Primer, crRNA, kit and method for detecting respiratory viruses

Abstract

The present invention provides a primer, crRNA, kit and method for detecting respiratory viruses, wherein the primer and crRNA are used to detect influenza A virus, influenza B virus, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, respiratory syncytial virus, novel coronavirus, human parainfluenza virus serotype 3, and human metapneumovirus 9 different respiratory viruses at one time. On the one hand, the present invention utilizes RPA isothermal amplification technology to amplify multiple targets, and designs a crRNA combination of CRISPR‑Cas12a for target sequences, which has high specificity and sensitivity; on the other hand, the present invention uses a multi-channel microfluidic chip as a carrier for simultaneous amplification and identification, which effectively simplifies the operation and shortens the detection time.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a primer, crRNA, a kit and a method for detecting respiratory viruses. Background Art

[0002] Respiratory tract infection (RTI) is one of the main causes of acute and fatal diseases in humans. It can occur in any gender, age and region, and the disease burden caused is higher than that caused by other causes. The latest data released by the World Health Organization (WHO) shows that lower respiratory tract infection ranks fourth among the major causes of death in the world. In 2019, 2.6 million people died from respiratory tract infection, a decrease of 460,000 compared to 2000. Although the number of deaths has dropped significantly in the past decade, the harm is still serious.

[0003] Viral infection is a very important cause of RTI. About 80% of RTI are caused by viruses. In developed countries, acute viral respiratory infection is the leading cause of hospitalization for infants and children. In developing countries, acute viral respiratory infection is the main cause of death. Respiratory viruses refer to a type of virus that can invade the respiratory tract and cause local lesions in the respiratory tract or only use the respiratory tract as an invasion portal, mainly causing lesions in respiratory tissues and organs. Typical respiratory viruses include influenza A virus (FLUAV), influenza B virus (FLUBV), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), respiratory syncytial virus (HRSV), new coronavirus (SARS-CoV-2), human parainfluenza virus serotype 3 (HPIV-3) and human metapneumovirus (HMPV).

[0004] Respiratory viruses are mostly transmitted in the form of aerosols and air droplets, which are highly contagious. Patients mainly show symptoms of upper respiratory tract infection such as fever, cough and runny nose, and the condition is mild and can heal itself. But at the same time, there are also some diseases caused by respiratory viruses, which are highly contagious and lethal. Patients show lower respiratory tract infection and induce diseases such as bronchiolitis, pneumonia and asthma, and even lead to systemic multiple organ failure. Since the clinical manifestations and biochemical indicators of patients after infection are very similar, differential diagnosis is very difficult. In addition, about 5% to 10% of patients have mixed infections of multiple respiratory viruses, which makes etiological diagnosis difficult. Therefore, early rapid and accurate identification of infectious viruses, especially those that are highly contagious and prone to cause severe RTI, and rapid screening of a large number of pathogens through multiple pathogen detection can provide the best intervention opportunity for clinical treatment, reduce the abuse of antibiotics, and inhibit the spread of viruses through corresponding preventive measures, providing strong support for disease treatment, epidemiological investigation and epidemic control.

