A primer set for monkeypox virus whole genome amplification and its application

Abstract

The present invention belongs to the field of gene detection, and specifically relates to a primer set for whole genome amplification of monkeypox virus and its application. The present invention provides a primer set for whole genome amplification of monkeypox virus, the primer set comprising: a first primer set, which comprises at least two selected from the sequences shown in SEQ ID NO:1-178; a second primer set, which comprises at least two selected from the sequences shown in SEQ ID NO:179-358. The primer set provided by the present invention for monkeypox virus has better sensitivity, can achieve whole genome amplification of monkeypox virus, the proportion of fragments with gene coverage of more than 30× is more than 99.9%, and the sequencing depth can reach up to 90000×. The present invention has broad application prospects in monitoring monkeypox virus subtypes.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and in particular relates to a primer set for monkeypox virus whole genome amplification and an application thereof. Background Art

[0002] Monkeypox is a zoonosis caused by infection with the monkeypox virus (MPXV). The monkeypox virus belongs to the genus Orthopoxvirus of the Poxviridae family. It is a double-stranded DNA virus with a genome length of approximately 197 kb. The virus was first discovered in monkeys in 1958. The monkeypox virus is divided into two evolutionary branches: (1) Branch I, formerly known as the Central African branch or the Congo Basin branch; (2) Branch II, formerly known as the West African branch, is further divided into branches IIa and IIb. The strain that caused the global non-endemic monkeypox outbreak in 2022 is branch IIb. Branch IIb has now developed many subbranches, including subbranches A.1, A.2, and B.1.

[0003] In order to guide all localities to carry out scientific and precise monkeypox prevention and control work, organize epidemiological investigations in a timely manner, and analyze the transmission chain and characteristics, it is necessary to conduct a whole genome analysis of the monkeypox virus.

[0004] Chinese patent application CN116987821A discloses a fluorescent quantitative PCR kit for detecting monkeypox virus, including a primer set, wherein the primer set includes an upstream primer of a target gene and a downstream primer of a target gene. The application designs a specific primer probe for monkeypox virus, uses RNase P as an internal standard, combines fluorescent quantitative PCR technology, and uses specific probes to identify quantitative molecules.

[0005] Chinese patent application CN101407850A discloses a PCR rapid detection kit for monkeypox virus and a detection method thereof, comprising a kit consisting of reagents A, B, PCR reagent tubes, TaqDNA polymerase, a positive control of pUC57 plasmid containing a monkeypox virus-specific F3L gene fragment, and a sterilized double distilled water negative control. A simple, rapid and sensitive PCR detection kit is used, specific primers are designed based on a conserved sequence unique to monkeypox virus, and amplification comparison is performed, and the monkeypox virus detection result can be obtained within 3-4 hours, with high sensitivity, and the detection limit is as low as 0.3pg of DNA concentration, ensuring the accuracy and specificity of the detection.

[0006] Currently, most methods in this field use fluorescent quantitative PCR to detect monkeypox virus. Although it can quickly identify whether a sample contains monkeypox virus, false positives are common, the detection is not accurate enough, and the specific branch of monkeypox virus cannot be determined, which is not conducive to monitoring the mutation and prevalence of monkeypox virus. Summary of the invention

[0007] Among the sequencing technologies for the whole genome of the virus, amplicon sequencing is still the most widely used method. However, the monkeypox genome is large, and it is easy to have uneven amplification, which in turn affects the final sequencing results and the accuracy of identification. There is an urgent need for a monkeypox whole genome amplification primer set with uniform amplification and good coverage.

[0008] In view of this, the present invention specifically provides a primer set for amplifying the whole genome of monkeypox virus.

[0009] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0010] One of the purposes of the present invention is to provide a primer set for amplifying the whole genome of monkeypox virus, the primer set comprising:

[0011] (1) a first primer set comprising at least two selected from the sequences shown in SEQ ID NOs: 1-178; and

[0012] (2) A second primer set comprising at least two selected from the sequences shown in SEQ ID NOs: 179-358.

[0013] In some examples, the first primer set includes sequences shown as SEQ ID NO: 1-178.

[0014] In some examples, the second primer set includes a sequence selected from the group consisting of SEQ ID NOs: 179-358.

[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 larger than 600 bp.

[0016] 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 600-2500 bp.

[0017] A second object of the present invention is to provide a use of any of the aforementioned primer sets in the preparation of a detection reagent for monkeypox virus whole genome amplification.

[0018] A third object of the present invention is to provide a detection reagent for monkeypox virus whole genome amplification, wherein the detection reagent comprises any of the aforementioned primer sets.

[0019] In some examples, the detection reagent further comprises at least one of the following components:

[0020] PCR amplification reagents, such as DNA polymerase, preferably a high-fidelity enzyme; reaction solvents, such as sterile water, distilled water, etc.; buffers, etc.

[0021] A fourth object of the present invention is to provide a kit for amplifying the whole genome of monkeypox virus, the kit comprising any of the aforementioned primer sets or any of the aforementioned detection reagents.

