A primer set for novel coronavirus whole genome targeted detection and its application

Abstract

The present invention belongs to the field of gene detection, and specifically relates to a primer set and its application for the whole genome targeted detection of new coronavirus. The primer set includes: a first primer set; and a second primer set; the first primer set includes at least two selected from the sequences shown in SEQ ID NO: 1-30; the second primer set includes at least two of the sequences shown in SEQ ID NO: 31-58. The primer set provided by the present invention can achieve full coverage of the new coronavirus genome, and the sequencing depth of different subtypes is ≥100× or more, and the whole genome coverage can reach more than 99%, and the sequencing depth of some subtypes can reach up to 150000×. The detection sensitivity of the present invention is extremely high, and the whole genome coverage of the sequencing of samples with lower concentrations can also reach more than 99%. The present invention uses only 29 pairs of primers, and the problems of amplification differences and uneven coverage are avoided by reducing primer pairs and increasing the length of amplified fragments.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and specifically relates to a primer set and application thereof for whole-genome targeted detection of the new coronavirus. Background Art

[0002] At present, there are two main sequencing technology solutions in the field of pathogen detection: macrotranscriptome sequencing (mNGS) and targeted genome capture sequencing (tNGS). Macrotranscriptome sequencing directly sequences the total nucleic acid of clinical samples, and the host nucleic acid contained in the samples accounts for a high proportion, which means a large amount of waste of new crown detection data, reduced detection sensitivity, and a significant reduction in sequencing cost-effectiveness. Targeted genome sequencing designs full or near-full coverage of multiple amplification primers to specifically amplify the target gene, amplify various regions of its genome, and then obtain the complete viral genome sequence through high-throughput second-generation sequencing or nanopore single-molecule sequencing technology. The application of tNGS avoids the disadvantage of host gene sequences occupying a large amount of data, focuses the detection focus on the target genome, improves the sensitivity of detection, and is extremely cost-effective.

[0003] Chinese patent application 202110097754.3 divides the reference genome sequence of the new coronavirus into multiple windows with a window range of 200-300bp, designs primers for amplifying products in overlapping regions (50-100bp) in the windows, and obtains the full genome sequence of the new coronavirus through a multiplex PCR method. The multiplex amplification products are mostly 200-300bp. After obtaining the multiplex amplification products, a second-generation sequencing library is constructed using a matching second-generation library construction method to achieve detection of the new coronavirus full genome.

[0004] Chinese patent application 202010219661.9 also uses a variety of amplification primers designed for the entire genome sequence, 98 pairs of shingled multiple primers to quickly amplify the whole genome of the new coronavirus, and the PCR reaction obtains coronavirus gene fragments of different sizes, which can then be directly used for second-generation sequencing library construction experiments and third-generation sequencing platforms.

[0005] Most of the amplicon product fragments designed by the existing technology are short, the number of amplicon primers is large, and there are amplification differences between different amplicons. For samples with low viral load, uneven coverage is prone to occur, and the detection sensitivity is low. The entire library construction process involves fragmentation, adding adapters, and library amplification steps, with many operation steps and a long process time. Summary of the invention

[0006] In view of the defects of the prior art, the present invention designs and synthesizes specific primers for the whole genome of the new coronavirus, which are used to target and capture the genome fragments of the new coronavirus. This design designs 29 sets of shingled primers for the reference genome sequence of the new coronavirus, and uses the detection primers provided by the present invention to amplify the sample to be tested. The obtained amplified product is purified to obtain a sequencing fragment, and the sequencing fragment is sequenced to detect and identify the new coronavirus. These primers should be able to target and capture all fragments of the viral genome to avoid missing important mutation sites.

[0007] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0008] One of the purposes of the present invention is to provide a primer set for targeted detection of the whole genome of the new coronavirus, the primer set comprising:

[0009] (1) a first primer set; and

[0010] (2) a second primer set;

[0011] The first primer set includes at least two selected from the sequences shown in SEQ ID NOs: 1-30;

[0012] The second primer set includes at least two selected from the sequences shown in SEQ ID NOs: 31-58.

