A primer combination for detecting Nobecovirus subgenus coronaviruses and its application

Abstract

This invention relates to the field of virus detection technology, and more particularly to a detection method. Nobecovirus Primer combinations for subgenus coronaviruses and their applications. The primer combinations comprise the nucleotide sequences shown in SEQ ID NO.1–SEQ ID NO.14. The applications include: universal detection. Nobecovirus This invention studies and screens a set of subgenus coronaviruses, or distinguishes between HKU9 coronavirus, GCCDC1 coronavirus, or C704 coronavirus, or prepares kits for the aforementioned detections. Nobecovirus Primer combinations for detecting subgenus coronaviruses and different viral species can accurately and efficiently achieve detection of different viruses within a single reaction system. Nobecovirus The specific subtypes of coronaviruses have important application value.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and more particularly to a detection method. Nobecovirus Primer combinations for subgenus coronaviruses and their applications. Background Technology

[0002] Bats, as natural hosts of many coronaviruses, play a crucial role in the ecology and evolution of coronaviruses. Nobecovirus The subgenus belongs to the genus β-coronavirus, which includes three viral species: Eidolon bat coronavirus C704, Rousettus bat coronavirus GCCDC1, and Rousettus bat coronavirus HKU9. Nobecovirus Subgenus coronaviruses exhibit high genetic diversity, exist stably in multiple bat species, and have a wide host distribution, closely related to humans, poultry, and livestock. Nobecovirus Subgenus coronaviruses may evolve into novel, highly pathogenic coronaviruses and pose a risk of cross-species transmission. Currently, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Nobecovirus Detection methods for subgenus coronaviruses have not yet been established, therefore, for Nobecovirus Establishing simple and specific laboratory nucleic acid detection methods for subgenus coronaviruses is essential. This is particularly important for bats. Nobecovirus Effective monitoring and research are crucial for understanding the origins and evolution of coronaviruses and for early warning of emerging infectious diseases. In this research field, highly specific and sensitive nucleic acid detection methods are fundamental to achieving accurate monitoring.

[0003] Currently regarding bats Nobecovirus For the detection and identification of viruses, the academic and industrial communities have not yet established standardized and efficient specialized methods. Current technological approaches mainly rely on metagenomics based on high-throughput sequencing or RT-PCR detection combined with sequencing verification methods based on broad-spectrum primers. However, these methods have limitations. Metagenomics based on high-throughput sequencing is currently the main method for discovering new viruses and studying viral diversity. This method does not require prior knowledge of the viral sequence; sequencing alone can obtain all nucleic acid information in the sample. However, this method is costly, complex in operation, and has a long data analysis cycle. It also requires a high viral load, making it unsuitable for rapid screening and routine monitoring of large-scale samples, especially in field settings or laboratories with limited resources. RT-PCR detection combined with sequencing verification methods based on broad-spectrum primers have poor specificity; their amplification products may originate from various coronaviruses, or even non-target viruses, resulting in high background noise. Furthermore, it requires subsequent sequencing steps to determine whether it is a specific virus. Nobecovirus The process is cumbersome and has low throughput, making it impossible to achieve rapid and high-throughput qualitative detection. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a detection method. Nobecovirus Primer combinations for subgenus coronaviruses and their applications.

[0005] In a first aspect, the present invention provides a primer combination comprising any one or more of the following: (1) Primer pair 1 comprising the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; (2) Primer pair 2 comprising the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4; (3) Primer pair 3 comprising the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6; (4) Primer pair 4 comprising the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8; (5) Primer pair 5 comprising the nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10; (6) Primer pair 6 comprising the nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.12; (7) Primer pair 7 comprising the nucleotide sequences shown in SEQ ID NO.13 and SEQ ID NO.14.