[0022] In some examples, the kit further comprises at least one of the following components:

[0023] Amplification templates, positive quality control products, negative quality control products, etc.

[0024] A fifth object of the present invention is to provide a method for constructing a monkeypox virus sequencing library, the method comprising:

[0025] 1) Isolating nucleic acid from the sample to be tested;

[0026] 2) amplifying the nucleic acid using any of the aforementioned primer sets, detection reagents or kits to obtain an amplified product;

[0027] 3) Purifying the amplified product in step 2) to obtain a monkeypox virus sequencing library.

[0028] In some examples, the sample to be tested is a sample containing or suspected of containing monkeypox virus.

[0029] In some preferred embodiments, the sample to be tested includes, but is not limited to, at least one of a rash cover, a scab, blister fluid, pustular fluid, anticoagulated whole blood and serum.

[0030] In some examples, the purification in step 3) comprises the following steps:

[0031] (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;

[0032] (ii) separating the solid support from the hybridization product to obtain a purified amplification product.

[0033] In some examples, the solid support includes, but is not limited to, magnetic beads.

[0034] In some examples, step 3) further comprises fragmenting the purified amplification product.

[0035] A sixth object of the present invention is to provide a method for determining a monkeypox virus sequence, the method comprising:

[0036] (I) sequencing a monkeypox virus sequencing library obtained by any of the aforementioned methods for constructing a monkeypox virus sequencing library to obtain a sequencing result;

[0037] (II) Analyze the sequencing results to determine the monkeypox virus sequence.

[0038] In some examples, the sequencing in step (I) is genomic sequencing, preferably second-generation sequencing or third-generation sequencing.

[0039] The primer set provided by the present invention for monkeypox virus can realize the whole genome amplification of monkeypox virus, the proportion of fragments with gene coverage of more than 30× is more than 99.9%, and the sequencing depth can reach up to 90000×. At the same time, the primer set provided by the present invention has better sensitivity. When amplifying and sequencing with a template with a Ct value of 32, the coverage with a sequencing depth of more than 30× can still reach 99.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The depth results of the next generation sequencing of the sequencing library constructed with the S1 (hMpxV) template with a Ct value of 25. The genomic coordinate unit is bp.

[0041] Figure 2 The depth results of the second generation sequencing of the sequencing library constructed with the S2 (hMpxV) template with a Ct value of 28. The genomic coordinate unit is bp.

[0042] Figure 3 The depth results of the third-generation sequencing of the sequencing library constructed with the S1 (hMpxV) template with a Ct value of 25. The unit of the genomic coordinates is bp.

[0043] Figure 4 The depth results of the third-generation sequencing of the sequencing library constructed with the S2 (hMpxV) template with a Ct value of 28. The genomic coordinate unit is bp.

[0044] Figure 5 The depth results of the second generation sequencing of the sequencing library constructed with template S1 (hMpxV) with a Ct value of 32. The unit of genomic coordinates is bp.

[0045] Figure 6 The depth results of the second generation sequencing of the sequencing library constructed with template S1 (hMpxV) with a Ct value of 33. The unit of genomic coordinates is bp. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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".

[0050] 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.

[0051] 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.

[0052] The reagents and materials designed by the present invention are shown in Table 1:

[0053] Table 1

[0054]

[0055] 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.

[0056] Example 1 Construction of Monkeypox Virus Sequencing Library

[0057] The samples used were collected from damaged areas of the patient's skin. Nucleic acid was extracted using a nucleic acid extraction reagent (Zhuocheng Huisheng, catalog number CN8033-48T) to obtain a monkeypox genomic DNA template.

[0058] Two tubes of different primer pools (pool 1 (primer set 1) and pool 2 (primer set 2)) were configured for multiple amplification systems, the reaction system is shown in Table 2, and the reaction procedure is shown in Table 3. The sequences of the primer sets are shown in Table 4.

[0059] After amplification on the machine, two tubes of amplification products were obtained for each sample.

[0060] Library construction: The library was constructed using Illumina's Nextera XT Library Preparation Kit (Cat. No.: FC-131-1024). The specific process is as follows:

[0061] (1) Combine the two tubes of amplification products and purify them with 180 μL of AMPure XP beads (Beckman, Catalog No. A63881);

[0062] (2) adding the purified products from the previous round to ATM and TD components for fragmentation;

[0063] (3) The fragmented sequences are further amplified through the Index to complete library construction;

[0064] (4) Perform library quality inspection: The dsDNA HS Assay Kit double-stranded fluorescence quantitative kit was used for library quantification (the qualified library output should be above 50 ng). The library quality inspection results are shown in Table 2, where S1 (hMpxV) and S2 (hMpxV) are different samples.

[0065] Table 2

[0066]

[0067] Sequencing was performed on the machine, and the monkeypox genome assembly was completed based on the off-machine sequencing data.

[0068] Table 3

[0069]

[0070] Table 4

[0071]

[0072] Table 5

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Example 2 Sequencing Data Analysis

[0086] This monkeypox virus sequencing evaluation conducted a full-process evaluation and verification of 2 samples (S1 (hMpxV) and S2 (hMpxV)) using both NGS (second-generation sequencing) and TGS (third-generation sequencing) sequencing methods. The evaluation and verification results are as follows.