[0013] In some examples, the first primer set includes sequences shown as SEQ ID NOs: 1-30.

[0014] In some examples, the second primer set includes sequences shown as SEQ ID NOs: 31-58.

[0015] In some examples, the amplification product of the primer pair that is the upstream primer and the downstream primer of each other in the primer set is between 1100-1200 bp.

[0016] The primer mixing ratio provided by the present invention can make the genome detection coverage more uniform by using the primer mixing ratio.

[0017] In some examples, the primer set includes the following amounts of primers, measured in parts by volume:

[0018]

[0019]

[0020]

[0021] In some examples, the volume fractions are in μL.

[0022] The second object of the present invention is to provide the use of any of the aforementioned primer sets in the preparation of detection reagents for whole genome targeted detection of the new coronavirus.

[0023] The third object of the present invention is to provide a detection reagent for targeted detection of the whole genome of the new coronavirus, wherein the detection reagent includes any of the aforementioned primer sets.

[0024] In some examples, the detection reagent further comprises at least one of the following components:

[0025] Reverse transcription reagents, such as reverse transcriptase; PCR amplification reagents, such as DNA polymerase, preferably a high-fidelity enzyme; reaction solvents, such as sterile water, distilled water, etc.; buffers, etc.

[0026] The fourth object of the present invention is to provide a kit for targeted detection of the whole genome of the new coronavirus, wherein the kit comprises any of the aforementioned primer sets or any of the aforementioned detection reagents.

[0027] In some examples, the kit further comprises at least one of the following components:

[0028] Amplification templates, positive quality control products, negative quality control products, etc.

[0029] A fifth object of the present invention is to provide a method for constructing a novel coronavirus sequencing library, the method comprising:

[0030] 1) Isolating nucleic acid from the sample to be tested;

[0031] 2) amplifying the nucleic acid using any of the aforementioned primer sets, detection reagents or kits to obtain an amplified product;

[0032] 3) Purify the amplified product described in step 2) to obtain a novel coronavirus sequencing library.

[0033] In some instances, the sample to be tested is a sample that contains or is suspected of containing the new coronavirus.

[0034] In some preferred examples, the sample to be tested includes, but is not limited to at least one of sputum, throat swab, nasal swab, and alveolar lavage fluid.

[0035] In some examples, the purification in step 3) comprises the following steps:

[0036] (i) contacting the solid support to which the capture medium is bound with the amplification product in step 2) under hybridization conditions, so that the capture medium and the amplification product are hybridized to obtain a hybridization product;

[0037] (ii) separating the solid support from the hybridization product to obtain a purified amplification product.

[0038] In some examples, the solid support includes, but is not limited to, magnetic beads.

[0039] In some examples, the primer set includes the following amounts of primers, measured in parts by volume:

[0040]

[0041]

[0042] In some examples, the volume fractions are in μL.

[0043] The sixth object of the present invention is to provide a method for determining the sequence of a novel coronavirus, the method comprising the following steps:

[0044] (I) sequencing a novel coronavirus sequencing library obtained by any of the aforementioned methods for constructing a novel coronavirus sequencing library to obtain a sequencing result;

[0045] (II) Analyze the sequencing results to determine the sequence of the new coronavirus.

[0046] In some examples, the sequencing in step (I) is genomic sequencing, preferably second-generation sequencing or third-generation sequencing.

[0047] The present invention designs two sets of primers for the genomes of different subtypes of the new coronavirus by comprehensively considering the RNA integrity and primer specificity of the sample to be tested, and the amplification product of each pair of amplification primers is between 1100-1200bp. There is an overlapping region of 90bp-300bp between the adjacent primer amplification regions of the amplification primers in the detection primer set, which has a strong multi-type coverage capability, and sequencing is performed for different types, with a sequencing depth of ≥100× or more, and the whole genome coverage can reach more than 99%, with high coverage uniformity, and the sequencing depth of some subtypes can reach up to 150000×. In addition, for different types of samples with lower concentrations, the sequencing coverage can reach more than 99% of the whole genome coverage, so the detection sensitivity of the present invention is extremely high.