[0006] This invention has yielded a set of methods for amplification. Nobecovirus Primer pairs with subgenus coronavirus-specific nucleotide sequences not only exhibit no cross-reactivity but can also achieve multiple reactions in a single reaction system. Nobecovirus Differentiation of subgenus coronaviruses. In this primer combination, there is no mutual interference between different primer pairs, and it has high specificity, sensitivity, and coverage, which can efficiently and accurately detect viruses such as HKU9, GCCDC1, and C704.

[0007] Furthermore, primer pair 1 is used for detection. Nobecovirus Subgenus coronavirus; Primer pair 2 and primer pair 3 were used to detect nine types of coronaviruses in HKU; Primer pair 4 and primer pair 5 were used to detect one coronavirus from GCCDC; Primer pair 6 and primer pair 7 were used to detect coronavirus C704.

[0008] Secondly, the present invention provides a kit comprising: the aforementioned primer combination; Preferably, the kit is a multiplex PCR detection kit or a real-time quantitative PCR detection kit.

[0009] The primer combinations provided by this invention are applicable to various PCR detection scenarios, and may also include: DNA polymerase, reverse transcriptase, dNTPs, and Mg. 2+ These reagents, along with buffer solutions, can be used directly for multiplex PCR detection of target nucleic acid fragments, or further include probes, internal standards, and other components in a real-time quantitative PCR detection kit.

[0010] Furthermore, it also includes features for specific identification. Nobecovirus The probe is one or more of the following: subgenus coronavirus, HKU9 coronavirus, GCCDC1 coronavirus, or C704 coronavirus; preferably, the probe carries a fluorescent tag sequence.

[0011] Thirdly, the present invention provides the application of the aforementioned primer combinations in any of the following: (1) General testing Nobecovirus Subgenus coronaviruses, or to differentiate between HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (2) Prepare the kit for (1).

[0012] Furthermore, none of the applications provided by this invention include disease treatment or diagnosis. Those skilled in the art will know that, in addition to disease diagnosis, such primer combinations can also be applied to scenarios such as drug screening, vaccine efficacy evaluation, biomaterial construction, and targeted enrichment of feature fragments before sequencing.

[0013] Fourthly, the present invention provides the application of the aforementioned primer combinations in any of the following: (1) Screening for universal inhibitors Nobecovirus Subgenus coronaviruses, or reagents that specifically inhibit HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (2) Assess general prevention Nobecovirus Subgenus coronaviruses, or the efficacy of vaccines that specifically prevent infection with HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (3) Constructing a system containing general... Nobecovirus Biological materials containing nucleotide fragments of subgenus coronaviruses, or nucleotide fragments of specific HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (4) Targeted enrichment general Nobecovirus Nucleotide fragments of subgenus coronaviruses, or nucleotide fragments of specific HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (5) Prepare a kit for any of the applications shown in (1)-(4).

[0014] The primer combinations provided by this invention can not only be directly applied to the qualitative identification of target viruses, but also, based on their high specificity, high sensitivity and amplification characteristics covering both the genus and species levels, can be used as molecular biology tools in downstream fields such as antiviral drug development, vaccine evaluation, genetic engineering and high-throughput sequencing.

[0015] For example, after administering the drug and inoculating it with the corresponding virus in an in vitro cell model or in vivo animal model, the primer combination of the present invention can be used to perform real-time quantitative PCR detection on culture or host tissue samples. The amplification efficiency of the primers for a specific target sequence directly reflects the amount of viral nucleic acid amplified in the sample. If the candidate drug is effective, it will inhibit... Nobecovirus The replication of subgenus or specific species of coronaviruses (HKU9, GCCDC1, or C704) resulted in a significantly lower nucleic acid copy number after amplification using the primers of this invention compared to the untreated control group. Quantitative screening of the inhibitory efficacy of antiviral agents can be achieved by calculating the magnitude of the decrease in viral load.