[0005] In recent years, nucleic acid-based human respiratory virus detection methods have continued to emerge and develop, and the outbreak of the new coronavirus (SARS-CoV-2) has further promoted the emergence of some new technologies. Recombinase polymerase amplification (RPA) is a nucleic acid isothermal amplification technology that has emerged in recent years. It only takes about 37°C and 15 minutes to amplify nucleic acids at a single molecule concentration level to a concentration level that can be detected by existing methods. Moreover, this technology has very low requirements for hardware equipment, so it plays an important role in the detection of pathogenic bacteria, viruses and other microorganisms. However, although the amplification method of RPA is relatively simple, since RPA is an emerging technology, unlike PCR, there is no corresponding software to assist in the design of primers, so its primer design often has the risk of failure. However, there are some generally recognized experiences in RPA primer design: the primer length is generally 30-35nt; the 3-5 nucleotides at the 5' end should avoid polyguanine, and the 3 nucleotides at the 3' end, if they are guanine and cytosine, will help stabilize the binding of the polymerase and improve the amplification performance of the primer; it is best not to have long strings of polypurine or polypyrimidine in the primer, and GC content that is too high (>70%) or too low (<30%) is not conducive to RPA amplification; in addition, when designing primers, the formation of secondary structures, primer-primer interactions, and hairpin structure sequences should be avoided as much as possible to minimize the formation of primer dimers. However, even with the guidance of the above primer design rules, effective primers mainly rely on large-scale synthesis and screening, especially when detecting multi-target nucleic acids, more tests are needed to ensure that each target can be successfully amplified. The company that invented the RPA technology has made great progress in their products ( DNA amplification kit) analysis design manual states: "Oligonucleotides of different sequences are There are different performances in the reaction, but there is no fixed rule to predict the performance of a specific amplification primer based on the order and composition of the nucleotides. "Even small changes in the oligonucleotide sequence can sometimes lead to significant changes in primer activity." It can be seen that many attempts must be made to obtain RPA primers with good amplification effects.

[0006] In order to achieve RPA multi-target (>4) amplification and detection, many difficulties need to be overcome. In addition to the above primer design and target region selection, the total amount of primers in the RPA reaction should also be paid attention to when performing multi-target amplification; as long as more than two pairs of amplification primers are used in the reaction, the amount of different primers should be controlled and optimized, otherwise primer dimers are likely to appear, leading to amplification failure.

[0007] Recent studies have shown that Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology can be used for low-cost, portable molecular detection of pathogens. CRISPR and CRISPRassociated genes (Cas) genes are the immune systems of bacteria to resist exogenous viral infections. The modular nature of CRISPR makes this technology widely used in genome engineering. CRISPR-based molecular diagnosis of pathogen nucleic acids depends on the targeting activity of Cas nuclease RNA or DNA. In addition, based on CRISPR-Cas12a pathogen detection technology, a variety of in vitro detection methods have been developed (Biosensors and Bioelectronics, 2021, 187, 113292; Nature Reviews Molecular Cell Biology, 2019, 20, 490), all of which show that this method has the advantages of high sensitivity, accurate detection, and easy operation; further combining it with technologies such as constant temperature amplification, it can achieve as low as 10 for target nucleic acids in non-laboratory environments. -18 M level detection. However, these studies mainly target one virus or bacteria. Faced with complex and changeable real-world needs, multi-target nucleic acid detection is of greater significance. For multi-target detection, especially subtypes with similar target sequences, special attention should be paid when designing crRNA to avoid cross-recognition of another subtype by crRNA designed for one subtype, resulting in erroneous detection. Therefore, the design of crRNA for respiratory viruses must not only meet some requirements for identifying the corresponding viruses, but also ensure its high specificity and avoid misidentifying other viruses. Certain difficulties need to be overcome to achieve the expected goals. Summary of the invention

[0008] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and to provide a primer, crRNA, kit and method for detecting respiratory viruses that do not rely on large-scale detection instruments, can detect a variety of respiratory viruses under mild conditions, and have high specificity and sensitivity.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] An RPA primer combination for amplifying nucleic acids of respiratory viruses, wherein the respiratory viruses include influenza A virus, influenza B virus, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, respiratory syncytial virus, novel coronavirus, human parainfluenza virus serotype 3, and human metapneumovirus;

[0011] Among them, the nucleotide sequence for amplifying influenza A virus is shown in SEQ ID 1-2, the nucleotide sequence for amplifying influenza B virus is shown in SEQ ID 3-4, the nucleotide sequence for amplifying human coronavirus NL63 is shown in SEQ ID 5-6, the nucleotide sequence for amplifying human coronavirus OC43 is shown in SEQ ID 7-8, the nucleotide sequence for amplifying human coronavirus HKU1 is shown in SEQ ID 9-10, the nucleotide sequence for amplifying respiratory syncytial virus is shown in SEQ ID 11-12, the nucleotide sequence for amplifying new coronavirus is shown in SEQ ID 13-14, the nucleotide sequence for amplifying human parainfluenza virus serotype 3 is shown in SEQ ID 15-16, and the nucleotide sequence for amplifying human metapneumovirus is shown in SEQ ID 17-18.

