Novel coronavirus screening and confirmation detection reagent, detection kit and detection method based on rt-raa

Abstract

This invention relates to the field of biological detection technology, specifically to RT-RAA-based primary screening and confirmatory detection reagents, kits, and methods for novel coronavirus. It includes RT-RAA reaction reagent I, which comprises an RT-RAA-based ORF gene and N gene amplification reaction solution. The amplification reaction solution contains primer pairs, including N-F primer pairs and / or N-R primer pairs and / or ORF-F primer pairs and / or ORF-R primer pairs, with nucleotide sequences as follows: NO: 1~4. The RT-RAA-based primary screening detection method for novel coronavirus includes the following steps: (1) amplification of the novel coronavirus ORF gene and N gene; (2) RT-RAA-MPs experiment. The RT-RAA-based confirmatory detection method for novel coronavirus is characterized by including an RT-RAA-F-PAgo detection method or an RT-RAA-L-PAgo detection method. This invention enables the establishment of a low-cost, rapid primary screening experiment for novel coronavirus, providing support for rapid pathogen screening or home testing, and establishing pathogen detection suitable for low-resource environments.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to RT-RAA-based primary screening and confirmatory detection reagents, test kits, and detection methods for the novel coronavirus. Background Technology

[0002] Real-time quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for COVID-19 testing. However, RT-qPCR testing requires specialized equipment. Reverse Transcription Recombinase Aided Amplification (RT-RAA) technology, as a nucleic acid amplification technique that does not require thermal cycling, has attracted much attention due to its significant reduction in experimental equipment requirements and testing time. The required reaction conditions are easy to achieve, especially for remote areas.

[0003] The core mechanism of RT-RAA technology lies in the specific amplification of target sequences using various enzymes and proteins (including recombinases, single-strand binding proteins, and strand displacement DNA polymerases) under isothermal conditions. In the presence of ATP, recombinases (such as T4 UvsX and UvsY) bind to primers, forming a recombinase-primer complex. This complex can accurately locate homologous sequences of the primers in the double-stranded DNA template and, with the strand displacement activity of the recombinase, insert the primers into the corresponding homologous sites. Subsequently, single-strand binding proteins rapidly bind to the displaced single-stranded DNA, effectively preventing it from reforming into a double-stranded structure. Then, the recombinase dissociates from the complex, and the DNA polymerase binds to the 3'-OH end of the primer, initiating strand extension to form a new complementary strand. The entire reaction process is carried out within a temperature range of 37°C to 42°C and typically takes only 20 to 40 minutes to complete. However, it is currently recognized that RT-RAA still needs improvement in terms of sensitivity and specificity, thus limiting its widespread application due to the current technological limitations. Summary of the Invention

[0004] To address the above shortcomings, this invention provides RT-RAA-based reagents, test kits, and detection methods for the initial screening and confirmation of the novel coronavirus, thereby overcoming the deficiencies in sensitivity and specificity of existing RT-RAA technologies.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The novel coronavirus detection reagent based on RT-RAA includes RT-RAA reaction reagent I, which includes an RT-RAA-based ORF gene and N gene amplification reaction solution. The amplification reaction solution includes primer pairs, which include: NF primer pairs and / or NR primer pairs and / or ORF-F primer pairs and / or ORF-R primer pairs, with their nucleotide sequences as follows: NO: 1~4.

[0006] The novel coronavirus screening test reagent based on RT-RAA includes the novel coronavirus test reagent based on RT-RAA as described above, and also includes flocculation reaction reagent II; the flocculation reaction reagent II includes PEG 8000, magnetic beads, and acetic acid solution, with a pH less than 6.0; the magnetic beads are negatively charged under acidic conditions.

[0007] Preferably, the pH of the flocculation reagent II is 4.0~4.5 or 4.5 < pH ≤ 5.0.

[0008] The novel coronavirus confirmatory test reagent based on RT-RAA includes the novel coronavirus test reagent based on RT-RAA as described above, and also includes PfAgo reaction reagent; the PfAgo reaction reagent includes PfAgo protein, gDNA and probe, the probe is a fluorescent probe or a dual-labeled probe, the dual-labeled probe is modified with a fluorescent group at one end and biotin at the other end; The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA2, with nucleotide sequences as follows: NO: 5~8.