[0016] For example, in animal challenge protection experiments, the primer sets of this invention can be used to dynamically monitor animal models that have been immunized and challenged with a specific virus. The primer sets of this invention can specifically track and quantify... Nobecovirus The residual nucleic acid level of subgenus-specific coronaviruses, such as HKU9, GCCDC1, and C704. The degree of decline or the rate of zeroing of viral nucleic acid copy number can directly reflect the immune protective efficacy of candidate vaccines against homologous or heterologous coronavirus infections.

[0017] For example, by using nucleic acids from environmental or clinical isolates as templates and performing routine PCR amplification with the aforementioned primers, it is possible to specifically clone from a complex nucleic acid background. Nobecovirus Subgenera-wide fragments or specific identification fragments for HKU9, GCCDC1, and C704. These amplification products, after purification, can be inserted into plasmid vectors, expression vectors, or lentivirus / adenovirus packaging systems to construct biomaterials such as pseudoviruses, standards, laboratory quality control references, or engineered bacteria expressing recombinant antigens.

[0018] For example, the primer combination of the present invention can serve as a core component of a targeted amplification primer library. During the library construction stage, using this primer combination to perform multiple targeted pre-amplification of the sample enables the use of exponential amplification principles to amplify extremely small amounts of... Nobecovirus The abundance of subgenus-wide common fragments, as well as HKU9, GCCDC1, and C704-specific nucleic acid fragments, was significantly increased. After enriching the products and connecting them to sequencing adapters, the detection rate, sequencing depth, and assembly integrity of second- or third-generation sequencing technologies for these coronaviruses can be greatly improved.

[0019] Fifthly, the present invention provides a detection method. Nobecovirus Subgenus coronavirus methods include: For the sample to be tested, use the aforementioned primer combination or the aforementioned kit for detection.

[0020] Furthermore, the detection is a single-system PCR amplification reaction, and the reaction system comprises, in a total volume of 10 μL: 4-6 μL of RT-PCR premix, 0.3-0.8 μL of RT-PCR enzyme mixture, 8-15 μM of each primer, 1-3 μL of RNA template, and the remainder is water.

[0021] Furthermore, the detection reaction procedure includes: Reverse transcription at 45-55 ℃ for 10-20 min; Pre-denaturation: 93-98 °C, 25-50 sec; 93~98 ℃, 3~10 sec, 55~65 ℃, extension 15~25 sec, 35~55 cycles; The temperature was increased from 55-65 ℃ to 93-98 ℃ at intervals of 0.4-0.8℃, and the signal was collected.

[0022] Based on the reaction system and reaction procedure provided by this invention, the primer combination of this invention can be realized in a single reaction system. Nobecovirus The identification of subgenus coronaviruses and the classification of three viral species were developed, and the detection methods have high specificity, sensitivity and coverage.

[0023] Furthermore, it also includes: If primer pair 1 detects the target fragment, then the sample to be tested is determined to contain it. Nobecovirus Subgenus coronavirus components; If primer pair 2 and primer pair 3 detect the target fragment simultaneously, it is determined that the sample to be tested contains HKU9 coronavirus components; If primer pair 4 and primer pair 5 detect the target fragment simultaneously, it is determined that the sample to be tested contains GCCDC1 coronavirus components. If primer pair 6 and primer pair 7 detect the target fragment simultaneously, it is determined that the sample to be tested contains C704 coronavirus components.

[0024] The present invention has the following beneficial effects: This invention provides a universal detection method. Nobecovirus Subgenus coronaviruses, primer combinations for distinguishing and detecting HKU9 coronavirus, GCCDC1 coronavirus, or C704 coronavirus can specifically detect... NobecovirusSubgenus coronaviruses, avoiding nonspecific amplification. They also possess the advantages of high efficiency and high sensitivity, making them suitable for rapid screening of large-scale samples. Furthermore, they have a broad detection range, capable of covering... Nobecovirus Reduce missed detections of most or major viral members within the subgenus. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 The results are the cross-reactivity and standard curve of the Nobecovirus-NSP14 primer pair provided in this embodiment of the invention.