[0012] A crRNA combination for detecting respiratory viruses, wherein the nucleotide sequence for detecting influenza A virus is shown in SEQ ID 19, the nucleotide sequence for detecting influenza B virus is shown in SEQ ID 20, the nucleotide sequence for detecting human coronavirus NL63 is shown in SEQ ID 21, the nucleotide sequence for detecting human coronavirus OC43 is shown in SEQ ID 22, the nucleotide sequence for detecting human coronavirus HKU1 is shown in SEQ ID 23, the nucleotide sequence for detecting respiratory syncytial virus is shown in SEQ ID 24, the nucleotide sequence for detecting new coronavirus is shown in SEQ ID 25, the nucleotide sequence for detecting human parainfluenza virus serotype 3 is shown in SEQ ID 26, and the nucleotide sequence for detecting human metapneumovirus is shown in SEQ ID 27.

[0013] A kit for detecting respiratory viruses, comprising the aforementioned RPA primer combination and crRNA combination.

[0014] The kit also includes a Cas12a protein and a reporter molecule, wherein the reporter molecule is one of a single-stranded DNA, a DNA hairpin structure, a DNA G-triplex, and a DNA G-quadruplex.

[0015] The kit also includes a multi-channel microfluidic chip, wherein an injection hole is provided at the center of the microfluidic chip, and a plurality of outlet holes are provided at the periphery of the microfluidic chip, and the injection hole is connected with the outlet holes through a plurality of microchannels.

[0016] The kit also includes an amplification reaction solution and an enzyme digestion buffer.

[0017] A detection method for a kit for detecting respiratory viruses, comprising: first amplifying a sample to be tested using the RPA primer combination, then mixing the RPA amplification product, Cas12a protein, a reporter molecule and a crRNA combination, and then performing an enzyme cleavage reaction, and then judging the type of respiratory virus according to the change in the fluorescence signal of the reporter molecule.

[0018] During amplification, the total concentration of the RPA primer combination is 0.5-1.5 μM.

[0019] The kit also includes a multi-channel microfluidic chip, wherein an injection hole is provided at the center of the microfluidic chip, and a plurality of outlet holes are provided at the periphery of the microfluidic chip, and the injection hole is connected to the outlet holes through a plurality of microchannels;

[0020] The Cas12a protein, reporter molecule and crRNA combination are preloaded on the outlet hole, and the sample to be tested and the RPA primer combination are injected from the injection hole and diverted to each outlet hole, and then mixed with the Cas12a protein, reporter molecule and crRNA combination for amplification and enzyme cleavage reactions at the same time.

[0021] The amplification temperature is 37-40°C and the time is 15-25 minutes;

[0022] The temperature of the enzyme cleavage reaction is 36-38° C., and the time is 15-20 minutes.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides an RPA primer combination for amplifying nucleic acid of respiratory viruses, which targets influenza A virus, influenza B virus, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, respiratory syncytial virus, new coronavirus, human parainfluenza virus serotype 3, and human metapneumovirus. The constant temperature amplification technology of RPA is used, and no complex PCR instrument is required. Only a simple heating device is required to perform multi-target amplification under mild conditions, which can effectively amplify the corresponding subtype fragments and will not cross-amplify non-target subtypes. Therefore, the present invention does not rely on large-scale detection instruments, and can achieve specific detection of multiple respiratory viruses under mild conditions.

[0025] 2. The present invention provides a crRNA combination for detecting respiratory viruses, which designs the crRNA combination of CRISPR-Cas12a for the target sequence, and uses crRNA to specifically recognize the target, which can further improve the specificity of the detection, while Cas12a protein achieves the effect of amplifying the signal by shearing the reporter molecule, so that the sensitivity and specificity are greatly improved. Therefore, the present invention has high specificity and sensitivity.

