Virus nucleic acid detection probe based on amplification-free single molecule technology, virus detection system and application

Abstract

The invention provides a virus nucleic acid detection probe based on an amplification-free single molecule technology, a virus detection system and application, and belongs to the technical field of virus detection. The nucleic acid sequences of a capture probe and a report probe in the virus nucleic acid detection probe are respectively one or more of SEQ ID NO.5 to SEQ ID NO.6, SEQ ID NO.7 to SEQ ID NO.8, SEQ ID NO.9 to SEQ ID NO.10 and SEQ ID NO.11 to SEQ ID NO.12. The invention further discloses a kit for detecting the virus nucleic acid. The virus nucleic acid detection probe can realize rapid, specific, accurate and sensitive identification of SARS-CoV-2, IAV and IBV, when the virus nucleic acid detection probe is used for detecting viruses, target nucleic acid does not need constant-temperature or variable-temperature amplification, and the operation is simple and easy to implement.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a virus nucleic acid detection probe based on amplification-free single-molecule technology, a virus detection system and applications. Background Art

[0002] Respiratory tract infection (RTI) is one of the main causes of human illness and even death. It is an infectious disease caused by a variety of pathogenic microorganisms including bacteria, viruses, mycoplasma, fungi, parasites, etc., but most of them are caused by viruses. Common respiratory viruses, such as the new coronavirus, influenza A virus and influenza B virus, are highly contagious, spread quickly, have a short incubation period, develop rapidly, and lack long-lasting immunity after the disease. This type of viral infection not only causes harm to the human body, but also brings huge economic burdens and humanitarian disasters to the world.

[0003] Symptoms of respiratory viral infections vary but are not specific. The three viruses mentioned above can cause upper and lower respiratory tract infections, as well as overlapping clinical manifestations, making it difficult for doctors to distinguish pathogens without reliable laboratory analysis. Accurate and rapid diagnosis of pathogenic viral pathogens is very important for choosing appropriate treatments, saving people's lives, preventing epidemics, and reducing unnecessary use of antibiotics. Therefore, it is of great significance to study and establish fast and accurate virus detection methods.

[0004] Regarding virus detection methods, there are traditional detection methods and new nucleic acid molecule detection methods. Traditional diagnostic techniques (such as virus culture, hemagglutination inhibition test, enzyme immunoassay) were once the main means of pathogen detection. However, these methods are insensitive, time-consuming, and require amplification or reliance on professional technicians. In the context of the rapid development of viruses, faster detection methods are needed. Faced with a large number of clinical needs, new technologies have emerged to overcome the limitations of some traditional detection methods.

[0005] Magnetic beads are nanoscale magnetic materials that can aggregate under an applied electric field and can be easily separated from the reaction system. Quantum dots, as a semiconductor nanomaterial, are composed of chemical elements from Group III-IV or Group VI in the periodic table. They have superior fluorescence and optical stability, and are therefore widely used in the field of biological detection. Single molecule detection technology, as a new type of ultra-sensitive detection method, can study the conformational changes of biomolecules and the interactions between molecules at the single molecule level, and has broad application prospects in the field of molecular diagnosis. Compared with traditional analytical methods, single molecule detection technology can monitor the signals of single fluorescent molecules, allowing the observation of basic molecular properties in complex mixtures. It has significant advantages such as ultra-high sensitivity, good selectivity, fast analysis time and low sample consumption, and is regarded as an ideal method for rapid and simple quantification of low-concentration biomolecules. Since high-sensitivity detection can be achieved without nucleic acid amplification of the target gene, single molecule detection technology can greatly reduce the detection time and aerosol contamination risk of molecular diagnosis, and shows great potential in respiratory virus detection. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a virus nucleic acid detection probe, a virus detection system and application based on amplification-free single-molecule technology. The virus nucleic acid detection probe does not require constant temperature or variable temperature amplification of the target nucleic acid and can detect viruses simply and quickly.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a viral nucleic acid detection probe based on amplification-free single-molecule technology, wherein the viral nucleic acid detection probe is one or more of the following (1) to (4):

[0009] (1) The nucleic acid sequences of the capture probe and the reporter probe in the influenza A virus nucleic acid detection probe are shown in SEQ ID NO. 5 to SEQ ID NO. 6 respectively;