[0048] The present invention uses only 29 pairs of primers, which significantly reduces the production cost. By reducing the number of primer pairs and increasing the length of the amplified fragment, the problems of amplification difference and uneven coverage are avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The gene coverage of the BF.7 subtype of the novel coronavirus detected using the primer set ratio (test group) of the present invention is shown. The genome coordinate unit is bp.

[0050] Figure 2The figure shows the gene coverage of the BF.7 subtype of the new coronavirus detected using other primer set ratios (control group). The genome coordinate unit is bp.

[0051] Figure 3 The gene coverage of the BA.5 subtype of the novel coronavirus detected using the primer set ratio (test group) of the present invention is shown, wherein the genome coordinate unit is bp.

[0052] Figure 4 The figure shows the gene coverage of the BA.5 subtype of the new coronavirus detected using other primer set ratios (control group). The genome coordinate unit is bp.

[0053] Figure 5 The gene coverage of the XBB subtype of the novel coronavirus detected by the primer set ratio (test group) of the present invention is shown. The genome coordinate unit is bp.

[0054] Figure 6 The figure shows the gene coverage of the XBB subtype of the new coronavirus detected using other primer set ratios (control group). The genome coordinate unit is bp.

[0055] Figure 7 The gene coverage of the novel coronavirus EG5.1 subtype detected by the primer set ratio (test group) of the present invention is shown. The genome coordinate unit is bp.

[0056] Figure 8 The figure shows the gene coverage of the EG5.1 subtype of the novel coronavirus detected using other primer set ratios (control group). The genome coordinate unit is bp. DETAILED DESCRIPTION

[0057] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0058] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0059] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0060] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0061] The terms "optional", "either", "any", or "any" as used herein mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. As used herein, "a" and "an" are used herein to refer to one or more than one of the grammatical objects.

[0062] The term "and / or" used herein should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0063] The terms "new coronavirus", "novel coronavirus", "SARS-CoV-2", "Corona Virus" or "2019n-COV" used in this article all refer to severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2) (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5, 536-544 (2020).).

[0064] The term "uniformity" as used herein refers to a measure of the uniformity of the sequencing depth of sequenced reads in a target region. In some examples, uniformity refers to the proportion of base sites with a depth greater than 20% of the average depth to the total number of base sites in the target region.

[0065] The reagents and materials designed by the present invention are shown in Table 1:

[0066] Table 1

[0067] name Supplier / Brand Part Number HiScriptIII 1st Strand cDNA Synthesis Kit Vazyme R312 2×Phanta Max Master Mix Vazyme P515

[0068] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as " Molecular Cloning Experiment Guide (Fourth Edition) " (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0069] Example 1 Detection of the BF.7 subtype of the novel coronavirus

[0070] The present invention extracts nucleic acid from the isolated novel coronavirus BF.7 strain to obtain RNA of the sample to be tested.

[0071] 1.1 Single-strand cDNA synthesis

[0072] The reaction system and reaction procedure for cDNA synthesis are shown in Tables 2 and 3. The following cDNA synthesis reaction was performed to obtain a cDNA template.

[0073] Table 2

[0074]

[0075] Table 3

[0076]

[0077]

[0078] 1.2 Targeted capture amplification

[0079] The first primer set and the second primer set are respectively configured into two reaction systems, and the cDNA template obtained above is amplified. The reaction system and reaction procedure are shown in Table 4 and Table 5. The first primer set includes primers with nucleotide sequences as shown in SEQ ID NO: 1-30, and the second primer set includes primers with nucleotide sequences as shown in SEQ ID NO: 31-58. See Table 6 for primer sequences and configuration volumes. The configuration method is as follows: pool 1 (primer set (grouping) 1 mixture) and pool 2 (primer set (grouping) 2 mixture) are respectively configured according to the volumes shown in Table 6, and 1 μL is taken from pool 1 and pool 2 respectively during amplification.