[0026] 3. The present invention provides a kind of test kit for detecting respiratory virus, the test kit includes RPA primer combination, crRNA combination and multi-channel microfluidic chip, the center of microfluidic chip is provided with injection hole, the periphery of microfluidic chip is provided with multiple outlet holes, injection hole is communicated with outlet hole by multiple microchannels, Cas12a protein, reporter molecule and crRNA combination can be preloaded on outlet hole, sample to be tested, RPA primer combination are injected by injection hole and shunted to each outlet hole, and Cas12a protein, reporter molecule and crRNA combination are mixed and amplified and enzyme digestion reaction is carried out simultaneously, compared with the conventional nucleic acid detection process of amplification and detection first, by making amplification and detection reaction simultaneously on chip, it is conducive to simplifying operation, shortening detection time, and at the same time, Cas12a protein and crRNA combination can be preloaded in outlet hole and freeze-dried, so as to facilitate storage, transportation and on-site use. Therefore, the present invention not only effectively simplifies operation, shortens detection time, but also is easy to transport and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the PAGE diagram of the amplification products of the first round of RPA primers for 9 respiratory virus subtypes.

[0028] Figure 2 This is the PAGE diagram of the RPA amplification products of the 9 screened respiratory viruses.

[0029] Figure 3 This is the predicted secondary structure of candidate crRNA of influenza A virus.

[0030] Figure 4 This is the predicted secondary structure of candidate crRNA of influenza B virus.

[0031] Figure 5 This is the predicted secondary structure of the candidate crRNA of human coronavirus NL63.

[0032] Figure 6 This is the predicted secondary structure of the candidate crRNA of human coronavirus OC43.

[0033] Figure 7 This is the predicted secondary structure of candidate crRNA of respiratory syncytial virus.

[0034] Figure 8 This is the predicted secondary structure of the candidate crRNA of human coronavirus HKU1.

[0035] Fig. 9 This is the predicted secondary structure of the candidate crRNA of the new coronavirus.

[0036] Fig.10 This is the predicted secondary structure of the candidate crRNA of human parainfluenza virus serotype 3.

[0037] Fig.11 This is the predicted secondary structure of candidate crRNA for human metapneumovirus.

[0038] Fig.12 This is a cross-test result diagram of the first crRNA and the fifth crRNA of FLUAV.

[0039] Fig.13 The results of simultaneous amplification and crRNA identification of 9 respiratory viruses.

[0040] Fig.14 The results of simultaneous amplification and crRNA recognition detection of 8 respiratory viruses on a microfluidic chip. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0042] The present invention provides a kit and a detection method, which can achieve complete and interactive specific detection of influenza A virus, influenza B virus, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, respiratory syncytial virus, novel coronavirus, human parainfluenza virus serotype 3 and human metapneumovirus while ensuring sensitivity. The method has the advantages of simple operation, small sample dosage, fast analysis speed, and no reliance on expensive instruments, and is suitable for screening and diagnosis of respiratory virus infection in various medical institutions, especially grassroots hospitals and clinics.

[0043] The present invention realizes the identification of multiple respiratory viruses and mainly involves two core parts:

[0044] 1) Design and screening of RPA primers. We compared the sequences of the respiratory virus genes to be tested, found the characteristic regions in the gene sequences of the respiratory viruses, and then designed multiple pairs of RPA primers in a targeted manner. These primers should not only effectively amplify the corresponding subtype fragments, but also prevent cross-amplification of non-target subtypes. At the same time, they should also meet the crRNA design principles of downstream CRISPR-Cas12a. We repeatedly verified and screened to find the best RPA primers.

[0045] 2) Design and verification of crRNA. It is necessary to find suitable regions in the fragment regions of multiple subtypes to be amplified to design a combination of crRNAs. Since crRNAs may cross-recognize other targets (especially when the nucleic acid sequences of respiratory viruses are highly similar, it is easy for primers or crRNAs designed for one subtype to cross-recognize another or multiple subtypes), and a PAM sequence (TTTN) is required upstream of the target recognition region, a large number of comparisons and optimization designs are required. After the design, verification is also required to ensure that it can recognize the corresponding virus while not mistakenly identifying other viruses.

