Primer set of beta coronavirus, library construction method and application thereof
Through targeted primer collection and enrichment methods that enrich β-genomic coronavirus, the problems of high data volume, high cost and primer preference in the whole genome detection of β-genomic coronavirus are solved, and the detection effect of high sensitivity and high coverage is achieved , suitable for low-concentration samples and detection of new coronavirus.
Patent Information
- Application Number
- CN202310734025.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-20
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art has problems such as high data requirements, high cost, long cycle and primer preference in the whole genome detection of β-genus coronaviruses, especially the difficulty of detecting low-concentration samples, and severe interference with the human background of the sample.
A set of primer sets and corresponding enrichment methods targeted to enrich β-genus coronaviruses are provided, and reverse transcription, terminal repair, linker ligation and magnetic bead purification are carried out through specific and random primers binding, and combined with PCR amplification. Build a full genome library of β-genus coronaviruses, reduce data volume requirements, and improve detection sensitivity and coverage.
Effectively enrich β-type coronaviruses, including the new coronavirus, improve detection sensitivity, reduce human-source background interference in the sample, reduce the amount of sequencing data, achieve high coverage and uniform sequence distribution for the whole genome detection of β-type coronaviruses, and avoid expansion The preference problems caused by the increase.
Smart Images
Figure CN119162378A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of virus detection, and in particular to a set of primers for targeted enrichment of β-coronavirus, a targeted enrichment method for β-coronavirus, a method for constructing a β-coronavirus whole genome detection library, and a kit. Background Art
[0002] Compared with traditional Sanger sequencing, the second-generation sequencing technology can achieve whole genome sequencing of species more efficiently. With the maturity of sequencing tools and analysis algorithms, whole genome detection is becoming more and more widely used in viral genome sequencing. However, with the increasing demand for whole genome sequencing of viruses, its shortcomings are becoming more and more obvious. Due to the interference of human background and other microorganisms in the samples being tested, whole genome detection technology generally requires high-depth sequencing to overcome the problems of low coverage and sensitivity, which leads to a huge amount of data, thereby increasing the sequencing cost, especially for low-concentration and high-complexity samples, the sequencing difficulty will be greater.
[0003] At present, the commonly used detection methods mainly include metagenomic sequencing technology, probe hybridization technology and multiplex PCR technology. Metagenomic technology uses ultra-high depth sequencing to obtain a large amount of data and achieve coverage of the whole genome of the virus by sequencing data; probe hybridization technology uses targeted probes to capture large fragments of viral nucleic acids, thereby enriching viral gene sequences on a large scale and having strong coverage of the whole genome sequence of the virus. Multiplex PCR technology uses multiple primers to perform multiple amplification of viral genes during the genome library construction process, thereby enriching the target sequence of the virus, with the characteristics of low cost, high sensitivity and convenient process. However, metagenomic sequencing technology has the disadvantages of high data volume requirements, high sequencing depth requirements and serious interference from other background pathogens. Probe hybridization technology has the disadvantages of complex probe design process, high production cost, cumbersome experimental operation and long experimental cycle. In addition, this technology is prone to hybridization failure when facing low-concentration samples. Multiplex PCR technology has the disadvantages of high primer preference and uneven distribution of detected sequences. Summary of the invention
[0005] Based on the above situation, the present invention provides a set of solutions through in-depth and meticulous research on β-coronavirus and a large number of experimental studies to overcome the problems of high data volume requirements, high costs, long cycles and high primer preference in the prior art when performing whole genome detection of β-coronavirus.
[0006] In the first aspect, the present invention provides a primer set for targeting and enriching beta coronaviruses, the primer set comprising a plurality of specific primers, specifically, the plurality of specific primers are nucleic acid sequences shown in SEQ ID NO.1-SEQ ID NO.49 in Table 1 below. Furthermore, the primer set also comprises at least one random primer.
[0007] In a second aspect, the present invention provides a method for targeted enrichment of β-coronavirus, using the primer set provided in the first aspect to perform targeted enrichment of the whole genome of β-coronavirus in the sample to be tested, thereby obtaining the target sequence of the virus.