[0010] (2) The nucleic acid sequences of the capture probe and the reporter probe in the influenza B virus nucleic acid detection probe are shown in SEQ ID NO.7 to SEQ ID NO.8 respectively;

[0011] (3) The nucleic acid sequences of the capture probe and the reporter probe in the novel coronavirus nucleic acid detection probe targeting the N gene are shown in SEQ ID NO.9 to SEQ ID NO.10 respectively;

[0012] (4) The nucleic acid sequences of the capture probe and the reporter probe in the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene are shown in SEQ ID NO.11 to SEQ ID NO.12, respectively.

[0013] Preferably, the 5' end of the capture probe in the viral nucleic acid detection probe is modified with streptavidin magnetic beads; and the 3' end of the reporter probe in the viral nucleic acid detection probe is modified with streptavidin quantum dot microspheres.

[0014] The present invention provides a virus detection system, which comprises the above-mentioned virus nucleic acid detection probe.

[0015] Preferably, the detection system further comprises an RNA hybridization buffer.

[0016] Preferably, the detection system is 19-21 μL of RNA hybridization buffer, 0.8-1.2 μL of 0.8-1.2 μM capture probe, 0.8-1.2 μL of 0.8-1.2 μM reporter probe, and 0.8-1.2 μL of 0.8-1.2 μM RNA target sample, supplemented to 25 μL with DEPC; the capture probe and reporter probe are any one of the above-mentioned (1) to (4).

[0017] The present invention provides an application of the above-mentioned virus nucleic acid detection probe or virus detection system in the preparation of a virus detection product.

[0018] Preferably, the virus includes one or more of influenza A virus, influenza B virus and new coronavirus.

[0019] Preferably, the product comprises a reagent or a kit.

[0020] The present invention provides a method for detecting a virus, which is used for non-diagnostic purposes and comprises the following steps:

[0021] The above virus detection systems are mixed and reacted at 45°C for 2 hours to obtain hybridization products. The fluorescent signals are detected by non-denaturing polyacrylamide gel electrophoresis or an enzyme reader to determine whether the virus exists.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a virus nucleic acid detection probe, virus detection system and application based on amplification-free single-molecule technology. The virus nucleic acid detection probe of the present invention can realize the rapid, specific, accurate and sensitive identification of SARS-CoV-2, IAV (influenza A) and IBV (influenza B). The experimental results show that the detection limits of the novel coronavirus nucleic acid detection probe targeting the N gene and the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene are 81.419fmol / L and 157.85fmol / L respectively, and the detection limits of the influenza A virus nucleic acid detection probe for IAV and the influenza B virus nucleic acid detection probe for IBV are 176.359fmol / L and 148.229fmol / L respectively, and the clinical sample detection results show a good linear relationship with the virus RNA level detected by the virus nucleic acid detection probe. In short, the virus detection method using the virus nucleic acid detection probe of the present invention is a fast and reliable method. The target nucleic acid does not need constant temperature or variable temperature amplification, which can make the operation relatively simple and easy, reduce aerosol pollution, and is of great significance for clinical differential diagnosis of three respiratory viruses and epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Electrophoresis of the hybridization product of influenza A virus nucleic acid detection probe and target RNA, lane 1: capture probe; lane 2: reporter probe; lane 3: capture probe + reporter probe; lane 4: capture probe + target RNA; lane 5: reporter probe + target RNA; lane 6 or lane 7: capture probe + reporter probe + target RNA;

[0025] Figure 2 Electrophoresis of the hybridization product of influenza B virus nucleic acid detection probe and target RNA, lane 1: capture probe; lane 2: reporter probe; lane 3: capture probe + reporter probe; lane 4: capture probe + target RNA; lane 5: reporter probe + target RNA; lane 6 or lane 7: capture probe + reporter probe + target RNA;

[0026] Figure 3 Electrophoresis of the hybridization product of the novel coronavirus nucleic acid detection probe targeting the N gene and the target RNA, lane 1: capture probe; lane 2: reporter probe; lane 3: capture probe + reporter probe; lane 4: capture probe + target RNA; lane 5: reporter probe + target RNA; lane 6 or lane 7: capture probe + reporter probe + target RNA;

