combining the detailed contents of the technical solutions.
In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1
This example provides a primer probe combination for detecting SARS-CoV-2, which is based on the published full sequence of SARS-CoV-2 and selects highly conserved Orf1ab gene for primer probe design. Through a large amount of sequence analysis, primer optimization and manual screening, the optimal primer and probe combination is finally determined, and the sequences are shown in table 1:
TABLE 1 sequences of primer-probe combinations
Modifications in the sequence are described below, in which Biotin (Biotin) is labeled at the 5 'end of the reverse primer, fluorescein-labeled 6-FAM is labeled at the 5' end of the probe, a base gap (dSpacer) is formed between the 32 th and 33 th bases from the 5 'end of the probe by inserting Tetrahydrofuran (THF), and C3-spacer is modified at the 3' end of the probe.
Among them, the extension can be blocked by C3-spacer and THF modification on the probe, and only when the nfo enzyme is added to the system, the nfo enzyme recognizes the THF site and cleaves to form 3' -OH, so that the extension can be continued. And the reverse primer and the 5' end of the probe are modified by Biotin and 6-FAM respectively, so that an amplification product is simultaneously marked by the Biotin and the 6-FAM, and a colloidal gold test strip in the kit is matched for detection, and the result is directly judged by naked eyes.
The embodiment also provides a kit for detecting SARS-CoV-2, which comprises a detection kit and a nucleic acid extraction kit, wherein the composition of the detection kit is shown in Table 2:
TABLE 2 assay kit composition
The specification of the detection kit is 48T. The reaction system of the detection kit is 50 mu l, firstly, 25 mu l of 2 times reaction buffer solution is mixed with dry powder of amplification reagent to dissolve the dry powder, and the nucleic acid sample is added and then ddH is used2O filled the reaction system to 50. mu.l.
The reaction buffer solution is used for providing a proper pH value and a proper salt ion concentration for an amplification system, ensures the effective progress of an amplification reaction, and can comprise components such as Tris, polyethylene glycol, dNTP, ATP, potassium acetate and the like.
The amplification reagent (dry powder) comprises recombinase, single-stranded DNA binding protein, strand displacement DNA polymerase, nfo enzyme, reverse transcriptase and the like which are necessary for RPA reaction, and is packaged and lyophilized after being dissolved according to concentration. The primer probe combination can be used as a component in an amplification reagent in a dry powder form, and can also be added additionally.
The test paper is double antibody sandwich colloidal gold test paper. The test paper comprises a sample pad, a combination pad, a detection membrane and a water absorption pad which are attached in sequence, wherein the mixed solution after the amplification reaction is diluted by chromatographic solution and then contacts with the sample pad, and whether SARS-CoV-2 is contained in the sample or not is detected by chromatography. The combination pad is coated with colloidal gold particles, and the colloidal gold particles are marked with the mouse anti-6-FAM monoclonal antibody. The detection film is provided with a detection line and a quality control line, the detection line is coated with a mouse anti-biotin monoclonal antibody, and the quality control line is coated with a rabbit anti-mouse IgG antibody.
When the sample is positive, amplification products marked with biotin and 6-FAM exist in the mixed solution after the amplification reaction, and are combined with the marked colloidal gold when passing through the combination pad, and are combined with the mouse anti-biotin monoclonal antibody on the detection line when continuously flowing to the detection line, so that the colloidal gold particles are enriched on the detection line to form a macroscopic red color. When the sample is negative and the mixed solution does not contain the amplification product, the free biotin is combined with the mouse anti-biotin monoclonal antibody on the detection line, but the 6-FAM is also in a free state, so that a biotin-amplification product-FAM-FAM monoclonal antibody-colloidal gold compound cannot be formed, and the detection line cannot be combined with gold particles, so that color development is avoided. In both cases, the rabbit anti-mouse IgG antibody coated on the quality control line can adsorb the colloidal gold particles to develop color. Therefore, the color development of the quality control line represents that the detection test paper is normal, and the colloidal gold particles flow through the detection line to reach the quality control line.
For safety testing, a plasmid containing the sequence of the Orf1ab test region was synthesized as a positive control, and the sequence of Orf1ab is shown below:
GTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTAAAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTACAATGAT(SEQ ID No.4)。
specificity detection assay
(1) The above positive control plasmid was synthesized with 4. mu.g of the starting plasmid in dry powder form using 20. mu.l of ddH2After O dissolution, 5. mu.l (1. mu.g) of the resulting mixture was digested and incubated at 37 ℃ for 4 hours to obtain linear plasmid DNA. The reaction system is shown in Table 3.
TABLE 3 plasmid digestion reaction System
(2) Linear plasmid DNA was transcribed with T7 transcription kit to obtain RNA, which was incubated at 37 ℃ for 4h, and the transcription system is shown in Table 4.
TABLE 4 transcription reaction System
(3) RNA purification
1. Mu.l of 70% ethanol was mixed with 20. mu.l of the reaction solution obtained in step (2).
2. Transferring all the uniformly mixed liquid into a CR3 adsorption column at 12000rpm for 1min, and discarding the waste liquid.
3. Adding 350. mu.l RW1 protein cleaning solution for washing at 12000rpm for 1min, and discarding the waste liquid.
4. Add 80. mu.l DNaseI working solution and let stand at room temperature for 15 min.
5. Adding 350. mu.l RW1 protein cleaning solution for washing at 12000rpm for 1min, and discarding the waste liquid.
6. Adding 500. mu.l RW washing solution, 12000rpm for 1min, and discarding the waste liquid.
7. Idling at 12000rpm for 2min to remove residual liquid in the adsorption column, and air drying at room temperature for several minutes.