[0008] In a third aspect, the present invention provides a method for constructing a beta coronavirus whole genome library, using the primer set mentioned in the first aspect to enrich the beta coronavirus in the sample to be tested. Further, the construction method provided by the present invention comprises the following steps:
[0009] The extracted nucleic acid of the sample to be tested is mixed with the primer set and incubated.
[0010] Reverse transcription and end repair: After incubation, enrichment reaction buffer and enrichment reaction enzyme were added and mixed for incubation; enrichment reaction buffer was a mixed solution of 5x first strand buffer, dNTP Mixture, DTT and Tris-HCI Buffer; enrichment reaction enzyme was a mixed solution of RNase Inhibitor and SuperScriptⅡReverse Transcriptase. After reverse transcription, end repair buffer and end repair enzyme were added and mixed for incubation; end repair buffer was a mixed solution of 10x second strand buffer, 10x T4 polynucleotide phosphate kinase buffer and dNTP Mixture; end repair enzyme was a mixed solution of DNA polⅠ, TaKaRa Taq, BST 3.0DNA Polymerase and T4 polynucleotide kinase.
[0011] Connector ligation and magnetic bead purification: After end repair, ligation buffer, ligase, and platform connector are added to the product for incubation, and then magnetic beads are added for purification and recovery; the ligation buffer is a mixed solution of Tris-HCI Buffer, MgCl2, ATP and PEG, the ligase is 1000-2000U T4 DNA ligase; the magnetic beads are sorting magnetic beads.
[0012] PCR amplification and magnetic bead purification: the recovered product is added to the PCR reaction solution and amplification primers for PCR amplification, and after amplification, magnetic beads are added for purification and recovery; the PCR reaction solution is 2x Hi-Fi readymix; the magnetic beads are sorting magnetic beads; after magnetic bead purification, the nucleic acid is eluted and recovered from the magnetic beads, and the dissolving solution used is Nuclease-free Water.
[0013] In a fourth aspect, the present invention provides a kit for detecting the whole genome of a beta coronavirus, the kit comprising the primer set of the first aspect. Further, the kit of the present invention also comprises an enrichment reaction buffer, an enrichment reaction enzyme, an end repair buffer, an end repair enzyme, a ligation buffer, a ligase, a PCR reaction solution, magnetic beads, and a dissolving solution as mentioned in the construction method of the third aspect.
[0014] The beneficial effect of the present invention is that the primer set for targeted enrichment of β-coronavirus provided by the present invention has been verified by a large number of experiments, and can effectively enrich β-coronavirus including the new coronavirus, thereby improving the detection sensitivity of the virus, weakening the human background interference of the sample, and thus reducing the sequencing data volume requirement.
[0015] Based on the enrichment primer set and enrichment method, the present invention further optimizes the construction method of the β-coronavirus whole genome library and provides a corresponding library construction kit. Compared with the widely used metagenomic technology, probe hybridization technology and multiplex PCR technology, the construction method and kit of the present invention can improve the coverage of the detected sequence on the β-coronavirus genome, achieve the effect of detecting the whole genome of the β-coronavirus, and at the same time ensure the uniform distribution of the detected sequence on the whole genome, avoid the preference problem caused by amplification, and effectively solve the existing problems existing in the detection of the whole genome of the coronavirus genus, especially the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Schematic diagram of the detection process and corresponding library construction kit components used in Example 1;
[0017] Figure 2 The results of human coronavirus HKU1 detection using the kit of the present invention in Example 1, wherein the horizontal axis is the length of the whole genome, and the vertical axis is the number of reads at different positions of the viral genome;
[0018] Figure 3 The results of human coronavirus HKU1 detection using conventional methods in Example 1, wherein the horizontal axis is the length of the whole genome, and the vertical axis is the number of reads at different positions of the aligned viral genome;
[0019] Figure 4The results of the novel coronavirus 2019-nCoV detection using the kit of the present invention in Example 2, wherein the horizontal axis is the length of the whole genome, and the vertical axis is the number of reads at different positions of the viral genome;
[0020] Figure 5 The results of the detection of the new coronavirus 2019-nCoV using the conventional method in Example 2, wherein the horizontal axis is the length of the whole genome, and the vertical axis is the number of reads at different positions of the aligned viral genome. DETAILED DESCRIPTION
[0021] The new coronavirus belongs to the β coronavirus, and is of the same genus as several other pathogenic coronaviruses HCoV-OC43 and HCoV-HKU1, SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), with high sequence similarity. The present invention designs a set of primers for targeted enrichment of β coronaviruses through a large number of meticulous studies on β coronaviruses, which can not only effectively detect the whole genome of each mutant strain of the new coronavirus, but also detect other pathogenic coronaviruses, thus expanding the applicability of the primer set.