[0027] Figure 4Electrophoresis of hybridization products of the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene and the target RNA, lane 1: capture probe; lane 2: reporter probe; lane 3: capture probe + reporter probe; lane 4: capture probe + target RNA; lane 5: reporter probe + target RNA; lane 6 or lane 7: capture probe + reporter probe + target RNA;

[0028] Figure 5 It is a standard curve for detecting RNA target samples of different concentrations using influenza A virus nucleic acid detection probe;

[0029] Figure 6 It is a standard curve for detecting RNA target samples of different concentrations using influenza B virus nucleic acid detection probe;

[0030] Figure 7 A standard curve for detecting different concentrations of RNA target samples for the novel coronavirus nucleic acid detection probe targeting the N gene;

[0031] Figure 8 A standard curve for detecting different concentrations of RNA target samples for the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene;

[0032] Fig. 9 Heat map analysis of positive samples, negative samples, and interference samples for influenza A virus nucleic acid detection probes;

[0033] Fig.10 Heat map analysis of positive samples, negative samples, and interference samples detected by influenza B virus nucleic acid detection probe;

[0034] Fig.11 Heat map analysis of positive samples, negative samples, and interference samples for the novel coronavirus nucleic acid detection probe targeting the N gene;

[0035] Fig.12 Heat map analysis of positive samples, negative samples, and interference samples for the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene;

[0036] Fig.13 To analyze the correlation between the results of influenza A virus detection using influenza A virus nucleic acid detection probes and commercial RT-PCR kits;

[0037] Fig.14 To analyze the correlation between the results of influenza A virus detection using influenza B virus nucleic acid detection probes and commercial RT-PCR kits;

[0038] Fig.15To analyze the correlation between the results of novel coronavirus nucleic acid detection probe targeting the N gene and commercial RT-PCR kits for novel coronavirus detection;

[0039] Fig.16 To analyze the correlation between the results of detecting the new coronavirus using the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene and the commercial RT-PCR kit. DETAILED DESCRIPTION

[0040] The present invention designs capture probes and reporter probes for each virus based on the conserved sequences of influenza A virus M gene, influenza B virus NA gene, novel coronavirus N gene and Orf1ab gene.

[0041] The present invention provides a viral nucleic acid detection probe based on amplification-free single-molecule technology, wherein the viral nucleic acid detection probe is one or more of the following (1) to (4):

[0042] (1) The nucleic acid sequences of the capture probe and the reporter probe in the influenza A virus nucleic acid detection probe are shown in SEQ ID NO. 5 to SEQ ID NO. 6 respectively;

[0043] (2) The nucleic acid sequences of the capture probe and the reporter probe in the influenza B virus nucleic acid detection probe are shown in SEQ ID NO.7 to SEQ ID NO.8 respectively;

[0044] (3) The nucleic acid sequences of the capture probe and the reporter probe in the novel coronavirus nucleic acid detection probe targeting the N gene are shown in SEQ ID NO.9 to SEQ ID NO.10 respectively;

[0045] (4) The nucleic acid sequences of the capture probe and the reporter probe in the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene are shown in SEQ ID NO.11 to SEQ ID NO.12, respectively.

[0046] The 5' end of each capture probe in the above-mentioned viral nucleic acid detection probe of the present invention is modified with streptavidin magnetic beads; the 3' end of each reporter probe in the viral nucleic acid detection probe is modified with streptavidin quantum dot microspheres. The present invention uses the above-mentioned viral nucleic acid detection probe to detect viruses (such as one or more of the new coronavirus, influenza A virus and influenza B virus) with high accuracy, specificity and sensitivity, and does not require PCR amplification, making the detection simpler and faster.

[0047] The present invention provides a virus detection system, which comprises the above-mentioned virus nucleic acid detection probe.