[0009] Preferably, the PfAgo reaction reagent is PfAgo reaction reagent III, and its probe is a fluorescent probe; The fluorescent probes include NPF and / or ORF-PF, whose nucleotide sequences are as follows: NO: 9~10.

[0010] Preferably, the PfAgo reaction reagent is PfAgo reaction reagent IV, and its probe is a double-labeled probe; The dual-labeled probe includes ORF-PL and / or NPL, whose nucleotide sequences are as follows: NO: 11~12.

[0011] The novel coronavirus detection kit based on RT-RAA includes: an RT-RAA detection kit. The RT-RAA test kit includes the novel coronavirus test kit based on RT-RAA as described above; It also includes reverse transcriptase, recombinase, single-strand binding protein, DNA polymerase, buffer, magnesium ions, template solution, and sterile distilled water, wherein the primer pair concentration is 10 μmol / L, and the template occupies 1 / 50 to 1 / 5 of the total volume; thus forming the amplification system; The RT-RAA detection kit is an RT-RAA-MPs detection kit, which further includes: flocculation reaction reagent II; the flocculation reaction reagent II includes PEG 8000, magnetic beads, and acetic acid solution, with a pH less than 6.0, and the magnetic beads are negatively charged under acidic conditions; or The RT-RAA detection kit is an RT-RAA-PAgo detection kit, which further includes: PfAgo reaction reagent; the PfAgo reaction reagent includes PfAgo protein, gDNA and probe, the probe is a fluorescent probe or a chromatography probe, and the dual-labeled probe is modified with a fluorescent group at one end and biotin at the other end; The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA2, whose nucleotide sequences are as follows: NO: 5~8; The RT-RAA-PAgo detection kit is either an RT-RAA-F-PAgo detection kit or an RT-RAA-L-PAgo detection kit; wherein: (1) The probes of the RT-RAA-F-PAgo detection kit are fluorescent probes; the fluorescent probes include ORF-PF and / or NPF, and their nucleotide sequences are as follows: NO: 9~10; (2) The probes of the RT-RAA-L-PAgo detection kit are dual-labeled probes; the dual-labeled probes include ORF-PL and / or NPL, and their nucleotide sequences are as follows: NO: 11~12.

[0012] Preferably, the RT-RAA-MPs detection kit includes a housing, the lower part of the detection slot of the housing is filled with the flocculation reaction reagent II, the liquid surface of the flocculation reaction reagent II is covered with a heat-melting separator layer, and the amplification system is filled above the heat-melting separator layer.

[0013] The initial screening method for novel coronavirus based on RT-RAA includes the following steps: (1) The novel coronavirus ORF gene and N gene were amplified using the novel coronavirus detection reagent based on RT-RAA as described above: the amplification system also added reverse transcriptase, recombinase, single-strand binding protein, DNA polymerase, buffer, magnesium ions, template solution and sterile distilled water, wherein the primer concentration was 10 μmol / L, the template accounted for 1 / 50-1 / 5 of the total volume, the reaction temperature of the isothermal amplification was 20-45℃, and the time was 15-50 min; (2) RT-RAA-MPs experiment: The flocculation reaction reagent II is used, which includes PEG 8000, magnetic beads, and acetic acid solution with a pH less than 6.0. The magnetic beads are negatively charged under acidic conditions. Mix the flocculation reaction reagent II with the amplification product in step (1), and after the mixture is left to stand for 3-15 minutes, use a magnetic rack to adsorb magnetic beads for 3-15 minutes. Finally, shake the mixture and observe the state of the magnetic beads. (3) Result determination The magnetic beads were remixed, and the result was negative. The aggregation of magnetic beads indicates a positive result.

