Varicella-zoster virus whole genome capturing method, primer group and kit

Abstract

The invention discloses a varicella-zoster virus whole genome capturing method, a primer group and a kit. The primer combination for amplifying the whole genome sequence of the varicella-zoster virus disclosed by the invention can effectively amplify the whole genome sequence of the varicella-zoster virus, is high in coverage rate, and is beneficial to further research on pathogenic mechanisms, molecular epidemiology, etiology and the like of the varicella-zoster virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to a method, a primer set and a kit for capturing the whole genome of varicella-zoster virus. Background Art

[0002] Varicella-zoster virus (VZV), also known as human herpes virus-3 (HHV3), belongs to the α-herpesvirus subfamily of the Herpesviridae family. It is a DNA virus and its genes have good genetic stability, but there may still be genetic variations between different strains. VZV has only one serotype and is genetically stable. Its genome variation generally depends on single nucleotide variation. Internationally, single nucleotide polymorphisms (SNPs) based on different ORFs of the VZV genome are usually used to genotype VZV. According to the international universal nomenclature of VZV genotypes published by Breuer et al., VZV can be divided into five major genotypes (clade 1-5) and two tentative genotypes to be confirmed (clade VI and VII), and the SNP virus genotyping method based on ORF22 is currently the most widely used. Next-generation sequencing (NGS) is a highly sensitive method for virus detection and analysis using nucleic acids. It can provide complete viral genome information and monitor viral evolution.

[0003] VZV primary infection often occurs in childhood and can cause chickenpox characterized by systemic vesicular eruptions. After infection, the virus can remain dormant in the host's neuronal cells. With age, decreased immune function, or other factors, the latent virus can be reactivated and spread along the peripheral nerves, causing herpes zoster (HZ). With the popularization of vaccines, the incidence of chickenpox has been basically controlled, but HZ is common in many elderly people. For people who are infected with VZV for the first time and have low immunity or VZV reactivation, they are often accompanied by complications such as meningitis, stroke, and post-HZ neuralgia, which reduce the patient's quality of life and even endanger their lives.

[0004] Therefore, in order to better determine the genome sequence and detect genome variations of varicella-zoster virus, it is very important to provide primers that can amplify varicella-zoster virus. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method, a primer set and a kit for capturing the whole genome of varicella-zoster virus.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: In a first aspect, the present invention provides a primer combination for amplifying the whole genome sequence of varicella-zoster virus.

[0007] Furthermore, the primer combination includes 5 sets of primers.

[0008] Furthermore, the sequence of the primer combination is shown in SEQ ID No. 1-10.

[0009] In the present invention, primer combinations and primers can be used interchangeably, and primers refer to short nucleic acid molecules. The primers disclosed in the present invention are about 22 nucleotides in length. Can be annealed with complementary target nucleic acid molecules by nucleic acid hybridization, forming a hybrid between the primer and the target nucleic acid chain. Primers can be extended along the target nucleic acid molecule by polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, wherein the sequence of the primer is specific to the target nucleic acid molecule.

[0010] Target nucleic acid molecule refers to a nucleic acid molecule intended to be detected, quantitatively, qualitatively detected or a combination thereof. Nucleic acid molecules are not necessarily in a purified form. Various other nucleic acid molecules may also coexist with the target nucleic acid molecule. For example, the target nucleic acid molecule may be a specific nucleic acid molecule intended to be amplified. If desired, the purification or separation of the target nucleic acid molecule may be carried out by methods known to those skilled in the art, for example, using commercially available purification kits etc.

[0011] In some embodiments, primers and / or probes corresponding to sequences having more than 70% homology with the primer and / or probe sequences described in the present invention are also included in the protection scope of the present invention, that is, it can be understood that the primers and / or probes obtained after changes are made on the basis of the primers and / or probes provided by the present invention also fall within the protection scope of the present invention.

[0012] In some embodiments, the primer combination can be labeled with a labeling substance. The labeling substance includes but is not limited to a fluorescent substance, a radioisotope or an enzyme. Among them, the fluorescent substance includes but is not limited to TAMRAT TM , Alexa555, Alexa647, Cy3, Cy5 of the cyanine dye series, and fluorescein. Radioactive isotopes include but are not limited to 32 P. 33 P. 35 S. Enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase.

[0013] In the primers of the present invention labeled with a labeling substance, the labeling substance may be directly bound to the primer or may be bound via a linker. As a linker, any linker commonly used in the field may be used, specifically, for example, preferably a nucleic acid of 1 to 3 bases, more preferably a DNA of 1 to 3 bases, further preferably a DNA of 2 bases, and particularly preferably the 2 bases of adenine (A)-adenine (A).