[0048] In the present invention, the detection system also includes an RNA hybridization buffer. The RNA hybridization buffer is an RNA hybridization buffer containing deionized formamide, and the RNA hybridization buffer is purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091. As one embodiment, the detection system is 19-21 μL of RNA hybridization buffer, 08-1.2 μL of 0.8-1.2 μM capture probe, 0.8-1.2 μL of 0.8-1.2 μM reporter probe, 0.8-1.2 μL of 0.8-1.2 μM RNA target sample, supplemented to 25 μL with DEPC; the capture probe and the reporter probe are any one of the above (1) to (4). Further, the detection system is 20 μL of RNA hybridization buffer, 1 μL of 1 μM capture probe, 1 μL of 1 μM reporter probe, 1 μL of 1 μM RNA target sample, supplemented to 25 μL with DEPC; the capture probe and the reporter probe are any one of the above (1) to (4).

[0049] The present invention provides an application of the above-mentioned virus nucleic acid detection probe or virus detection system in the preparation of a virus detection product.

[0050] In the present invention, the virus includes one or more of influenza A virus, influenza B virus and novel coronavirus. The product includes a reagent or a kit.

[0051] The present invention provides a method for detecting a virus, which is used for non-diagnostic purposes and comprises the following steps:

[0052] The above virus detection systems are mixed and reacted at 45°C for 2 hours to obtain hybridization products. The fluorescent signals are detected by non-denaturing polyacrylamide gel electrophoresis or an enzyme reader to determine whether the virus exists.

[0053] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] Log in to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and download the M gene sequences of different subtypes of influenza A virus (IAV), the NA gene sequences of different subtypes of influenza B virus (IBV), the N gene sequences and Orf1ab gene sequences of different subtypes of the new coronavirus (SARS-CoV-2), and other respiratory virus sequences. Use DNAMAN software for multiple sequence alignment to find out the conservative and virus-specific regions and confirm the sequences. The conservative sequences of IAV, IBV and SARS-CoV-2 were respectively designed and synthesized using Primer5 software according to the basic principles of probe design to obtain capture probes and reporter probes for IAV, IBV and SARS-CoV-2.

[0057] The sequence information of influenza A virus M gene, influenza B virus NA gene, novel coronavirus N gene and Orf1ab gene are shown in Table 1.

[0058] Table 1 Sequence information of different target genes

[0059]

[0060] Capture probes and reporter probes for IAV, IBV, and SARS-CoV-2 are as follows:

[0061] For targeting the M gene, the nucleic acid sequence of the influenza A virus nucleic acid detection probe is:

[0062] Capture probe: 5'-CGTGCCCAGTGAGCGAGGACTGCA-3' (SEQ ID NO. 5);

[0063] Reporter probe: 5'-CGGCATCTTAGGATTTGTGTTCACGCTCA-3' (SEQ ID NO. 6).

[0064] For the targeted NA gene, the nucleic acid sequence of the influenza B virus nucleic acid detection probe is:

[0065] Capture probe: 5'-TGCAATTGCATCGGGGGAGAT TGTTAT-3' (SEQ ID NO. 7);

[0066] Reporter probe: 5'-AACAACATCCTAAGAACACAAGAAAGTGCC-3' (SEQ ID NO. 8).

[0067] Targeting the N gene, the nucleic acid sequence of the novel coronavirus nucleic acid detection probe is:

[0068] Capture probe: 5'-ACAATTTGCCCCCAGCGCTTCAG-3' (SEQ ID NO. 9);

[0069] Reporter probe: 5'-CGTTCTTCGGAATGTCGCGCATTGGCATGG-3' (SEQ ID NO. 10).

[0070] Targeting the Orf1ab gene, the nucleic acid sequence of the novel coronavirus nucleic acid detection probe is:

[0071] Capture probe: 5'-AGATGTCTTGTGCTGCCGGTA-3' (SEQ ID NO. 11);

[0072] Reporter probe: 5'-CTACACAAACTGCTTGCACTGATGACAAT-3' (SEQ ID NO. 12).

[0073] At the same time, the 5' end of the capture probe of each nucleic acid sequence such as SEQ ID NO. 5, 7, 9 and 11 is modified with streptavidin magnetic beads, and the 3' end of the reporter probe of each nucleic acid sequence such as SEQ ID NO. 6, 8, 10 and 12 is modified with streptavidin quantum dot microspheres.