[0022] In a specific embodiment of the present invention, the primer set includes specific primers and random primers, wherein the specific primers are 49 primers as shown in SEQ ID NO.1-SEQ ID NO.49, specifically see the primer set sequence in Table 1. Wherein, the degenerate base sequence Y=C,T in the table.
[0023] The random primer can be expressed as NNNNNN (SEQ ID NO.50). Specifically, the random primer is composed of 6 nucleotides randomly and has no fixed sequence. There can be multiple random primers as long as they maintain a certain ratio with the specific primer. Its main function is to provide primers for conventional reverse transcription reactions. In this kit, it is mixed with the specific primer for use, and the molar ratio of the random primer to the specific primer is 1 / 20-1 / 5.
[0024] Table 1 Primer set sequences
[0025] Primer name sequence Primer name sequence SEQ ID NO.1 TCTTTGGTTTCAC SEQ ID NO.26 TCTCTAGCACTCC SEQ ID NO.2 TCAGATAGTAGAG SEQ ID NO.27 YACATATTCCCAC SEQ ID NO.3 GAGAATGGTCATA SEQ ID NO.28 GGAGTCAAAATTA SEQ ID NO.4 GACAAATCCAAAA SEQ ID NO.29 TCAATGCATTTGG SEQ ID NO.5 TTGGTGTAATTCA SEQ ID NO.30 TTGAACACAGTTG SEQ ID NO.6 TGGACAATAATCC SEQ ID NO.31 TGGATCAAAATGA SEQ ID NO.7 TTGGAGATGATGG SEQ ID NO.32 TGGAAGATGAACT SEQ ID NO.8 TGTCAAATCTATG SEQ ID NO.33 TTCCTCCTGATAA SEQ ID NO.9 TCCCGGTATTTAG SEQ ID NO.34 TTGTGTGAACTTT SEQ ID NO.10 TCCGCATCTCAAT SEQ ID NO.35 TTTGCTTCTGTTG SEQ ID NO.11 TCAATAACCTTGT SEQ ID NO.36 TTGTTGCCATGAG SEQ ID NO.12 TTTAGGACTAGTG SEQ ID NO.37 GAGAATGGTCATA SEQ ID NO.13 TGCAATACATCTA SEQ ID NO.38 TGCATCCACTCTT SEQ ID NO.14 TTCTTCTTCATCA SEQ ID NO.39 TGTTGACTTCTTT SEQ ID NO.15 TTTACCGATAGCT SEQ ID NO.40 TTGCTTCCAAATT SEQ ID NO.16 TGGTTCTTGATTC SEQ ID NO.41 TGTGCTCTATGTT SEQ ID NO.17 TTCTTCCTCAAGC SEQ ID NO.42 YATGATGGATACC SEQ ID NO.18 GCCAGCTCGTTTA SEQ ID NO.43 AATTGTTCCTGTT SEQ ID NO.19 TTGAATGGTGTAT SEQ ID NO.44 TTAGCTCAAGAGC SEQ ID NO.20 TGCAACACAACTG SEQ ID NO.45 TTGAAAACTTGGC SEQ ID NO.21 CCAAACAAGAGAA SEQ ID NO.46 AAACCCAAATGAA SEQ ID NO.22 TTAAGAAAAGGCT SEQ ID NO.47 TTGGGATAGAGAT SEQ ID NO.23 TTTGGGCTTCATG SEQ ID NO.48 TGTTTTGTTGCCT SEQ ID NO.24 TCAAGACCAGGAG SEQ ID NO.49 CAACACCAACAAA SEQ ID NO.25 CAGATTCTTAATC
[0026] In combination with the above primer set, the present invention has developed a method for targeted enrichment of β coronavirus, as well as a method for constructing a β coronavirus whole genome library and a corresponding kit. The present invention adds targeted enrichment primers during the normal library construction process to effectively reduce the amount of data, increase the coverage of the detected sequence on the whole genome of the new coronavirus, and ensure the uniform distribution of the detected sequence on the whole genome. At the same time, it does not affect the detection of other pathogens in the sample, has low cost, convenient operation, and short experimental cycle.