[0027] Figure 2 The results are the cross-reaction and standard curve of the HKU9-NSP3 primer pair provided in this embodiment of the invention.

[0028] Figure 3 These are the cross-reaction and standard curve results of the HKU9-N primer pair provided in this embodiment of the invention.

[0029] Figure 4 The results are the cross-reaction and standard curve of the GCCDC1-NSP16 primer pair provided in this embodiment of the invention.

[0030] Figure 5 The results are the cross-reaction and standard curve of the GCCDC1-E primer pair provided in this embodiment of the invention.

[0031] Figure 6 These are the cross-reaction and standard curve results of the C704-M primer pair provided in the embodiments of the present invention.

[0032] Figure 7 These are the cross-reactivity and standard curve results of the C704-N primer pair provided in the embodiments of the present invention.

[0033] Figure 8 This is the specific detection result provided in the embodiments of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0036] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0037] Example 1 1. This invention designs and obtains multiple primer pairs, including the following: Nobecovirus-NSP14-F: 5`-GCATATTCWTGCTGGAGTCACCATAG-3` (SEQ ID No. 1).

[0038] Nobecovirus-NSP14-R: 5`-CCTACATAACCCCATTGCTGYACATC-3` (SEQ ID No. 2).

[0039] HKU9-NSP3-F-1: 5`-ACCTTTGGTGATGAGGAGGT-3` (SEQ ID No. 3).

[0040] HKU9-NSP3-R-1: 5`-ACYTGAAAAGGCGCCATGAC-3` (SEQ ID No. 4).

[0041] HKU9-NF-1: 5`-ACGTTCAAACAAGAATCTGAYGGTTC-3` (SEQ ID No. 5).

[0042] HKU9-NR-1: 5`-GCAWACCARGAAACATTCTGMGGAG-3` (SEQ ID No. 6).

[0043] GCCDC1-NSP16-F-1:5`-GCGATTCTCACTCCGTGCTA-3` (SEQ ID No. 7).

[0044] GCCDC1-NSP16-R-1:5`-AACAACCTCCCTGCCTTGAG-3` (SEQ ID No. 8).

[0045] GCCDC1-EF-1: 5`-CTGGTGGGCACGGATACATC-3` (SEQ ID No. 9).

[0046] GCCDC1-ER-1: 5`-TGCAGTATAAGCAAGACCGCA-3` (SEQ ID No. 10).

[0047] C704-MF: 5`-GCCCGAGACCAATATGCTGC-3` (SEQ ID No. 11).

[0048] C704-MR: 5`-AAYTGAACCTGGCCYCTGAG-3` (SEQ ID No. 12).

[0049] C704-NF-1:5`-AAGGATGCTGCYGACCAGAC-3` (SEQ ID No. 13).

[0050] C704-NR-1: 5`-KGATTGGCGTCTCTGAGCCTT-3` (SEQ ID No. 14).

[0051] These primer pairs are capable of passing through 49 known lines. Nobecovirus Sequences (including 22 full-length sequences of BtCoV-HKU9, 14 full-length sequences of BtCoV-GCCDC1, 6 full-length sequences of BtCoV-C704, and unclassified sequences) Nobecovirus Accurate and specific identification was achieved from 7 full-length sequences. The Nobecovirus-NSP14 primer pair can accurately identify these 49 known sequences. Nobecovirus The sequences, HKU9-NSP3, GCCDC1-NSP16 and C704-M can accurately identify BtCoV-HKU9, BtCoV-GCCDC1 and BtCoV-C704 respectively.

[0052] 2. Experimental samples.

[0053] Select an appropriate dilution of 10. 5Plasmid standards (BtCoV-HKU9: NC009020, BtCoV-GCCDC1: NC009021, BtCoV-C704: MG693168 sequences derived from NCBI and synthesized by Sangon Biotech) were prepared using the Vazyme Q221 OneStep SYBR qRT-PCR kit. Nobecovirus Cross-testing was conducted on three viral species within the subgenus.