[0074] Example 2

[0075] Optimization of conditions for detection using capture probes and reporter probes:

[0076] 100 μL of the mixed streptavidin-modified magnetic beads were taken out and coupled with 4 μL of 100 μM biotinylated capture probe (SEQ ID NO.5). The reaction buffer selected was Binding and washing buffer (B&W, purchased from Beijing Bio-Ray Biotechnology Co., Ltd., catalog number: BN20502). The reaction time was 10 min, and the coupling rate reached 89.02%.

[0077] 200 μL of streptavidin quantum dot microspheres were drawn into a 1.5 mL centrifuge tube, 5 μL of 100 μM biotinylated reporter probe (SEQ ID NO.6) was added, PBS buffer was added to 1 mL, and the mixture was reacted on a shaker in the dark for 20 min. The coupling rate was 84.19%.

[0078] Example 3

[0079] A virus nucleic acid detection system, comprising the following components:

[0080] 20 μL of RNA hybridization buffer containing deionized formamide (purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091), 1 μL of 1 μM capture probe, 1 μL of 1 μM reporter probe, 1 μL of 1 μM RNA target sample, and DEPC water were added to 25 μL;

[0081] The capture probe and the reporter probe are one of the following (1) to (4):

[0082] (1) When detecting whether the target RNA is influenza A virus, the nucleic acid sequences of the capture probe and the reporter probe are as shown in SEQ ID NOs. 5 to 6;

[0083] (2) When detecting whether the target RNA is influenza B virus, the nucleic acid sequences of the capture probe and the reporter probe are as shown in SEQ ID NOs. 7 to 8;

[0084] (3) When detecting whether it is a novel coronavirus target RNA, the nucleic acid sequences of the capture probe and the reporter probe are as shown in SEQ ID NOs. 9 to 10;

[0085] (4) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequences of the capture probe and the reporter probe are as shown in SEQ ID NOs. 11 to 12.

[0086] The RNA target sample is: the RNA artificially synthesized in Table 1.

[0087] Comparative Example 1

[0088] The difference between this comparative example and Example 3 is that this comparative example does not contain 1 μL of 1 μM reporter probe, and the detection system of this comparative example is composed of the following components:

[0089] 20 μL of RNA hybridization buffer containing deionized formamide (purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091), 1 μL of 1 μM capture probe, 1 μL of 1 μM RNA target sample were added, and DEPC water was added to 25 μL.

[0090] The capture probe is one of the following (1) to (4):

[0091] (1) When detecting whether the target RNA is influenza A virus, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.5;

[0092] (2) When detecting whether the target RNA is influenza B virus, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.7;

[0093] (3) When detecting whether it is a novel coronavirus target RNA, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.9;

[0094] (4) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.11.

[0095] The RNA target sample is shown in Example 3.

[0096] Comparative Example 2

[0097] The difference between this comparative example and Example 3 is that this comparative example does not contain 1 μL of 1 μM capture probe, and the detection system of this comparative example is composed of the following components:

[0098] 20 μL of RNA hybridization buffer containing deionized formamide (purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091), 1 μL of 1 μM reporter probe, 1 μL of 1 μM RNA target sample, and DEPC water were added to 25 μL;

[0099] The reporter probe is one of the following (1) to (4):

[0100] (1) When detecting whether it is influenza A virus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.6;

[0101] (2) When detecting whether it is influenza B virus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.8;

[0102] (3) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.10;

[0103] (4) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.12.

[0104] The RNA target sample is shown in Example 3.

[0105] Comparative Example 3

[0106] The difference between this comparative example and Example 3 is that this comparative example does not contain 1 μL of 1 μM RNA target sample, and the detection system of this comparative example is composed of the following components:

[0107] 20 μL of RNA hybridization buffer containing deionized formamide (purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091), 1 μL of 1 μM capture probe, 1 μL of 1 μM reporter probe, and DEPC water added to 25 μL.

[0108] Comparative Example 4

[0109] The difference between this comparative example and Example 3 is that this comparative example does not contain 1 μL of 1 μM capture probe and 1 μL of 1 μM RNA target sample. The detection system of this comparative example is composed of the following components:

[0110] 20 μL of RNA hybridization buffer containing deionized formamide (purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091), 1 μL of 1 μM reporter probe, and DEPC water added to 25 μL;

[0111] The reporter probe is one of the following (1) to (4):

[0112] (1) When detecting whether it is influenza A virus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.6;

[0113] (2) When detecting whether it is influenza B virus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.8;

[0114] (3) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.10;

[0115] (4) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequence of the reporter probe is as shown in SEQ ID NO.12.