[0087] The sequencing data obtained in Example 1 were subjected to bioinformatics analysis, and the sequencing fragment sequences were aligned to the genome (OR459779.1). The full length of the monkeypox genome is 197193 bp, and then the coverage depth of each genomic site (i.e., how many sequencing fragments cover each site) was calculated. The results showed that the number of genomic sites with a sequencing depth of more than 30× was x bp, the coverage = x / 197193×100%, the coverage ≥99.9%, and the amplification efficiency was uniform (Table 5 and Figure 1-Figure 4 ).

[0088]

[0089] Comparative Example 1

[0090] This comparative example uses other primer sets as controls to compare the sequencing effects of the primer set of the present invention and other primer sets. The primers of the control group (other primer sets) are shown in Table 7.

[0091] Table 7

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] The sequencing library construction and sequencing data analysis of this comparative example are the same as those of Example 1 and Example 2. The libraries constructed using the primer sets of the present invention (primer sets 1 and 2) and the control primer sets (primer sets 3 and 4) were analyzed, and the results are shown in Table 8.

[0101] Table 8

[0102]

[0103] The experimental results in Table 8 show that the 30× coverage of the primer set of the present invention is much higher than that of the primers in the control group.

[0104] Test example

[0105] The meaning of the threshold cycle number (Cycle threshold, Ct value) is: the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold. There is a linear relationship between the Ct value and the logarithm of the starting copy number of the template. The higher the starting template concentration, the smaller the Ct value; the lower the starting template concentration, the larger the Ct value.

[0106] In order to test the detection limit of the primer set provided by the present invention, this test example uses starting templates of different concentrations (ie, different Ct values) for amplification, constructs sequencing libraries, and then measures coverage at a depth of more than 30×.

[0107] After testing the sample concentration gradient, the nucleic acid extracted from the S1 (hMpxV) sample was gradiently diluted, and the Ct values ​​were Ct25, Ct28, Ct30, Ct32, Ct33, Ct34, and Ct35. The experimental results are shown in Table 9. The coverage of the Ct32 sample at 30× is still 99.90%, and the coverage of the Ct33 sample at 30× is 86.39%, which is significantly reduced. Finally, it is confirmed that the detection limit of the reagent of the present invention is Ct32.

[0108] Table 9

[0109]

[0110] The sequencing depth diagrams of the sequencing libraries constructed with templates having a Ct value of 32 (i.e., Ct32) and a Ct value of 33 (i.e., Ct33) are shown in FIG. Figure 5 and Figure 6 shown.

[0111] 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 whole genome amplification of monkeypox virus, characterized in that: The primer set consists of a first primer set and a second primer set; the first primer set is the sequence shown in SEQ ID NO: 1-178; the second primer set is the sequence shown in SEQ ID NO: 179-358.

2. Use of the primer set according to claim 1 in preparing a detection reagent for monkeypox virus whole genome amplification.

3. A detection reagent for monkeypox virus whole genome amplification, characterized in that: The detection reagent comprises the primer set according to claim 1.

4. The detection reagent according to claim 3, characterized in that The detection reagent further comprises at least one of the following components: a PCR amplification reagent, a reaction solvent or a buffer.

5. The detection reagent according to claim 4, characterized in that The PCR amplification reagent is DNA polymerase; the reaction solvent is sterile water.

6. The detection reagent according to claim 5, characterized in that The DNA polymerase is a high-fidelity enzyme.

7. A kit for amplifying the whole genome of monkeypox virus, characterized in that: The kit comprises the primer set according to claim 1 or the detection reagent according to any one of claims 3-6.

8. The kit according to claim 7, characterized in that The kit further comprises at least one of the following components: Amplification template, positive control or negative control.

9. A method for constructing a monkeypox virus sequencing library, characterized in that: The method comprises: 1) Isolation of 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 to 6, or the kit of any one of claims 7 to 8 to obtain an amplification product; 3) Purify the amplified product described in step 2) to obtain a monkeypox virus sequencing library.

10. The method according to claim 9, characterized in that The sample to be tested is a sample containing or suspected of containing monkeypox virus.

11. The method according to claim 9 or 10, characterized in that: The sample to be tested includes at least one of the upper cover of the rash, scab, blister fluid, pustular fluid, anticoagulated whole blood and serum.

12. The method according to claim 9, characterized in that The purification in step 3) comprises the following steps: (i) contacting the solid support with the capture medium 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.

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

14. The method according to claim 13, characterized in that The step 3) further comprises fragmenting the purified amplification product.

15. A method for determining a monkeypox virus sequence, characterized in that: The method comprises: (I) sequencing the monkeypox virus sequencing library obtained by the method of claims 9 to 14 to obtain sequencing results; (II) Analyze the sequencing results to determine the monkeypox virus sequence.

16. The method according to claim 15, characterized in that The sequencing described in step (I) is genome sequencing.

17. The method according to claim 16, characterized in that The genome sequencing is second generation sequencing or third generation sequencing.

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