[0054] The three plasmid standards were mixed in equal volumes to obtain the sample to be tested.

[0055] 3. Detection methods.

[0056] Reaction system: The Vazyme Q221 OneStep SYBR qRT-PCR kit was used. The system consisted of 5 μL of 2X One Step SYBR Green Mix, 0.5 μL of One Step SYBR Green Enzyme Mix, 0.2 μL each of F / R primers (10 μM), 2 μL of RNA template, and ddH2O to bring the volume to 10 μL.

[0057] Reaction procedure: Reverse transcription at 50 ℃ for 15 min; Pre-denaturation at 95 °C for 30 seconds; 95 ℃, 5 sec, 60 ℃, extension 20 sec, 45 cycles.

[0058] The melting process involves increasing the temperature from 65°C to 95°C at 0.5°C intervals, and then collecting the signal.

[0059] 4. Experimental results.

[0060] The results are as follows Figures 1-7 As shown in the left figure, from Figure 1 It can be seen that there is no cross-reaction among these 7 primer pairs.

[0061] Example 2 This embodiment further uses the primer pairs from Example 1 to construct a standard curve to test their sensitivity and amplification efficiency. The specific procedure is as follows: Plasmid standards (BtCoV-HKU9: NC009020, BtCoV-GCCDC1: NC009021, BtCoV-C704: MG693168 sequences derived from NCBI and synthesized by Sangon Biotech) were selected at 10-fold serial dilutions and tested using the Vazyme Q221 OneStep SYBR qRT-PCR kit. NobecovirusSensitivity tests were conducted on three viral species within the subgenus.

[0062] The reaction system and reaction procedure are the same as in Example 1.

[0063] Experimental results: The results are as follows Figures 1-7 As shown in the right figure, the primer combinations provided by this invention all have high detection sensitivity: The detection sensitivity of primer pair 1 is 10 copies / μL.

[0064] The detection sensitivity of primer pair 2 is 10 copies / μL.

[0065] The detection sensitivity of primer pair 3 is 10 copies / μL.

[0066] The detection sensitivity of primer pair 4 is 10 copies / μL.

[0067] The detection sensitivity of primer pair 5 is 10 copies / μL.

[0068] The detection sensitivity of primer pair 6 is 10 copies / μL.

[0069] The detection sensitivity of primer pair 7 is 10 copies / μL.

[0070] Example 3 This invention selects bat swabs from Nobecovirus Subgenus-positive nucleic acid samples were tested using primer pairs of SEQ ID No. 1-2; nucleic acid samples from BtCoV-HKU9-positive bats were tested using primer sets of SEQ ID No. 3-6; nucleic acid samples from BtCoV-GCCDC1-positive bats were tested using primer sets of SEQ ID No. 7-10; and nucleic acid samples from BtCoV-C704-positive bats were tested using primer sets of SEQ ID No. 11-14. The detection method of Example 1 was used to verify the coverage and detection capability of each primer set for actual samples. The results are shown in Table 1.

[0071] Table 1 Nobecovirus Quantitative methods for detecting positive nucleic acid samples in subgenus and representative species within subgenus

[0072] The results above demonstrate that the primer combination provided by this invention has a high positive rate in complex sample environments (real detection scenarios), enabling accurate detection of the target virus.

[0073] Example 4 This embodiment further cross-tested representative strains of common respiratory pathogens, enteric pathogens, and other subgenera of coronaviruses. The detection process is as follows: 1. Experimental materials.

[0074] The samples shown in Table 2 were mixed at a dose of 100 copies / μL for subsequent testing to verify specificity.

[0075] Table 2. Samples of non-Nobecovirus subgenus pathogens

[0076] 2. Detection methods.

[0077] Same as Example 3.

[0078] 3. Experimental results.