[0027] Regarding the above experimental library construction and sequencing process and the corresponding use of the kit components, Figure 1 As shown. As described below, the sequencing process of the present invention includes: extraction of nucleic acid from the sample to be tested, mixing and incubating the extracted nucleic acid of the sample to be tested with a primer set (enrichment primer binding), reverse transcription and end repair, adapter connection and magnetic bead purification, PCR amplification and magnetic bead purification, platform sequencing, and data analysis. Among them, the reagents involved in the steps of enrichment primer binding, reverse transcription and end repair, adapter connection and magnetic bead purification, PCR amplification and magnetic bead purification constitute the kit components provided by the present invention, and the relevant components include: primer set (enrichment primer set), enrichment reaction buffer, enrichment reaction enzyme, end repair buffer, end repair enzyme, connection buffer, ligase, PCR reaction solution, magnetic beads, and dissolution solution.
[0028] It should be noted that in the present invention, the enrichment reaction buffer is a mixed solution of 5x first strand buffer, dNTP Mixture, DTT and Tris-HCI Buffer, wherein compared with the conventional process, the enrichment reaction buffer is additionally added with Tris-HCI Buffer to optimize the enrichment reaction system, and the input amount of dNTP Mixture is increased to improve the enrichment reaction efficiency. The enrichment reaction buffer and the enrichment reaction enzyme are mainly used for viral RNA reverse transcription and targeted enrichment primer amplification. The enrichment reaction enzyme is a mixed solution of RNase Inhibitor and SuperScriptⅡReverseTranscriptase. The end repair buffer is a mixed solution of 10x second strand buffer, 10x T4 polynucleotide phosphate kinase buffer and dNTP Mixture. The end repair enzyme is a mixed solution of DNA polⅠ, TaKaRa Taq, BST 3.0DNA Polymerase and T4 polynucleotide kinase. The above end repair buffer and end repair enzyme are used for two-strand synthesis and end repair. The connection buffer is a mixed solution of Tris-HCI Buffer, MgCl2, ATP and PEG. The ligase was T4 DNA ligase.
[0029] In a specific embodiment of the present invention, two more common viruses in the genus β coronavirus are used as detection objects: human coronavirus HKU1 sample and new coronavirus 2019-nCoV.
[0030] In a specific embodiment of the present invention, the kit is to amplify the nucleic acid sample containing human coronavirus HKU1 or novel coronavirus 2019-nCoV by using beta coronavirus targeted enrichment primers, and target enrichment of human coronavirus HKU1 or novel coronavirus 2019-nCoV genes. The process is: nucleic acid extraction of the sample to be tested, combination of enrichment primer sets, reverse transcription and end repair, connector connection, magnetic bead purification, PCR amplification, magnetic bead purification and MGISEQ platform sequencing, and finally the whole genome data of human coronavirus HKU1 or novel coronavirus 2019-nCoV is analyzed. The sequencing platform matched in this embodiment is the MGISEQ platform, which has the characteristics of low sequencing cost, compatibility with multiple sequencing fluxes and compatibility with multiple sequencing read lengths, and the sequencing cycle of 4-5 hours of the platform can also meet the needs of rapid detection. Therefore, the MGISEQ sequencing platform is a high-quality choice in this embodiment, but it should be understood that the present invention can also be adapted to other sequencing platforms, and the same effect can be achieved by adjusting its library construction adapter and PCR primer according to different sequencing platforms.