[0014] RT-RAA-based confirmatory detection methods for the novel coronavirus include the RT-RAA-F-PAgo detection method or the RT-RAA-L-PAgo detection method; The RT-RAA-F-PAgo detection method includes the following steps: (1) The novel coronavirus ORF gene and N gene were amplified using the novel coronavirus detection reagent based on RT-RAA as described above: the amplification system also added reverse transcriptase, recombinase, single-strand binding protein, DNA polymerase, buffer, magnesium ions, template solution and sterile distilled water, wherein the primer concentration was 10 μmol / L, the template accounted for 1 / 50-1 / 5 of the total volume, the reaction temperature of the isothermal amplification was 20-45℃, and the time was 15-50 min; (2) Add 2-6 μL of the amplification product obtained in step (1) to the PfAgo reaction reagent; the PfAgo reaction reagent includes 1-4 μL of PfAgo protein, 1-4 μL of gDNA, 1-4 μL of buffer, and 1-4 μL of Lmn. 2+ Add 2–6 μl of fluorescent probe and sterile water to a final volume of 10–50 μL; react on a fluorescence instrument at 85–98 °C for 10–50 min, collecting fluorescence every 30 s. The fluorescent probe includes ORF-PF and / or NPF, whose nucleotide sequences are as follows: NO: 9~10; The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA2, whose nucleotide sequences are as follows: NO: 5~8; (3) Result determination No fluorescence curve was observed, and the reaction tube did not emit light under ultraviolet light, indicating a negative result. The reaction tube exhibits a fluorescence curve and emits light under ultraviolet light, indicating a positive result. The RT-RAA-L-PAgo detection method includes the following steps: The difference between this method and the RT-RAA-F-PAgo detection method is that a dual-labeled probe is used to replace the fluorescent probe. In step (2), 2-15 μL of the reaction product is added to 10-40 μL of enzyme-free sterile water and then inserted into the test strip. The dual-labeled probe includes ORF-PL and / or ORF-PF, with nucleotide sequences as shown in NO: 11-12. The results will be determined as follows: The test strip shows a band on line C and no band on line T, indicating a negative result. The test strip shows a band on line C and a band on line T, indicating a positive result. The test strip has no band on line C, indicating that the test strip is invalid and the test result is invalid.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) Establish a low-cost, rapid initial screening test for the novel coronavirus to support rapid screening or home testing of pathogens and to establish pathogen testing suitable for low-resource environments.

[0016] (2) After adding the sample to the RT-RAA-MPs detection kit, the lid is closed before performing related operations. The detection and results can be completed without opening the lid again, which can effectively reduce aerosol contamination during the detection operation.

[0017] (3) A highly sensitive and specific method for detecting Pfago protein was established. The detection solution system constructed in this invention exhibits higher sensitivity and specificity during the detection process. It can quickly and accurately detect the presence of the novel coronavirus even when the viral load is low.

[0018] (4) The detection method based on Pfago overcomes the limitations of CRISPR technology. Existing CRISPR technology is highly dependent on PAM sequences during the detection process, which greatly limits its detection range. However, the method of combining Pfago protein with RT-RAA in this technical solution allows Pfago protein to bind to specific nucleic acid sequences under the guidance of gDNA. This enables this technical solution to detect a wider range of viral nucleic acid sequences, significantly expanding the detection range.

[0019] (5) The detection of the novel coronavirus ORF gene and N gene by combining RT-RAA with Pfago protein can detect both genes simultaneously, which greatly improves the accuracy and reliability of the detection. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0021] Figure 1 This is a flowchart and result diagram of the detection process of the novel coronavirus standard sample by RT-RAA-MPs in Example 1.

[0022] Figure 2 This is an 18% Urea-PAGE electrophoresis image of the novel coronavirus O amplification product after gDNA cleavage in Example 2.

[0023] Figure 3 This is a graph showing the results of the novel coronavirus standard sample tested using RT-RAA-F-PfAgo in Example 3.

[0024] Figure 4 This is a graph showing the results of the novel coronavirus standard sample tested using RT-RAA-L-PfAgo in Example 4. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] In the description of this invention, it should be noted that the terms "inner", "front", "rear", "left", "right", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of this invention is usually placed when in use. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0027] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set" and "connection" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0028] As a further explanation of the present invention, the enzyme-free sterile water used in the following examples is sterile ultrapure water; A Buffer, B Buffer, enzyme-free sterile water, and lyophilized powder are products of Zhongce Biotechnology Co., Ltd. (Hangzhou); PfAgo protein, Buffer, and Mn 2+ These are products of Baoying Tonghui Biotechnology Co., Ltd. (Beijing); the primer pairs, gDAN pairs, fluorescent probe pairs, and chromatography probe pairs involved are all synthesized by Sangon Biotech.

[0029] Table 1: The primer sequences of each set of primers described in this invention are shown in the table below:

[0030] The novel coronavirus detection reagent based on RT-RAA includes RT-RAA reaction reagent I, which comprises RT-RAA-based ORF gene and N gene amplification reaction solution. The amplification reaction solution includes A Buffer, B Buffer, enzyme-free sterile water, lyophilized powder, and primer pairs. The primer pairs include: NF primer pairs and / or NR primer pairs and / or ORF-F primer pairs and / or ORF-R primer pairs, with their nucleotide sequences as follows: NO: 1~4.