8. Add 60. mu.l ddH2And O, standing for 3min, then eluting at 12000rpm for 2min to obtain RNA.
(4) RPA amplification
Adding the reagents into the PCR tube containing the amplification reagent (dry powder) in sequence according to the sequence in the table 5, covering the PCR tube with a cover after the reagents are added, turning upside down and uniformly mixing for 8-10 times, and reacting for 20min at 37 ℃. Wherein, 4 experimental groups with different RNA template concentrations and a control group without primer probe combination are respectively arranged, and the total number is 8. The concentration gradient of the RNA template was set to 0.05. mu.g, 0.1. mu.g, 0.25. mu.g, and 0.5. mu.g of the RNA template in 1. mu.l of the template solution, respectively.
TABLE 5 RPA amplification reaction System
(5) Reaction of test paper
1. Mu.l of the reaction mixture after RPA amplification was added with 95. mu.l of ddH2O to 1.5ml centrifuge tube.
2. After mixing, the mixture was centrifuged briefly and inserted into a test strip to react for about 5 min.
3. The results are shown in FIG. 1, wherein 1-4 respectively represent the contents of different RNA templates of 0.05. mu.g, 0.1. mu.g, 0.25. mu.g and 0.5. mu.g in the template solution, and the left side is the control group and the right side is the experimental group under the same number. As can be seen from the figure, under different template concentrations, the control group without primers and probes can not amplify the target gene, and further can not make the test strip detection line (T area) develop color; whereas the experimental group can amplify and color the detection line. Meanwhile, the quality control lines (zone C) of the experimental group and the control group are normally colored, and the representative results are credible. The experimental result proves that the kit provided by the embodiment of the application has better specificity.
Sensitivity detection experiment
Estimating the molecular weight of each copy to be about 100kDa according to the length (319bp) of positive RNA, diluting RNA extracted in a specificity detection experiment to obtain 7 kinds of RNA with different concentrations of 103、10210, 5, 2, 1 copy/. mu.l of RNA dilution, and using it as template for amplification. The reaction system was 50. mu.l each as in Table 5, wherein the RNA templates were 1. mu.l each of the 7 concentrations of RNA dilutions described above.
The results are shown in FIG. 2, the upper end of the test strip is the template concentration in the corresponding RNA diluent, which is 0, 10 from left to right2、1031, 2, 5, 10 copies/. mu.l. As can be seen, all the strips exhibited color along the control line (zone C) and no color along the test line (zone T), while the remaining 7 strips exhibited color, but 1 copy/. mu.l of the test strips exhibited no color along the control line (zone C)The test strip has no obvious color development, the color development of the test strip of 2 copies/mul and 5 copies/mul is darker than that of the test strip of 1 copy/mul but still not obvious enough compared with that of the test strip of 10 copies/mul, so the minimum detection limit in the test 50 mul reaction system is 10 copies/mul.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen Huaqi Biotech Limited
<120> primer probe combination for detecting SARS-CoV-2, kit and application
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<400> 1
taagtatgta caaataccta caacttgtgc 30
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<400> 2
ctgtatacga catcagtact agtgcctgtg 30
<210> 3
<211> 49
<212> DNA
<213> Artificial sequence
<400> 3
ctgcggtatg tggaaaggtt atggctgtag tttgatcaac tccgcgaac 49
<210> 4
<211> 319
<212> DNA
<213> Artificial sequence
<400> 4
gttgtctgta ctgccgttgc cacatagatc atccaaatcc taaaggattt tgtgacttaa 60
aaggtaagta tgtacaaata cctacaactt gtgctaatga ccctgtgggt tttacactta 120
aaaacacagt ctgtaccgtc tgcggtatgt ggaaaggtta tggctgtagt tgtgatcaac 180
tccgcgaacc catgcttcag tcagctgatg cacaatcgtt tttaaacggg tttgcggtgt 240
aagtgcagcc cgtcttacac cgtgcggcac aggcactagt actgatgtcg tatacagggc 300
ttttgacatc tacaatgat 319
Claims (10)
1. A primer probe combination for detecting SARS-CoV-2, comprising:
a forward primer: TAAGTATGTACAAATACCTACAACTTGTGC, respectively;
reverse primer: CTGTATACGACATCAGTACTAGTGCCTGTG, respectively;
and (3) probe: CTGCGGTATGTGGAAAGGTTATGGCTGTAGTTTGATCAACTCCGCGAAC are provided.
2. The primer probe combination according to claim 1, wherein a base analog is inserted into the probe at a distance of 30 to 35nt from the 5 'end, and the probe is labeled at the 3' end with a modifying group selected from any one of an amine group, a phosphate group and C3-spacer.
3. The primer probe combination of any one of claims 1 to 2, wherein the reverse primer is labeled at its 5 'end with a first binding label and the probe is labeled at its 5' end with a second binding label.
4. The primer probe combination of claim 3, wherein the first binding label is biotin and the second binding label is fluorescein.
5. A kit comprising the primer probe combination of any one of claims 1 to 4.
6. The kit of claim 5, further comprising a test strip.
7. The kit of claim 6, wherein the test strip is a colloidal gold assay strip.
8. The kit according to claim 7, wherein the colloidal gold chromatography test paper comprises a conjugate pad and a detection membrane which are sequentially attached, the conjugate pad is coated with colloidal gold, and the detection membrane is provided with a detection line and a quality control line.
9. Use of a primer-probe combination according to any one of claims 1 to 4 for the preparation of a kit for the detection of SARS-CoV-2.
A method for detecting SARS-CoV-2, which comprises the steps of:
providing a nucleic acid sample;
mixing the nucleic acid sample with the primer probe combination of any one of claims 1 to 4 to perform a recombinase polymerase amplification reaction;
analyzing the results of the recombinase polymerase amplification reaction;
the detection method is used for non-disease diagnostic purposes.
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