[0080] Table 4

[0081]

[0082] Table 5

[0083]

[0084] Table 6

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] 1.3 Capture fragment purification

[0091] The amplification product corresponding to the first primer set (25 μL) and the amplification product corresponding to the second primer set (25 μL) were combined into a 1.5 mL EP tube, totaling 50 μL. About 40 μL of Agencourt AMPure XP (Beckman, CAT#: A63880) magnetic beads were used for purification, and the purified product was eluted with nuclease-free water after rinsing twice with 200 μL of freshly prepared 80% ethanol.

[0092] The purified fragments were sequenced using the next-generation sequencing technology.

[0093] Example 2 Detection of BA.5 subtype of novel coronavirus

[0094] The present invention extracts nucleic acid from the isolated BA.5 strain of the new coronavirus to obtain RNA of the sample to be tested.

[0095] The remaining detection steps and primer sets are the same as those in Example 1.

[0096] Example 3 Detection of the XBB subtype of the novel coronavirus

[0097] The present invention extracts nucleic acid from the isolated novel coronavirus XBB strain to obtain RNA of the sample to be tested.

[0098] The remaining detection steps and primer sets are the same as those in Example 1.

[0099] Example 4 Detection of novel coronavirus EG 5.1 subtype

[0100] The present invention extracts nucleic acid from the isolated novel coronavirus EG 5.1 strain to obtain RNA of the sample to be tested.

[0101] The remaining detection steps and primer sets are the same as those in Example 1.

[0102] In order to ensure the quality of the samples to be tested, the Ct value of the extracted nucleic acid was detected by the SARS-CoV-2 qRT-PCR kit (ABT-X Beijing Zhuocheng Huisheng Biotechnology Co., Ltd., CAT#: Z-AQ322). The results are shown in Table 7.

[0103] Table 7

[0104]

[0105] From the above results, it can be seen that the nucleic acid extracted from the sample to be tested by the present invention has better quality.

[0106] The sequencing results of Examples 1-4 were subjected to bioinformatics analysis, and the results are shown in Table 8.

[0107] Table 8

[0108]

[0109]

[0110] The parameters in the table show the detection of the four subtypes in this system. Sequencing quality This article uses the Q20 parameter. For the second-generation sequencing data, the Q20 base must be above 95%. The Q20 standards tested in this example are all within the standard range, indicating that the sequencing data quality is good; the coverage ≥ 100× parameter refers to: the probability that the sequencing base has been read by more than 100 sequences. In this example, the coverage ≥ 100× parameters are all above 99%, indicating that the coverage rate is extremely high, that is, the entire genome of the new crown has been detected; bioinformatics typing: refers to the subtyping of sequencing data through bioinformatics analysis.

[0111] Analysis of the coverage of samples of different concentrations of each subtype (coverage ≥ 100×) shows that the whole genome data can be evenly covered in each subtype, and there will be no differential detection of subtypes. In the analysis of sample coverage, the detection limit of each subtype was tested to Ct32, that is, when the sample concentration was extremely low, the detection accuracy remained at a high level with high sensitivity. When subtyping based on bioinformatics data, the accurate subtype of the sample was accurately typed, that is, the specificity was high.

[0112] Test Example 1

[0113] The ratio of the primer combination of the present invention (test group) was tested and compared with the ratio of the primer combination of other concentrations (control group). The bioinformatics data was mapped to the new crown reference genome to generate a gene coverage map. The specific experimental steps are as follows:

[0114] The extraction, detection steps and primer sets of the SARS-CoV-2 BF.7, BA.5, XBB and EG 5.1 strains are the same as those in Examples 1-4.

[0115] Experimental group: The primers were configured according to the ratios in Table 6.

[0116] Control group: The primer groups were mixed in equal proportions (all primers were taken in 1 μL).

[0117] The same template and primer set (the primer sets are the same as those in Example 1, with the same sequences but different ratios) were used for amplification and sequencing, and the sequencing results were analyzed by bioinformatics. The results are shown in Table 9. The bioinformatics data were mapped to the new crown reference genome to generate a gene coverage map ( Figure 1-Figure 8 ).