[0031] The novel coronavirus screening test reagent based on RT-RAA includes the novel coronavirus test reagent based on RT-RAA as described above, and also includes flocculation reaction reagent II; the flocculation reaction reagent II includes 4% (w / v) PEG 8000, magnetic beads, enzyme-free sterile water, and acetic acid solution, with a pH less than 6.0; the magnetic beads are negatively charged under acidic conditions, and can be selected from carboxyl magnetic beads, sulfonic acid magnetic beads, phosphate magnetic beads, silanol magnetic beads, and toluenesulfonyl magnetic beads, etc., all of which can achieve the above dissociation in an acidic environment; the pH of the flocculation reaction reagent II is 4.0~4.5 or 4.5 < pH ≤ 5.0, and when using magnetic beads with carboxyl groups, the pH is preferably 4.5.

[0032] The novel coronavirus confirmatory test reagent based on RT-RAA includes the novel coronavirus test reagent based on RT-RAA as described above, and also includes a PfAgo reaction reagent; the PfAgo reaction reagent includes PfAgo protein, buffer, and Mn. 2 + The ingredients are: enzyme-free sterile water, gDNA, and a probe, wherein the probe is a fluorescent probe or a chromatographic probe, and the chromatographic probe is modified with a fluorescent group at one end and biotin at the other end. The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA2, with nucleotide sequences as follows: NO: 5~8.

[0033] Preferably, the PfAgo reaction reagent is PfAgo reaction reagent III, and its probe is a fluorescent probe; The fluorescent probes include NPF and / or ORF-PF, whose nucleotide sequences are as follows: NO: 9~10.

[0034] Preferably, the PfAgo reaction reagent is PfAgo reaction reagent IV, and the dual-labeled probe used is a chromatography probe; The chromatographic probes include ORF-PL and / or NPL, whose nucleotide sequences are as follows: NO: 11~12.

[0035] The novel coronavirus detection kit based on RT-RAA includes: an RT-RAA detection kit. The RT-RAA test kit includes the novel coronavirus test kit based on RT-RAA as described above; It also includes reverse transcriptase, recombinase, single-strand binding protein, DNA polymerase, buffer, magnesium ions, template solution, and sterile distilled water, wherein the primer pair concentration is 10 μmol / L, and the template occupies 1 / 50 to 1 / 5 of the total volume; thus forming the amplification system; The RT-RAA detection kit is an RT-RAA-MPs detection kit, which further includes: flocculation reaction reagent II; the flocculation reaction reagent II includes PEG 8000, magnetic beads, and acetic acid solution, with a pH less than 6.0, and the magnetic beads are negatively charged under acidic conditions; in this embodiment, carboxyl-containing magnetic beads (particle size range 1-10 μM) are preferred, and the pH is preferably 4.5.

[0036] Preferably, the RT-RAA-MPs detection kit includes a housing, the lower part of the detection slot of the housing is filled with the flocculation reaction reagent II, the liquid surface of the flocculation reaction reagent II is covered with a heat-melting separator layer, and the amplification system is filled above the heat-melting separator layer.

[0037] In this embodiment, an RT-RAA-PAgo detection kit is also provided. In addition to the amplification system, the RT-RAA-PAgo detection kit further includes: PfAgo reaction reagents; the PfAgo reaction reagents include PfAgo protein, buffer, and Mn. 2+ The ingredients are: enzyme-free sterile water, gDNA, and a fluorescent probe or a dual-labeled probe, wherein the dual-labeled probe is modified with a fluorescent group at one end and biotin at the other end; The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA2, whose nucleotide sequences are as follows: NO: 5~8; The RT-RAA-PAgo detection kit is either an RT-RAA-F-PAgo detection kit or an RT-RAA-L-PAgo detection kit; wherein: (1) The probes of the RT-RAA-F-PAgo detection kit are fluorescent probes; the fluorescent probes include NPL and / or NPF, and their nucleotide sequences are as follows: NO: 9~10; (2) The dual-labeled probes of the RT-RAA-L-PAgo detection kit are chromatographic probes; the chromatographic probes include ORF-PL and / or ORF-PF, and their nucleotide sequences are as follows: NO: 11~12.