[0079] The results are as follows Figure 8 As shown in the figure, it can be seen that in the experimental group, the primers provided by this invention do not cross-react with common respiratory pathogens and coronaviruses of other subgenus, and have high specificity.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer combination, characterized in that, Includes any one or more of the following: (1) Primer pair 1 comprising the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; (2) Primer pair 2 comprising the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4; (3) Primer pair 3 comprising the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6; (4) Primer pair 4 comprising the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8; (5) Primer pair 5 comprising the nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10; (6) Primer pair 6 comprising the nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.12; (7) Primer pair 7 comprising the nucleotide sequences shown in SEQ ID NO.13 and SEQ ID NO.

14.

2. The primer combination according to claim 1, characterized in that, Primer pair 1 is used for detection Nobecovirus Subgenus coronavirus; Primer pair 2 and primer pair 3 were used to detect nine types of coronaviruses in HKU; Primer pair 4 and primer pair 5 were used to detect one coronavirus from GCCDC; Primer pair 6 and primer pair 7 were used to detect coronavirus C704.

3. A reagent kit, characterized in that, include: The primer combination as described in claim 1; Preferably, the kit is a multiplex PCR detection kit or a real-time quantitative PCR detection kit.

4. The reagent kit according to claim 3, characterized in that, It also includes methods for specific identification. Nobecovirus The probe is one or more of the following: subgenus coronavirus, HKU9 coronavirus, GCCDC1 coronavirus, or C704 coronavirus; preferably, the probe carries a fluorescent tag sequence.

5. The use of the primer combination of claim 1 or 2 in any of the following: (1) General testing Nobecovirus Subgenus coronaviruses, or to differentiate between HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (2) Prepare the kit for (1).

6. The use of the primer combination of claim 1 or 2 in any of the following: (1) Screening for universal inhibitors Nobecovirus Subgenus coronaviruses, or reagents that specifically inhibit HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (2) Assess general prevention Nobecovirus Subgenus coronaviruses, or the efficacy of vaccines that specifically prevent infection with HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (3) Constructing a system containing general... Nobecovirus Biological materials containing nucleotide fragments of subgenus coronaviruses, or nucleotide fragments of specific HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (4) Targeted enrichment general Nobecovirus Nucleotide fragments of subgenus coronaviruses, or nucleotide fragments of specific HKU9 coronaviruses, GCCDC1 coronaviruses, or C704 coronaviruses; (5) Prepare a kit for any of the applications shown in (1)-(4).

7. A detection method Nobecovirus The method for subgenus coronaviruses is characterized by, include: For the sample to be tested, the primer combination described in claim 1 or 2, or the kit described in claim 3 or 4, is used for detection.

8. The method according to claim 7, characterized in that, The detection is a single-system PCR amplification reaction, with a total volume of 10 μL. The reaction system includes: 4-6 μL of RT-PCR premix, 0.3-0.8 μL of RT-PCR enzyme mixture, 8-15 μM of each primer, 1-3 μL of RNA template, and the remainder is water.

9. The method according to claim 7 or 8, characterized in that, The detection reaction procedure includes: Reverse transcription at 45-55 ℃ for 10-20 min; Pre-denaturation: 93-98 °C, 25-50 sec; 93~98 ℃, 3~10 sec, 55~65 ℃, extension 15~25 sec, 35~55 cycles; The temperature was increased from 55-65 ℃ to 93-98 ℃ at intervals of 0.4-0.8℃, and the signal was collected.

10. The method according to any one of claims 7-9, characterized in that, Also includes: If primer pair 1 detects the target fragment, then the sample to be tested is determined to contain it. Nobecovirus Subgenus coronavirus components; If primer pair 2 and primer pair 3 detect the target fragment simultaneously, it is determined that the sample to be tested contains HKU9 coronavirus components; If primer pair 4 and primer pair 5 detect the target fragment simultaneously, it is determined that the sample to be tested contains GCCDC1 coronavirus components. If primer pair 6 and primer pair 7 detect the target fragment simultaneously, it is determined that the sample to be tested contains C704 coronavirus components.

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