[0014] The method of labeling the primer involved in the present invention with a fluorescent substance can be carried out according to a method generally known in the art. Specifically, for example, a method of incorporating a nucleotide labeled with fluorescent substance into a primer according to a method known in the art can be mentioned.

[0015] As a method for labeling the primer involved in the present invention with a radioactive isotope, it can be carried out according to a method known per se that is usually carried out in this field. Specifically, for example, a method of labeling by incorporating a nucleotide labeled with a radioactive isotope can be mentioned. Specifically, a random primer method, a nick translation method, a 5' end labeling method based on T4 polynucleotide kinase, a 3' end labeling method based on terminal deoxynucleotidyl transferase, etc. can be mentioned. The primer set includes at least one modified nucleotide.

[0016] The method of labeling the primers involved in the present invention with an enzyme can be carried out according to a method known per se and generally performed in the field. Specifically, for example, there can be mentioned a direct labeling method in which an enzyme molecule such as alkaline phosphatase or horseradish peroxidase is directly covalently bonded to the primer to be labeled.

[0017] A second aspect of the present invention provides a kit.

[0018] Furthermore, the kit comprises the primer combination described in the first aspect of the present invention.

[0019] Furthermore, the kit also includes conventional PCR amplification reagents, PCR product purification reagents, DNA quantification reagents and DNA library construction reagents.

[0020] Preferably, the kit further comprises instructions.

[0021] In the present invention, a suitable amount of one or more primers is provided in one or more containers, or fixed on a substrate, and the primer can be provided as a suspension in an aqueous solution, or, for example, as a freeze-dried or lyophilized powder. Wherein, the container provided with the nucleic acid can be any conventional container capable of accommodating the provided form, such as a microcentrifuge tube, an ampoule or a bottle. The kit can contain labeled or unlabeled probes for detecting varicella-zoster virus sequences.

[0022] In some applications, one or more primers (as described above) may be provided in pre-measured, single-use amounts in separate, typically disposable tubes or equivalent containers. Using such a setup, a sample to be tested for the presence of varicella-zoster virus may be added to a separate tube and amplified directly.

[0023] The amount of nucleic acid primers provided in the kit can be any suitable amount and can depend on the target market for the product. For example, if the kit is suitable for research or clinical applications, the amount of each nucleic acid primer provided can be an amount sufficient to initiate several PCR amplification reactions. General guidance for determining suitable amounts can be found in the literature of Innis et al., Sambrook et al., and Ausubel et al. The kit can contain more than two primers to facilitate PCR amplification of larger amounts of varicella-zoster virus nucleotide sequences.

[0024] In certain embodiments, the kit may contain the necessary reagents for performing a PCR amplification reaction, including DNA sample preparation reagents, enzymes for PCR, buffers, Mg 2+ and deoxyribonucleotides (dNTPs).

[0025] The enzymes of PCR include DNA polymerase and / or RNA polymerase.

[0026] The DNA polymerase includes but is not limited to Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and Klenow fragment.

[0027] dNTP is a nucleoside source for DNA amplification by PCR, and four types of dATP, dGTP, dCTP, and dTTP are necessary. In addition, dNTP can be chemically modified for use in the hot start method, for example, CleanAmpTM dNTP manufactured by TriLink BioTechnologies, Inc. can be used.

[0028] In PCR-based DNA amplification, Mg 2+ It is necessary as Mg 2+ Sources include but are not limited to MgCl2, MgSO4, etc.

[0029] In the present invention, the kit may further include instructions, which may include guidance on obtaining samples and processing samples.

[0030] Furthermore, the kit may contain genomic DNA of varicella-zoster virus used as a positive control for PCR and sterile water used as a negative control.

[0031] When implementing the present invention, other necessary instruments include pipettes, pipette tips, 1.5 ml microtubes and other instruments widely used in molecular biology experiments, and equipment include PCR, clean benches, tube centrifuges and other instruments widely used in molecular biology experiments.

[0032] The third aspect of the present invention provides a method for obtaining the complete genome sequence of varicella-zoster virus.

[0033] Furthermore, the method comprises using the primer combination described in the first aspect of the present invention to amplify the whole genome sequence of varicella-zoster virus.

[0034] Furthermore, the amplification includes LCR, NASBA, SDA, TMA, bDNA, and PCR methods.

[0035] Preferably, the amplification is performed by a PCR method.

[0036] Preferably, the amplification is performed under similar amplification conditions.