[0038] The initial screening method for novel coronavirus based on RT-RAA includes the following steps: (1) The novel coronavirus RNA was amplified using the amplification system described above, and the reaction temperature of the isothermal amplification was 20-45℃ and the time was 15-50 min. (2) RT-RAA-MPs experiment: Using the above-mentioned flocculation reaction reagent II, mix flocculation reaction reagent II with the amplification product in step (1), and after the mixture is left to stand for 3-15 minutes, use a magnetic rack to adsorb magnetic beads for 3-15 minutes, and finally shake and observe the state of the magnetic beads. (3) Result determination The magnetic beads were remixed, and the result was negative. The aggregation of magnetic beads indicates a positive result.

[0039] The principle of the above-mentioned RT-RAA-MPs detection technology of the present invention is as follows: In an acidic environment, the negative charge of the long-chain DNA phosphate backbone obtained by amplification is partially neutralized, and the whole becomes close to electroneutrality and exposes hydrophobic bases; these hydrophobic segments simultaneously entangle multiple magnetic beads with carboxyl groups through hydrophobic adsorption, and the magnetic beads are woven into a three-dimensional network by means of the dual action of "hydrophobic-bridging", which rapidly forms a visible floc precipitate; while the magnetic beads in the targetless system still carry a net negative charge and repel each other, the precipitate formed by magnetic adsorption is easy to redisperse, and the solution remains turbid. Specific Implementation Example 1 In this embodiment, taking the EP tube as an example, during the specific preparation, 5-15 μL of carboxyl-containing magnetic beads can be added to PEG8000 and mixed with acetic acid buffer to adjust the pH to 4.5 for later use. Take a clean 1.5 mL EP tube, add 200-700 μL of the flocculation reaction reagent II to the bottom of the tube, drop a layer of paraffin in the middle, drop the amplification solution prepared according to the amplification system onto the paraffin, and add the novel coronavirus RNA sample to be tested. Amplify the template according to the above isothermal amplification conditions. For example, the reaction can be carried out at 42℃ for 30 minutes first, and then the temperature can be increased until the paraffin melts. Then the amplification product is mixed with the flocculation reaction reagent II. After the EP tube is placed for 3-15 minutes, the magnetic beads are adsorbed with a magnetic rack (2000-4200 Gauss (Gs)) for 3-15 minutes, and then shake it vigorously and observe the state of the magnetic beads at the bottom of the EP tube.

[0041] If the magnetic beads are remixed, the result is negative; if the magnetic beads aggregate, the result is positive.

[0042] Quality control: Use enzyme-free sterile water instead of the negative control sample for testing; Result interpretation refers to... Figure 1 .

[0043] RT-RAA-based confirmatory detection methods for the novel coronavirus include the RT-RAA-F-PAgo detection method or the RT-RAA-L-PAgo detection method.

[0044] The RT-RAA-F-PAgo detection method includes the following steps: (1) Perform ORF and N gene amplification according to step (1) of the novel coronavirus screening detection method based on RT-RAA; (2) Add 2-6 μL of the amplification product obtained in step (1) to the PfAgo reaction reagent; the PfAgo reaction reagent includes 1-4 μL of PfAgo protein, 1-4 μL of gDNA, 1-4 μL of buffer, and 1-4 μL of Ln. 2+ Add 2–6 μl of fluorescent probe and sterile water to a final volume of 10–30 μL; react on a fluorescence instrument at 85–98 °C for 10–50 min, collecting fluorescence every 30 s. The fluorescent probe includes NPL and / or NPF, whose nucleotide sequences are as follows: NO: 9~10; The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA2, whose nucleotide sequences are as follows: NO: 5~8; (3) Result determination No fluorescence curve was observed, and the reaction tube did not emit light under ultraviolet light, indicating a negative result. The reaction tube exhibits a fluorescence curve and emits light under ultraviolet light, indicating a positive result. The RT-RAA-L-PAgo detection method includes the following steps: The difference between this method and the RT-RAA-F-PAgo detection method is that: 2-15 μL of the reaction product in step (2) is added to 10-40 μL of enzyme-free sterile water and then inserted into the test strip; the chromatography probe includes ORF-PL and / or ORF-PF, and its nucleotide sequence is as follows: NO: 11~12; The results will be determined as follows: The test strip shows a band on line C and no band on line T, indicating a negative result. The test strip shows a band on line C and a band on line T, indicating a positive result. The test strip has no band on line C, indicating that the test strip is invalid and the test result is invalid.