[0046] In the present invention, Cas12a protein was purchased from Guangzhou Meige Biotechnology Co., Ltd., 9 respiratory virus gene plasmids, primers, and TBA11-FQ were provided by Beijing Qingke Xinye Biotechnology Co., Ltd., and crRNA was provided by Sangon Biotechnology Co., Ltd.

[0047] Example 1: Respiratory virus target sequence alignment

[0048] DNAMAN software was used to compare the target sequences of nine respiratory viruses to be detected, including influenza A virus, influenza B virus, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, respiratory syncytial virus, new coronavirus, human parainfluenza virus serotype 3, and human metapneumovirus. The comparison results showed that the similarity between the target sequences of the nine viruses was not high, and each target had a sufficiently specific region for us to design RPA primers and crRNA.

[0049] Example 2: RPA primer design and optimization

[0050] In this example, the target gene sequences of 9 respiratory viruses were compared and the corresponding regions were found. Based on this, 43 pairs of RPA primers were designed (as shown in Table 1). According to the instructions of the RPA kit (TwsitDx basic version), the target genes of 9 respiratory viruses were amplified (heated at 39°C for 20 minutes), and then the amplified products were analyzed by gel electrophoresis (PAGE). The results are shown in Figure 1 It can be seen that some primers can amplify a clear single band, while some primers cannot amplify a band or amplify two bands.

[0051] According to the PAGE analysis results, the primers corresponding to the singlest, clearest and thickest target bands were selected (as shown in Table 2), and these primers were used to amplify the corresponding targets. Then, the amplified products were analyzed by gel electrophoresis (PAGE). The results are shown in Figure 2 The results showed that the amplified bands of each respiratory virus were clear and single. Therefore, this RPA primer combination was used as the RPA primer for efficiently and specifically amplifying the corresponding targets of 9 respiratory viruses.

[0052] Table 1 RPA primers designed for 9 respiratory viruses

[0053]

[0054]

[0055]

[0056] Table 2 Screened RPA primers

[0057]

[0058]

[0059] Example 3: crRNA design and optimization of 9 respiratory viruses

[0060] This example uses CRISPR technology to detect the target nucleic acid RPA amplification product. Specifically, the CRISPR-Cas12a system can activate its trans-cleavage activity after crRNA is paired with the target, and the reporter molecule double-labeled with fluorescent and quenching groups is cleaved, thereby releasing strong fluorescence for detection.

[0061] Since the present invention is aimed at the detection of multiple respiratory viruses, multiple candidate sequences that meet the basic principles of crRNA design (i.e., containing the PAM sequence "TTTN" on the target chain) are found for each target amplification region; then check whether the 20 nucleotides after the PAM sequence do not contain "AAA". Based on this, we designed 48 crRNA sequences as shown in Table 3, and then used mFold to predict the secondary structures of these 48 crRNAs. The results are as follows Figure 3-Figure 11 shown.

[0062] Table 3 Respiratory virus crRNA candidate sequences

[0063]

[0064]

[0065]

[0066] Based on the secondary structure prediction results of 48 crRNAs, 9 crRNAs were selected in which the repeat region successfully formed a hairpin loop and the spacer region did not form a secondary structure as much as possible or only formed an unstable secondary structure (as shown in Table 4).

[0067] Table 4 9 kinds of crRNA screened

[0068]

[0069] After determining the crRNA corresponding to each virus, its recognition effect was verified through a 9×9 orthogonal experiment. In a 96-well plate, 1 μL of Cas12a protein (2 μM) and 27 μL of buffer (10 mM Tris, pH 7.9, 70 mM KCl, 10 mM MgCl2) were mixed with 2 μL of FLUBV PB1, HCoV-NL63 Orf1ab, HCoV-OC43 Orf1ab, HRSV M, HCoV-HKU1 Orf1ab, SARS-CoV-2 Orf1ab, HPIV-3M and HMPV F crRNA (1 μM) (three replicate wells were configured for each subtype), and incubated at 37 ° C for 10 min; then 10 μL of FLUBV PB1, HCoV-NL63 Orf1ab, HCoV-OC43 Orf1ab, HRSV M, HCoV-HKU1 Orf1ab, SARS-CoV-2 Orf1ab, HPIV-3M and HMPV F were added to the above system accordingly. F (synthetic plasmid, 40 ng / μL) and 10 μL TBA11-FQ (100 μM) were incubated at 37 ° C for 15 minutes; then heated at 65 ° C for 10 minutes to inactivate the Cas12a protein; finally, the fluorescence value was measured with a microplate reader (488 nm excitation, 500-600 nm collection), and the fluorescence at 518 nm was taken out (the average of 3 replicate wells for each subtype) for plotting.