[0116] Comparative Example 5

[0117] The difference between this comparative example and Example 3 is that this comparative example does not contain 1 μL of 1 μM reporter probe and 1 μL of 1 μM RNA target sample. The detection system of this comparative example is composed of the following components:

[0118] 20 μL of RNA hybridization buffer containing deionized formamide (purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091), 1 μL of 1 μM capture probe, and DEPC water added to 25 μL.

[0119] The capture probe is one of the following (1) to (4):

[0120] (1) When detecting whether the target RNA is influenza A virus, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.5;

[0121] (2) When detecting whether the target RNA is influenza B virus, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.7;

[0122] (3) When detecting whether it is a novel coronavirus target RNA, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.9;

[0123] (4) When detecting whether it is a new coronavirus target RNA, the nucleic acid sequence of the capture probe is as shown in SEQ ID NO.11.

[0124] Example 4 Application of viral nucleic acid detection probe

[0125] A method for detecting viruses using the detection system described in Example 3, comprising the following steps:

[0126] After mixing the components of each content in the detection system described in Example 3 and Comparative Examples 1 to 5, the mixture was reacted at 45° C. for 2 h to obtain a hybridization product, wherein the RNA target sample is the artificially synthesized RNA in Table 1, and the capture probe and the reporter probe are one of (1) to (4) in Example 3. The hybridization product was subjected to 10% non-denaturing polyacrylamide gel electrophoresis under the conditions of constant voltage 200 V and duration 90 min. Using the bubble staining method, 15 μL of nucleic acid dye was mixed with 50 mL of 0.7% sodium chloride to obtain a bubble staining solution, and then the gel was placed in the gel, and the gel was stained at room temperature for 30 min in a dark environment. The gel after bubble staining was observed on a UV imager.

[0127] Figure 1 to Figure 4 The results showed that the capture probe and reporter probe in the influenza A virus nucleic acid detection probe, influenza B virus nucleic acid detection probe, the new coronavirus nucleic acid detection probe targeting the N gene, and the new coronavirus nucleic acid detection probe targeting the Orf1ab gene can all bind to the target RNA, and the bands of the final hybridization products at 60bp, 57bp, 53bp and 50bp are the brightest and the migration speed is the slowest, indicating that the capture probe and the reporter probe have successfully hybridized with the target RNA. In addition, the third lane shows that the capture probe and the reporter probe are two separate bands, which will not produce non-specific binding, thereby avoiding interference with the background signal of subsequent experiments.

[0128] Example 5 Detection of the detection limit of viral nucleic acid detection probe

[0129] According to the RNA target samples described in Example 3, the RNA target samples of influenza A virus and influenza B virus were diluted into solutions with different concentration gradients, and the concentrations were diluted to 1 μM, 100nM, 10nM, 1nM, and 100pM. The RNA target samples of the new coronavirus were diluted into solutions with different concentration gradients, and the concentrations were diluted to 1 μM, 100nM, 10nM, 1nM, and 100pM for the detection of SARS-CoV-2 nucleic acid detection probes targeting the N gene. The RNA target samples of the new coronavirus were diluted into solutions with different concentration gradients, and the concentrations were diluted to 1 μM, 100nM, 1nM, 100pM, and 10pM for the detection of SARS-CoV-2 nucleic acid detection probes targeting the Orf1ab gene. At the same time, the detection target DEPC water was used as the blank control group. The detection system described in Example 3 was used to measure the fluorescence signal of different viruses using an ELISA instrument. The fluorescence difference between the positive sample and the blank sample was set as the ordinate, and the RNA target sample concentration was set as the abscissa to establish a standard curve.

[0130] In addition, 11 groups of blank control samples (target was replaced by DEPC water) were tested, and the ratio of their standard deviation to the slope of the standard curve was taken to calculate the detection limit (LOD). The calculation formula is as follows: LOD = σ / S, where σ is the standard deviation of the 11 groups of blank controls, and S is the slope of the standard curve, which is used to calculate and evaluate the detection limit of the virus nucleic acid detection probe method for detecting viruses described in Example 1.