[0031] It is further explained that in the above specific embodiment, the enrichment primer MIX is a primer set of β coronavirus specific primers and random primers. The sequences of the specific primers in the primer set are shown in Table 1. The concentration of the primer set is 50-150 μM, and the molar ratio of random primers to 49 specific primers is 1 / 20-1 / 5. The concentration of dNTP Mixture in the enrichment reaction buffer is preferably 0.5-1.5 mM, the concentration of DTT is 5-15 mM, and the concentration of Tris-HCI is 30-40 mM. The amount of RNase Inhibitor used in the enrichment reaction enzyme is preferably 40-80 U, and the amount of SuperScriptⅡReverse Transcriptase used is 100-200 U. The concentration of dNTP Mixture in the end repair buffer is preferably 0.5-1.5 mM. The preferred end repair enzyme is DNA polⅠ, with an amount of 10-50U, TaKaRa Taq, with an amount of 1-10U, BST 3.0 DNA Polymerase, with an amount of 5-15U, and T4 polynucleotide kinase, with an amount of 5-15U. The preferred connection buffer is Tris-HCl at a concentration of 20-50mM, MgCl2 at a concentration of 5-15mM, ATP at a concentration of 1-5mM, and PEG at a concentration of 5%-20%. The preferred ligase is 1000-2000U T4 DNA ligase. The preferred PCR reaction solution is 2x Hi-Fi readymix. The preferred magnetic beads are sorting magnetic beads. The preferred dissolving solution is Nuclease-free Water.
[0032] The present invention will be further described in detail below through specific implementation modes in conjunction with the accompanying drawings.
[0033] Example 1: Detection of human coronavirus HKU1
[0034] This embodiment takes the enrichment and detection of human coronavirus HKU1 as an example. The samples containing the virus are detected using conventional methods and the kit of the present invention respectively.
[0035] A. The overall detection process of the kit of the present invention is as follows Figure 1 As shown. It usually includes the following steps:
[0036] 1. Sample packaging: Divide the inactivated sample into two portions, 300 μL each.
[0037] 2. Sample nucleic acid extraction: Use the microbial extraction kit (magnetic bead method) to extract nucleic acid according to the instructions, and use RNase-Free ddH2O to dissolve the nucleic acid.
[0038] 3. Enrichment primer set combination: Take 10 μL of the extracted nucleic acid sample, add 12 μL of enrichment primer MIX (the primer set is as mentioned above, its specific primers are shown in Table 1, and the random primers are randomly generated as mentioned above, and the molar ratio of random primers to specific primers is 1:5), mix thoroughly, incubate at 94°C for 2 minutes, and cool at 4°C.
[0039] 4. Reverse transcription and end repair: Add 15 μL of enrichment reaction buffer and 10 μL of enrichment reaction enzyme, mix thoroughly, incubate at 25°C for 5 min, 37°C for 30 min, 85°C for 2 min, cool to 4°C and keep. Add 16 μL of end repair buffer and 10 μL of end repair enzyme, mix thoroughly, incubate at 37°C for 20 min, 72°C for 10 min, cool to 4°C and keep.
[0040] 5. Adapter ligation and magnetic bead purification: Add 36 μL of ligation buffer, 2 μL of ligase, and 2 μL of MGISEQ platform adapter to the product of step 4, incubate at 23°C for 15 min. Add 0.5x magnetic beads for purification.
[0041] 6. PCR amplification and magnetic bead purification: Recover 21 μL of purified product in step 5, add 25 μL of PCR reaction solution and 4 μL of MGISEQ platform amplification primer. 98℃ 2min, (98℃ 15sec, 55℃ 15sec, 72℃ 30sec) 16cycles, 72℃ 5min, 4℃ hold. After the reaction, add 1x magnetic beads for purification.
[0042] 6. MGISEQ platform sequencing: Sequencing was performed according to the MGISEQ platform sequencing instructions.
[0043] 7. Data analysis: The data from the machine were compared with the genome of human coronavirus HKU1, and the reads that could only be compared with the corresponding species were counted. The detection of human coronavirus HKU1 by the kit method and the conventional method was calculated, and the coverage of the detected reads on the whole genome was calculated (such as Figure 2 ), the results showed that the method of the present invention detected 2170 reads of human coronavirus HKU1 in the sample, and the coverage of the detected sequence in the whole genome was 94.24%, and it was evenly distributed.
[0044] B. The conventional method detection process used in this example is as follows:
[0045] 1. Sample processing: the other sample divided as above.