[0070] Fig.12 The cross-test results of the first crRNA (left figure, crRNA-1) and the fifth crRNA (left figure, crRNA-5) of FLUAV are shown. It can be seen from the figure that the recognition efficiency and specificity of crRNA-1 are not very good; for crRNA-5, when it coexists with the corresponding HPV subtype (i.e., at the diagonal), the fluorescence value is very strong, and when the other two are not corresponding, the fluorescence value is very low (although there are very few values ​​that are slightly higher, but it does not affect the judgment). It can be seen that the crRNA designed for 9 respiratory viruses has good specificity.

[0071] Example 4: Simultaneous amplification and crRNA recognition of 9 respiratory viruses

[0072] In this example, the mixed plasmid sample containing the above 9 respiratory viruses was amplified by RPA using the selected RPA primer combination (amplification at 39°C for 20 minutes, the total concentration of the RPA primer combination was 1.0 μM), and then the 9 selected crRNAs were identified (incubated at 37°C for 15 minutes). The results are as follows: Fig.13 shown.

[0073] Fig.13The fluorescence test results in the results show that when the corresponding target exists in the reaction system, it can be specifically recognized by the corresponding crRNA. This result once again proves that the primers we designed can accurately amplify 9 respiratory viruses, and the designed crRNA can achieve high specific recognition without obvious cross-recognition.

[0074] Example 5: Simultaneous amplification and crRNA recognition detection on a microfluidic chip

[0075] A plasmid mixture of 8 respiratory viruses, FLUBV PB1, HCoV-NL63 Orf1ab, HCoV-OC43 Orf1ab, HRSV M, HCoV-HKU1Orf1ab, SARS-CoV-2 Orf1ab, HPIV-3 M and HMPV F, was used as the sample to be tested, and 5 μL was added to the amplification system (the primers corresponding to the above 8 respiratory viruses were mixed at the same concentration so that the total primer concentration in the amplification solution was 1 μM), and Cas12a (5 μL, 2 μM) and crRNA of 8 respiratory viruses (10 μL, 1 μM) and 5 μL buffer were incubated at 37 ° C for 10 min, and TBA11-FQ reporter molecules (1 μL, 40 μM) were added to the incubated 8 solutions, and 4 μL of reaction solution was added to the outlet holes 1-24 in sequence after mixing (such as FLUBV was added to the outlet holes 1-3). PB1 solution, HCoV-NL63 Orf1ab subtype solution was added to wells 4-6); 4 μL of solution without any Cas12a / crRNA but containing equal concentration of TBA11-FQ reporter molecules was added to wells 25-27 as a negative control. Then the amplification system with the sample to be tested was injected into the microfluidic chip from the injection hole, and the chip was placed in a 37°C metal bath for incubation for 15 minutes; then the green fluorescence photos of each well were taken under a microscope and fluorescence analysis was performed. The results are as follows Fig.14 As shown. It can be seen that the primers we designed can accurately amplify 8 respiratory viruses, and the designed crRNA can achieve high specific recognition without obvious cross-recognition.