[0118] Table 9

[0119]

[0120] The bioinformatics results show that the mixing of primers in different ratios has a great influence on the uniformity of gene detection. The use of the primer set and configuration ratio provided by the present invention can better and more uniformly detect the whole genome data of each subtype. In the 4 subtype samples, compared with the control scheme, the ratio of base sites with an average depth greater than 20% in the test group (coverage ≥ 20%) was higher than that in the control group, namely, the BF.7 subtype test group (coverage ≥ 20% = 95.82%) was better than the control group (coverage ≥ 20% = 95.06%), the BA.5 subtype test group (coverage ≥ 20% = 99.21%) was better than the control group (coverage ≥ 20% = 91.28%), the XBB subtype test group (coverage ≥ 20% = 99.22%) was better than the control group (coverage ≥ 20% = 87.52%), and the EG5.1 subtype test group (coverage ≥ 20% = 95.82%) was better than the control group (coverage ≥ 20% = 79.97%). The uniformity was significantly improved in BA.5, BF.7, XBB, and EG5.1.

[0121] Map the bioinformatics data to the COVID-19 reference genome and generate a gene coverage map. Figure 1-Figure 8 It can be seen that the primer ratio provided by the present invention is more uniform in the coverage of different genes. Comparing the gene distribution map of the test group with that of the control group, the ratio of the primer group used in the test group, i.e., this scheme, is more uniformly distributed on different genes, which greatly improves the uneven distribution of sequencing of different gene sites in the control group, and has a significant effect on improving uniformity.

[0122] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A primer set for targeted detection of the whole genome of the new coronavirus, characterized in that: The primer set comprises: (1) a first primer set; and (2) a second primer set; The first primer set consists of the sequences shown in SEQ ID NO: 1-30; The second primer set consists of the sequences shown in SEQ ID NO: 31-58, The sequences of the first primer set and the second primer set are configured according to the following ratio:

2. Use of the primer set according to claim 1 in the preparation of a detection reagent for whole genome targeted detection of the new coronavirus.

3. A detection reagent for targeted detection of the whole genome of the new coronavirus, characterized in that: It comprises the primer set according to claim 1.

4. The detection reagent according to claim 3, characterized in that It also includes at least one of the following components: a reverse transcription reagent, a PCR amplification reagent, a reaction solvent, and a buffer.

5. The detection reagent according to claim 4, characterized in that The reverse transcription reagent is reverse transcriptase, the PCR amplification reagent is a high-fidelity enzyme, and the reaction solvent is sterile water or distilled water.

6. A kit for targeted detection of the whole genome of the new coronavirus, characterized in that: It comprises the primer set according to claim 1 or the detection reagent according to any one of claims 3-5.

7. The kit according to claim 6, characterized in that It also includes at least one of the following components: an amplification template, a positive quality control product, or a negative quality control product.

8. A method for constructing a novel coronavirus sequencing library, characterized in that: The method comprises: 1) Isolating nucleic acid from the sample to be tested; 2) amplifying the nucleic acid using the primer set of claim 1, the detection reagent of any one of claims 3-5, or the kit of any one of claims 6-7 to obtain an amplified product; 3) Purify the amplified product described in step 2) to obtain a novel coronavirus sequencing library.

9. The method according to claim 8, characterized in that The sample to be tested is a sample that contains or is suspected of containing the new coronavirus.

10. The method according to claim 9, characterized in that The sample to be tested includes at least one of sputum, throat swab, nasal swab, and alveolar lavage fluid.

11. The method according to any one of claims 8 to 9, characterized in that: Purification in step 3) comprises the following steps: (i) contacting the solid support to which the capture medium is bound with the amplification product in step 2) under hybridization conditions, so that the capture medium and the amplification product are hybridized to obtain a hybridization product; (ii) separating the solid support from the hybridization product to obtain a purified amplification product.

12. The method according to claim 11, characterized in that The solid support comprises magnetic beads.

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