[0045] The above-mentioned technical principles of the present invention: In the RT-RAA-F-Pfago detection technology described in this invention, after the fluorescent probe is cut, the luminescent group and the quenching group separate and generate fluorescence.

[0046] The RT-RAA-L-Pfago detection system of this invention employs a dual-labeled probe, with one end modified with a FAM fluorescent group and the other end modified with biotin. With both ends dual-labeled (fluorescent group + biotin), in the presence of the target, the RT-RAA amplification product is first cleaved by Pfago protein under the guidance of gDNA, releasing secondary gDNA (s-gDNA). The s-gDNA further guides Pfago to cleave the probe, causing FAM and biotin to physically separate. After cleavage, the free FAM group binds to the anti-FAM antibody coated on the detection line (T line), while the biotin binds to avidin on the control line (C line), resulting in visible bands on both the T and C lines, indicating a positive result. If no target is present, the probe remains intact, and the biotin on it only binds to avidin on the C line, resulting in color development only on the C line, indicating a negative result. Figure 4By preparing lyophilized powders from the RT-RAA amplification system and the Pfago cleavage system, and then detecting them using a temperature-controlled insulated cup, RT-RAA-Pfago can minimize the need for detection equipment and improve the applicability of the method. Specific Implementation Example 2 This method was used to identify the gDNA of the novel coronavirus. The amplification products were purified according to the above method. gDNA was designed based on the RT-RAA amplification products. The cleavage system and reaction were prepared according to Table 2. After cleavage, the gDNA cleavage products were screened by electrophoresis with 18% Urea-PAGE. Results interpretation is as follows. Figure 2 .

[0048] The marker lane contains the amplification product. When the amplification product is cleaved by the PfAgo protein gDNA, no band will appear at the marker location. Electrophoresis conditions are 250V for 20-50 minutes.

[0049]

[0050] Example 3: The standard RT-RAA-F-PfAgo was tested, and the specific process is as follows: (1) Amplify according to the above amplification system and react at 42℃ for 30 minutes; (2) Add the amplification product obtained in step (1) to the PfAgo reaction reagent. In this embodiment, a fluorescent probe is used, that is, the fluorescent cleavage system used is 2 μL PfAgo protein, 2 μL buffer, and 1 μL Mn. 2+ 2 μL of gDNA, 8 μL of the amplification product obtained in step (1) and 5 μL of ORF / N gene fluorescent probe were mixed, and sterile water was added to 50 μL. The cutting conditions were 95℃ for 30 min, and fluorescence was collected every 30 s. (3) Take the product from step (2) and observe the qPCR amplification curve and / or observe the fluorescence intensity under 490–495 nm ultraviolet light; Quality control: Use enzyme-free sterile water instead of the negative control sample for testing; Result interpretation refers to... Figure 4 .

[0051] Example 4: The standard RT-RAA-L-PfAgo was tested, and the specific process is as follows: (1) Amplify according to the above amplification system and react at 42℃ for 30 minutes; (2) Add the amplification product obtained in step (1) to the PfAgo reaction reagent. In this embodiment, a chromatography probe is used, and the chromatography cleavage system is 2 μL PfAgo protein, 2 μL buffer, and 1 μL Mn. 2+Add 2 μL of gDNA, take 8 μL of the amplification product obtained in step (1) and 5 μL of ORF / N gene chromatography probe, add sterile water to 50 μL, and react at 95℃ for 30 minutes.

[0052] (3) Take 3-10 μL of the product from step (2) and mix it with 30-50 μL of enzyme-free sterile water, insert the chromatographic test strip, and observe the results within 3-15 min.

[0053] Quality control: Use enzyme-free sterile water instead of the negative control sample for testing; Result interpretation refers to... Figure 4 .

[0054] Performance verification of this invention: 1. Measurement accuracy With the consent of the Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region and the informed consent of the patients, 23 clinical nasopharyngeal swab samples were collected, including 18 positive samples and 5 negative samples. These samples were tested using RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo, respectively, and the positive and negative concordance rates were calculated. The results showed that the study results were completely consistent with those obtained using the Novel Coronavirus (2019-nCoV) ORF1ab / N gene nucleic acid detection kit (Xi'an Tianlong Technology Co., Ltd., China), with both the positive and negative concordance rates reaching 100%.