Claims

1. An RPA primer combination for detecting respiratory viruses, characterized in that: The respiratory viruses include influenza A virus, influenza B virus, human coronavirus NL63, human coronavirus OC43, human coronavirus HKU1, respiratory syncytial virus, new coronavirus, human parainfluenza virus serotype 3, and human metapneumovirus; In the RPA primer combination, the nucleotide sequence for amplifying influenza A virus is shown in SEQ ID NOs.1-2, the nucleotide sequence for amplifying influenza B virus is shown in SEQ ID NOs.3-4, the nucleotide sequence for amplifying human coronavirus NL63 is shown in SEQ ID NOs.5-6, the nucleotide sequence for amplifying human coronavirus OC43 is shown in SEQ ID NOs.7-8, the nucleotide sequence for amplifying human coronavirus HKU1 is shown in SEQ ID NOs.9-10, the nucleotide sequence for amplifying respiratory syncytial virus is shown in SEQ ID NOs.11-12, the nucleotide sequence for amplifying new coronavirus is shown in SEQ ID NOs.13-14, the nucleotide sequence for amplifying human parainfluenza virus serotype 3 is shown in SEQ ID NOs.15-16, and the nucleotide sequence for amplifying human metapneumovirus is shown in SEQ ID NOs.17-18.

2. A kit for detecting respiratory viruses, comprising an RPA primer combination and a crRNA combination, characterized in that: The RPA primer combination is as shown in claim 1. In the crRNA combination, the nucleotide sequence for detecting influenza A virus is shown in SEQ ID NO.19, the nucleotide sequence for detecting influenza B virus is shown in SEQ ID NO.20, the nucleotide sequence for detecting human coronavirus NL63 is shown in SEQ ID NO.21, the nucleotide sequence for detecting human coronavirus OC43 is shown in SEQ ID NO.22, the nucleotide sequence for detecting human coronavirus HKU1 is shown in SEQ ID NO.23, the nucleotide sequence for detecting respiratory syncytial virus is shown in SEQ ID NO.24, the nucleotide sequence for detecting new coronavirus is shown in SEQ ID NO.25, the nucleotide sequence for detecting human parainfluenza virus serotype 3 is shown in SEQ ID NO.26, and the nucleotide sequence for detecting human metapneumovirus is shown in SEQ ID NO.

27.

3. A kit for detecting respiratory viruses according to claim 2, characterized in that: The kit also includes Cas12a protein and a reporter molecule, wherein the reporter molecule is TBA11-FQ.

4. A kit for detecting respiratory viruses according to claim 2, characterized in that: The kit also includes a multi-channel microfluidic chip, wherein an injection hole is provided at the center of the microfluidic chip, and a plurality of outlet holes are provided at the periphery of the microfluidic chip, and the injection hole is connected with the outlet holes through a plurality of microchannels.

5. A kit for detecting respiratory viruses according to claim 2, characterized in that: The kit also includes an amplification reaction solution and an enzyme digestion buffer.

6. A detection method for non-diagnostic purposes of the kit for detecting respiratory viruses according to claim 2, characterized in that: The detection method comprises: firstly amplifying the sample to be tested by using the RPA primer combination, then mixing the RPA amplification product, Cas12a protein, reporter molecule and crRNA combination for enzyme cleavage reaction, and then judging the type of respiratory virus according to the change of the fluorescence signal of the reporter molecule.

7. The non-diagnostic detection method of a kit for detecting respiratory viruses according to claim 6, characterized in that: During amplification, the total concentration of the RPA primer combination is 0.5-1.5 μM.

8. The non-diagnostic detection method of a kit for detecting respiratory viruses according to claim 6, characterized in that: The kit also includes a multi-channel microfluidic chip, wherein an injection hole is provided at the center of the microfluidic chip, and a plurality of outlet holes are provided at the periphery of the microfluidic chip, and the injection hole is connected to the outlet holes through a plurality of microchannels; The Cas12a protein, reporter molecule and crRNA combination are preloaded on the outlet hole, and the sample to be tested and the RPA primer combination are injected from the injection hole and diverted to each outlet hole, and then mixed with the Cas12a protein, reporter molecule and crRNA combination for amplification and enzyme cleavage reactions at the same time.

9. The non-diagnostic detection method of a kit for detecting respiratory viruses according to claim 6, characterized in that: The amplification temperature is 37-40°C and the time is 15-25 minutes; The temperature of the enzyme cleavage reaction is 36-38° C., and the time is 15-20 minutes.

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