[0131] from Figures 5 to 8 It can be seen that the concentration gradient of RNA target samples and the fluorescence difference show a good linear relationship.

[0132] Among them, the LODs of IAV nucleic acid detection probe, IBV nucleic acid detection probe, SARS-CoV-2 targeting N gene, and SARS-CoV-2 nucleic acid detection probe targeting Orf1ab gene were 176.359 fmol / L, 148.229 fmol / L, 81.419 fmol / L, and 157.85 fmol / L, respectively.

[0133] Example 6 Specificity detection of viral nucleic acid detection probes

[0134] In this example, positive and negative samples from clinically confirmed cases of influenza A and B and novel coronavirus infection were collected, and 5 nasal swab samples were taken from each case without contamination and repeated freezing and thawing.

[0135] In addition, 5 cases of clinical residual samples infected with Mycoplasma pneumoniae (MP), cytomegalovirus (CMV), parainfluenza virus (HPIV), Epstein-Barr virus, hepatitis B virus (HBV), hepatitis C virus (HCV), new Bunisia virus (SFTSV), monkeypox virus (MPXV), Mycobacterium tuberculosis (MTC), nontuberculous mycobacteria (NTM), Klebsiella pneumoniae (KP), Acinetobacter baumannii (ABA), and Pseudomonas aeruginosa (PA) were selected as interference samples. All samples used in this embodiment were provided by the Laboratory Department of Nanjing Second Hospital and approved by the Medical Ethics Committee of Nanjing Second Hospital (Ethics Approval No.: 2024-LS-ky-015).

[0136] 200 μL of the virus to be tested was extracted using a nucleic acid rapid extraction kit (magnetic bead method) (ShuoShi Bio, China) to obtain RNA target samples.

[0137] The nucleic acids of the positive samples, negative samples and interference samples were extracted respectively using the RNA target sample preparation method to obtain viral RNA.

[0138] Then, the mixture was mixed according to the detection system in Table 2, reacted in a 45°C metal bath for 120 min, and then placed on a magnetic separation rack. After standing for 120 s, the supernatant was discarded; 150 μL PBS buffer was added to wash the reactants, and the washing was repeated 3 times, and 150 μL PBS was added to form a complex to be detected.

[0139] Table 2 Detection system

[0140] Element concentration Volume (μL) Capture probe (MBs-CP) 1μM 75 Viral RNA - 24 Reporter probe (QDs-RP) 1μM 75 RNA Hybridization Buffer - Make up to 250 μL

[0141] In Table 2, the sequences of the capture probe and the reporter probe are respectively the influenza A virus nucleic acid detection probe, the influenza B virus nucleic acid detection probe, the novel coronavirus nucleic acid detection probe targeting the N gene, or the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene described in Example 1. The RNA hybridization buffer was purchased from Beijing Regen Biotechnology Co., Ltd., No. NH0091.

[0142] Fluorescence detection: The fluorescence signal of clinical positive samples, negative samples and interference samples was measured by using an ELISA instrument. The excitation wavelength of the fluorescence parameters was set to 488nm, and the emission wavelength was set to 610nm. The fluorescence signal intensity was scanned, measured and recorded. The data was collected and analyzed by heat map using GraphPad Prism software, which can intuitively show the differences in their fluorescence changes.

[0143] When the influenza A virus nucleic acid detection probe, the influenza B virus nucleic acid detection probe, the novel coronavirus nucleic acid detection probe targeting the N gene, or the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene described in Example 1 is used for detection, fluorescence appears at 610 nm, indicating that the sample to be tested contains influenza A virus, influenza B virus, or the novel coronavirus.

[0144] Figures 9 to 12 The results show that when the influenza A virus nucleic acid detection probe, influenza B virus nucleic acid detection probe, novel coronavirus nucleic acid detection probe targeting N gene or novel coronavirus nucleic acid detection probe targeting Orf1ab gene are used for detection, the difference between positive samples and other samples (negative samples and interference samples) is very obvious, and the data fluctuation within the group is not large, indicating that the influenza A virus nucleic acid detection probe, influenza B virus nucleic acid detection probe, novel coronavirus nucleic acid detection probe targeting N gene or novel coronavirus nucleic acid detection probe targeting Orf1ab gene have strong specificity and high stability in detecting the corresponding viruses.