[0055] 2. Analytical Sensitivity This invention employs three methods—RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo—to analyze the sensitivity of novel coronavirus positive samples, with each sample containing seven concentration gradients. The sensitivities of the three detection methods in this invention—RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo—are 10, ... 2 copies / μL, 5 copies / μL and 5 copies / μL.

[0056] 3. Analyze specificity This invention uses nine common respiratory viruses, including RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo, for detection. The results show that none of the nine common respiratory pathogens produce non-specific signals, indicating that RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo have high specificity.

Claims

1. A novel coronavirus detection reagent based on RT-RAA, characterized in that: The reagent includes RT-RAA reaction reagent I, which includes RT-RAA-based ORF and N gene amplification reaction solutions. The amplification reaction solution includes primer pairs, which include: NF primer pairs and / or NR primer pairs and / or ORF-F primer pairs and / or ORF-R primer pairs, with nucleotide sequences as follows: NO: 1~4.

2. A novel coronavirus primary screening test reagent based on RT-RAA, characterized in that: The novel coronavirus detection reagent based on RT-RAA as described in claim 1 also includes flocculation reaction reagent II; the flocculation reaction reagent II includes PEG8000, magnetic beads, and acetic acid solution with a pH less than 6.0; the magnetic beads are negatively charged under acidic conditions.

3. The novel coronavirus primary screening test reagent based on RT-RAA according to claim 2, characterized in that: The pH of the flocculation reagent II is 4.0~4.5 or 4.5 < pH ≤ 5.

0.

4. A novel coronavirus confirmatory test reagent based on RT-RAA, characterized in that: The novel coronavirus detection reagent based on RT-RAA as described in claim 1 also includes a PfAgo reaction reagent; the PfAgo reaction reagent includes PfAgo protein, gDNA and a probe, wherein the probe is a fluorescent probe or a dual-labeled probe, and the dual-labeled probe is modified with a fluorescent group at one end and biotin at the other end; The gDNA includes N-gDNA1 and N-gDNA2 and / or ORF-gDNA1 and ORF-gDNA1, with nucleotide sequences as follows: NO: 5~8.

5. The novel coronavirus confirmatory test reagent based on RT-RAA according to claim 4, characterized in that: The PfAgo reaction reagent is PfAgo reaction reagent III, and its probe is a fluorescent probe; The fluorescent probes include ORF-PF and / or NPF, whose nucleotide sequences are as follows: NO: 9~10.

6. The novel coronavirus confirmatory test reagent based on RT-RAA according to claim 4, characterized in that: The PfAgo reagent is PfAgo reagent IV, and its probe is a double-labeled probe; The dual-labeled probe includes ORF-PL and / or NPL, whose nucleotide sequences are as follows: NO: 11~12.

7. A novel coronavirus detection kit based on RT-RAA, characterized in that, include: An RT-RAA detection kit, wherein the RT-RAA detection kit comprises the novel coronavirus detection kit based on RT-RAA as described in claim 1; It also includes reverse transcriptase, recombinase, single-strand binding protein, DNA polymerase, buffer, magnesium ions, template solution, and sterile distilled water, wherein the primer pair concentration is 10 μmol / L, and the template occupies 1 / 50 to 1 / 5 of the total volume; thus forming the amplification system; The RT-RAA detection kit is an RT-RAA-MPs detection kit, which further includes: flocculation reaction reagent II; the flocculation reaction reagent II includes PEG 8000, magnetic beads, and acetic acid solution, with a pH less than 6.0, and the magnetic beads are negatively charged under acidic conditions; or The RT-RAA detection kit is an RT-RAA-PAgo detection kit, which further includes: PfAgo reaction reagent; the PfAgo reaction reagent includes PfAgo protein, gDNA and probe, the probe is a fluorescent probe or a chromatographic probe, the chromatographic probe is modified with a fluorescent group at one end and biotin at the other end; The gDNA includes N-gDNA1 and / or N-gDNA2 and / or ORF-gDNA1 and / or ORF-gDNA1, whose nucleotide sequences are as follows: NO: 5~8; The RT-RAA-PAgo detection kit is either an RT-RAA-F-PAgo detection kit or an RT-RAA-L-PAgo detection kit; wherein: (1) The probes of the RT-RAA-F-PAgo detection kit are fluorescent probes; the fluorescent probes include ORF-PF and / or NPF, and their nucleotide sequences are as follows: NO: 9~10; (2) The RT-RAA-L-PAgo detection kit uses a dual-labeled probe; the dual-labeled probe includes ORF-PL and / or NPL, and its nucleotide sequence is as follows: NO: 11~12.