[0046] 2. Sample nucleic acid extraction: Use the microbial extraction kit (magnetic bead method) to extract nucleic acid according to the instructions, and use RNase-Free ddH2O to dissolve the nucleic acid.
[0047] 3. Reverse transcription: Take 10μL of the extracted nucleic acid sample, add 2μL 6N primer (10μM), mix thoroughly, incubate at 94℃ for 2min, and cool at 4℃. Add 8μL 5x first strand buffer (Invitrogen), 1μL dNTPMixture (10mM each), 4μL 0.1M DTT (Abclonal), 40-80U RNase Inhibitor, 100-200U SuperScriptⅡReverse Transcriptase. Mix thoroughly, incubate at 25℃ for 5min, 37℃ for 30min, 85℃ for 2min, and cool to 4℃ for storage.
[0048] 4. End repair: Add 5-15U BST 3.0 DNA Polymerase, 10-50U DNA polⅠ, 1-10U TaKaRa Taq, 5-15U T4 polynucleotide kinase, 1μL Tris-HCl Buffer, 1μL dNTPMixture (10mM) to the reaction product in 3. Mix thoroughly, incubate at 37℃ for 20min, incubate at 72℃ for 10min, and cool to 4℃ for storage.
[0049] 5. MGISEQ platform library construction: Follow the MGISEQ platform library construction instructions to perform adapter connection, magnetic bead purification, PCR amplification, magnetic bead purification and other processes.
[0050] 6. Sequencing and data analysis on the MGISEQ platform are the same as the above process. The data from the machine is compared with the genome of human coronavirus HKU1, and only the reads that can be compared with the corresponding species are counted. The detection of human coronavirus HKU1 under the kit method and the conventional method is calculated, and the coverage of the detected reads on the whole genome is calculated (such as Figure 3 ), 199 reads were detected by conventional methods, and the coverage of the detected sequences in the whole genome was 34.97%.
[0051] right Figure 2 and Figure 3 The results were analyzed, and the results showed that the detection rate of the kit method of the present invention is 10.9 times that of the conventional method (reads increased by 10.9 times), the coverage of the whole genome is increased by 2.7 times, and the coverage is close to the whole genome, which meets the conditions for whole genome sequencing.
[0052] Example 2: Detection of the new coronavirus 2019-nCoV
[0053] This example takes enrichment detection of the novel coronavirus 2019-nCoV as an example. Samples containing the virus are detected using conventional methods and the kit described in the present invention, respectively.
[0054] The kit method of the present invention and the conventional method used in this example are the same as those in Example 1 (not described here again), and the only difference is the data analysis link, that is, the off-machine data is compared with the genome of the new coronavirus 2019-nCoV, and the reads that can only be compared with the corresponding species are counted, and the detection of the new coronavirus 2019-nCoV under the kit method and the conventional method is calculated, and the coverage of the detected reads on the whole genome is calculated (such as Figure 4 , 5 ), the results showed that the method of the present invention detected 1392 reads of the new coronavirus 2019-nCoV in the sample, and the coverage of the detected sequence in the whole genome was 92%, and it was evenly distributed. The conventional method detected 122 reads, and the coverage of the detected sequence in the whole genome was 22.36%.
[0055] right Figure 4 and Figure 5 The results were analyzed, and the results showed that the detection rate of the kit method of the present invention was 11.4 times that of the conventional method (reads increased by 11.4 times), the coverage of the whole genome was increased by 4.1 times, and the coverage was close to that of the whole genome, which met the conditions for whole genome sequencing.
[0056] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.
Claims
1. A set of primers targeting and enriching β coronavirus, characterized in that: The primer set includes 49 specific primers as shown in SEQ ID NO.1 to SEQ ID NO.49 in Table 1, Table 1 。 2. The primer set according to claim 1, characterized in that The primer set also includes at least one random primer, wherein the random primer is randomly composed of 6 nucleotides; Preferably, the molar ratio of the random primer to the specific primer is 1 / 20-1 / 5.
3. A targeted enrichment method for β coronavirus, characterized in that: The primer set as described in claim 1 or 2 is used to carry out targeted enrichment of the whole genome of the β coronavirus in the sample to be tested, thereby obtaining the target sequence of the virus.