[0145] Example 7

[0146] Ten cases of influenza A and B viruses and 10 cases of novel coronavirus were selected, and commercial RT-PCR fluorescence diagnostic kits (Cepheid, USA) were used to detect influenza A and B viruses, and commercial RT-PCR fluorescence diagnostic kits (Shuo Shi Biology, China) were used to detect novel coronavirus. Ct values ​​below 40 were considered positive. Viral nucleic acids with different Ct values ​​were collected and detected using the detection method described in Example 5 (single molecule amplification-free method). The excitation wavelength of the fluorescence parameters was set to 488 nm, and the emission wavelength was set to 610 nm. The fluorescence signal intensity was scanned, measured and recorded, and a curve was established with the Ct value obtained by the PCR method.

[0147] Figure 13 to Figure 16 The results show that the Ct values ​​of the influenza A virus nucleic acid detection probe, influenza B virus nucleic acid detection probe, novel coronavirus nucleic acid detection probe targeting N gene or novel coronavirus nucleic acid detection probe targeting Orf1ab gene detected by the present invention are well correlated with the commercial RT-PCR results, indicating that the virus nucleic acid detection probe of the present invention has high accuracy in detecting viruses, and the present invention does not require PCR amplification to directly detect sample RNA, and the detection method is simple and fast.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A viral nucleic acid detection probe based on amplification-free single-molecule technology, characterized in that: The viral nucleic acid detection probe is one or more of the following (1) to (4): (1) The nucleic acid sequences of the capture probe and the reporter probe in the influenza A virus nucleic acid detection probe are shown in SEQ ID NO.5 to SEQ ID NO.6 respectively; (2) The nucleic acid sequences of the capture probe and the reporter probe in the influenza B virus nucleic acid detection probe are shown in SEQ ID NO.7 to SEQ ID NO.8 respectively; (3) The nucleic acid sequences of the capture probe and the reporter probe in the novel coronavirus nucleic acid detection probe targeting the N gene are shown in SEQ ID NO.9 to SEQ ID NO.10 respectively; (4) The nucleic acid sequences of the capture probe and the reporter probe in the novel coronavirus nucleic acid detection probe targeting the Orf1ab gene are shown in SEQ ID NO.11 to SEQ ID NO.12, respectively.

2. The viral nucleic acid detection probe according to claim 1, characterized in that: The 5' end of the capture probe in the viral nucleic acid detection probe is modified with streptavidin magnetic beads; the 3' end of the reporter probe in the viral nucleic acid detection probe is modified with streptavidin quantum dot microspheres.

3. A virus detection system, characterized in that: The detection system comprises the viral nucleic acid detection probe according to claim 1 or 2.

4. The virus detection system according to claim 3, characterized in that: The detection system also includes an RNA hybridization buffer.

5. The virus detection system according to claim 3 or 4, characterized in that: The detection system comprises 19-21 μL of RNA hybridization buffer, 0.8-1.2 μL of 0.8-1.2 μM capture probe, 0.8-1.2 μL of 0.8-1.2 μM reporter probe, and 0.8-1.2 μL of 0.8-1.2 μM RNA target sample, which is supplemented to 25 μL with DEPC; the capture probe and reporter probe are any one of (1) to (4) of claim 1 or 2.

6. Use of the viral nucleic acid detection probe according to claim 1 or 2 or the virus detection system according to any one of claims 3 to 5 in the preparation of a virus detection product.

7. The use according to claim 6, characterized in that: The viruses include one or more of influenza A virus, influenza B virus and new coronavirus.

8. The use according to claim 6, characterized in that: The product comprises a reagent or a kit.

9. A method for detecting a virus, characterized in that: The method is for non-diagnostic purposes and comprises the following steps: The virus detection system according to any one of claims 3 to 5 is mixed, reacted at 45° C. for 2 hours to obtain a hybridization product, and the fluorescent signal is detected by non-denaturing polyacrylamide gel electrophoresis or an enzyme reader to determine whether the virus exists.

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