8. The novel coronavirus detection kit based on RT-RAA according to claim 7, characterized in that: The RT-RAA-MPs detection kit includes a housing, the lower part of the detection slot of the housing is filled with the flocculation reaction reagent II, the liquid surface of the flocculation reaction reagent II is covered with a heat-melting separator layer, and the amplification system is filled above the heat-melting separator layer.

9. A novel coronavirus primary screening and detection method based on RT-RAA, characterized in that, Includes the following steps: (1) The novel coronavirus ORF gene and N gene were amplified using the RT-RAA-based novel coronavirus detection reagent as described in claim 1: the amplification system also included reverse transcriptase, recombinase, single-stranded binding protein, DNA polymerase, buffer, magnesium ions, template solution and sterile distilled water, wherein the primer concentration was 10 μmol / L, the template occupied 1 / 50-1 / 5 of the total volume, the isothermal amplification reaction temperature was 20-45℃, and the time was 15-50 min; (2) RT-RAA-MPs experiment: Flocculation reagent II is used, which includes PEG 8000, magnetic beads, and acetic acid solution with a pH less than 6.

0. The magnetic beads are surface-modified and capable of dissociating H+. + The acidic functional groups of the compound carry a negative charge under acidic conditions. Mix the flocculation reaction reagent II with the amplification product in step (1), and after the mixture is left to stand for 3-15 minutes, use a magnetic rack to adsorb magnetic beads for 3-15 minutes. Finally, shake the mixture and observe the state of the magnetic beads. (3) Result determination The magnetic beads were remixed, and the result was negative. The aggregation of magnetic beads indicates a positive result.

10. A novel coronavirus confirmatory detection method based on RT-RAA, characterized in that, This includes the RT-RAA-F-PAgo detection method or the RT-RAA-L-PAgo detection method; The RT-RAA-F-PAgo detection method includes the following steps: (1) The novel coronavirus ORF gene and N gene were amplified using the RT-RAA-based novel coronavirus detection reagent as described in claim 1: the amplification system also included reverse transcriptase, recombinase, single-stranded binding protein, DNA polymerase, buffer, magnesium ions, template solution and sterile distilled water, wherein the primer concentration was 10 μmol / L, the template occupied 1 / 50-1 / 5 of the total volume, the isothermal amplification reaction temperature was 20-45℃, and the time was 15-50 min; (2) Add 2-6 μL of the amplification product obtained in step (1) to the PfAgo reaction reagent; the PfAgo reaction reagent includes 1-4 μL of PfAgo protein, 1-4 μL of gDNA, 1-4 μL of buffer, and 1-4 μL of Lmn. 2+ Add 2–6 μl of fluorescent probe and sterile water to a final volume of 10–50 μL; react on a fluorescence instrument at 85–98 °C for 10–50 min, collecting fluorescence every 30 s. The fluorescent probe includes NPL and / or NPF, whose nucleotide sequences are as follows: NO: 9~10; The gDNA includes N-gDNA1 and / or N-gDNA2 and / or ORF-gDNA1 and / or ORF-gDNA1, whose nucleotide sequences are as follows: NO: 5~8; (3) Result determination No fluorescence curve was observed, and the reaction tube did not emit light under ultraviolet light, indicating a negative result. The reaction tube exhibits a fluorescence curve and emits light under ultraviolet light, indicating a positive result. The RT-RAA-L-PAgo detection method includes the following steps: The difference between this method and the RT-RAA-F-PAgo detection method is that: a dual-labeled probe is used to replace the fluorescent probe. 2-15 μL of the reaction product in step (2) is added to 10-40 μL of enzyme-free sterile water and then inserted into the test strip. The dual-labeled probe includes ORF-PL and / or ORF-PF, and their nucleotide sequences are as follows: NO: 11~12. The results will be determined as follows: The test strip shows a band on line C and no band on line T, indicating a negative result. The test strip shows a band on line C and a band on line T, indicating a positive result. The test strip has no band on line C, indicating that the test strip is invalid and the test result is invalid.

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