4. A method for constructing a beta coronavirus whole genome library, characterized in that: The construction method comprises using the primer set as described in claim 1 or 2 to enrich the β coronavirus in the sample to be tested.
5. The construction method according to claim 4, characterized in that: The following steps are involved: Mixing and incubating the extracted nucleic acid of the sample to be tested with the primer set; After the incubation is completed, enrichment reaction buffer and enrichment reaction enzyme are added and mixed and incubated; the enrichment reaction buffer is a mixed solution of 5xfirst strand buffer, dNTP Mixture, DTT and Tris-HCl Buffer; the enrichment reaction enzyme is a mixed solution of RNase Inhibitor and SuperScriptⅡReverse Transcriptase; after enrichment, end repair buffer and end repair enzyme are added and mixed and incubated to complete end repair; the end repair buffer is a mixed solution of 10xsecond strand buffer, 10x T4 polynucleotide phosphate kinase buffer and dNTP Mixture, and the end repair enzyme is a mixed solution of DNA polⅠ, TaKaRa Taq, BST 3.0DNA Polymerase and T4 polynucleotide kinase; After the end repair, a connection buffer, a ligase, and a platform connector are added to the product for incubation, and then magnetic beads are added for purification and recovery to obtain a recovered product; the connection buffer is a mixed solution of Tris-HCI Buffer, MgCl2, ATP and PEG, and the ligase is 1000-2000U T4 DNA ligase; The magnetic beads are sorting magnetic beads; The recovered product is added to PCR reaction solution and amplification primers for PCR amplification, and after amplification, magnetic beads are added for purification and recovery; the PCR reaction solution is 2x Hi-Fi readymix; the magnetic beads are sorting magnetic beads; and the dissolving solution used for purification, recovery and elution is Nuclease-free Water.
6. A kit for detecting the whole genome of β coronavirus, characterized in that: Comprising the primer set as claimed in claim 1 or 2; Preferably, the primer set concentration is 50-150 μM; Preferably, the molar ratio of the random primers to the specific primers in the primer set is 1 / 20-1 / 5.
7. The kit according to claim 6, characterized in that It also includes enrichment reaction buffer, enrichment reaction enzyme, end repair buffer, end repair enzyme, connection buffer, connection enzyme, PCR reaction solution, magnetic beads, and dissolution solution.
8. The kit according to claim 7, characterized in that The enrichment reaction buffer is a mixed solution of 5x first strand buffer, dNTP Mixture, DTT and Tris-HCl Buffer; the enrichment reaction enzyme is a mixed solution of RNase Inhibitor and SuperScript II Reverse Transcriptase; Preferably, the concentration of dNTP Mixture is 0.5-1.5 mM, the concentration of DTT is 5-15 mM, and the concentration of Tris-HCl is 30-40 mM; Preferably, the dosage of RNase Inhibitor is 40-80U, and the dosage of SuperScriptⅡReverse Transcriptase is 100-200U.
9. The kit according to claim 7, characterized in that The end repair buffer is a mixed solution of 10x second strand buffer, 10x T4 polynucleotide phosphate kinase buffer and dNTP Mixture; the end repair enzyme is a mixed solution of DNA polⅠ, TaKaRa Taq, BST 3.0 DNA Polymerase and T4 polynucleotide kinase; Preferably, the concentration of dNTP Mixture is 0.5-1.5 mM; Preferably, the amount of DNA pol I used is 10-50U, the amount of TaKaRa Taq used is 1-10U, the amount of BST 3.0 DNA Polymerase used is 5-15U, and the amount of T4 polynucleotide kinase used is 5-15U.
10. The kit according to claim 7, characterized in that The connection buffer is a mixed solution of Tris-HCl Buffer, MgCl2, ATP and PEG; Preferably, the concentration of Tris-HCl is 20-50 mM, the concentration of MgCl2 is 5-15 mM, the concentration of ATP is 1-5 mM, and the concentration of PEG is 5%-20%; Preferably, the ligase is 1000-2000U T4 DNA ligase; Preferably, the PCR reaction solution is 2x Hi-Fi readymix; Preferably, the magnetic beads are sorting magnetic beads; Preferably, the dissolving solution is Nuclease-free Water.