[0037] Preferably, based on a total volume of 50 μL, the PCR amplification system includes: 25 μL of PCR reaction enzyme, 5 μL of primers, 1 ng to 1 μg of DNA sample, and the balance is water.

[0038] Preferably, the reaction procedure of the PCR amplification is pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 10s, extension at 72°C for 90s, denaturation, annealing and extension for 30 cycles; and finally extension at 72°C for 2min.

[0039] Furthermore, the sample includes blood, saliva, cerebrospinal fluid, and blister fluid.

[0040] In the present invention, the amplification includes but is not limited to ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), branched DNA signal amplification (bDNA), and polymerase chain reaction (PCR).

[0041] The amplification reactions were performed under similar amplification conditions.

[0042] The present invention embodiment part provides the exemplary embodiment of amplification condition.However, the term "amplification condition" used herein relates to the temperature and / or incubation time that are suitable for obtaining the detectable amount of target.Therefore, the term "similar amplification condition" means that if desired, each target can be assayed at a similar temperature.The term "similar amplification condition" also refers to if desired, each target can be assayed under similar incubation time.In some cases, the term "similar amplification condition" also relates to the number of amplification cycles.However, it is well known in the art that the number of cycles is not always strict.For example, some samples can be removed or left before other samples for additional amplification cycles.In other cases, the term "similar amplification condition" also relates to the properties of the buffer used and the amplification reagent (enzyme, nucleotide, salt, etc.).The term "similar amplification condition" also refers to that conditions (e.g., time, buffer, number of cycles, temperature, etc.) can be slightly changed or can be the same.

[0043] In some embodiments, the library amplification can be divided into two categories: variable temperature amplification and constant temperature amplification. Variable temperature amplification mainly includes the classic polymerase chain reaction (PCR) and ligase chain reaction (LCR), while constant temperature amplification includes strand displacement amplification (SDA), rolling circle amplification (RCA), loop mediated amplification (LAMP), helicase-dependent isothermal DNA amplification (HDA), nucleic acid sequence based amplification (NASBA), transcription-based amplification system (TAS). In the specific embodiment, PCR is used as an example for library construction.

[0044] In some embodiments, primers containing a tag sequence (Index) may be used in the library amplification. By using primers containing a tag sequence to amplify the ligation product, data from different sample sources in sequencing can be distinguished and the source of the sample can be identified. The design method of the tag sequence is conventionally known to those skilled in the art, and the design of primers containing a tag sequence (Index) is also conventionally known to those skilled in the art.

[0045] In some embodiments, the sequencing can be performed by a high-throughput sequencing platform including Illumina NovaSeq, HiSeq X Ten, Illumina HiSeq, Illumina MiSeq, PacBio Sequel, 10×Genomics, and MGISEQ-2000.

[0046] In a specific embodiment of the present invention, the sequencing is performed by Illumina MiSeq platform.

[0047] A fourth aspect of the present invention provides a method for detecting varicella-zoster virus in a sample.

[0048] Furthermore, the method comprises performing amplification using the primer combination described in the first aspect of the present invention, the kit described in the second aspect of the present invention, or the method described in the third aspect of the present invention to obtain an amplified product, and performing sequencing using the amplified product.

[0049] Preferably, the samples include human samples and environmental samples, and the human samples are in vitro human samples.

[0050] Preferably, the human body sample includes blood, saliva, cerebrospinal fluid, and blister fluid.

[0051] A fifth aspect of the present invention provides a library for detecting varicella-zoster virus.

[0052] Furthermore, the library comprises the primer combination described in the first aspect of the present invention.

[0053] In some embodiments, the library refers to a collection of DNA or RNA samples that stores a large number of gene sequences. These samples are collected, classified, cataloged, and labeled so that researchers can easily find and use them to study the function of genes or find clones of specific genes. The process of establishing a library is similar to establishing a library, both of which are for the efficient management and utilization of large amounts of information or material resources. In the detection of Mycobacterium tuberculosis, the library may contain a variety of gene sequences related to the bacteria, which helps to gain a deeper understanding of its biological characteristics, pathogenic mechanisms, drug resistance and other information.

[0054] The sixth aspect of the present invention provides any one of the following applications.

[0055] Furthermore, the applications include: 1) application of the primer combination described in the first aspect of the present invention in the preparation of a product for amplifying or detecting the whole genome sequence of varicella-zoster virus; 2) application of the primer combination described in the first aspect of the present invention or the kit described in the second aspect of the present invention in amplifying the whole genome sequence of varicella-zoster virus or in sequencing varicella-zoster virus.

[0056] Compared with the prior art, the present invention has the following advantages and beneficial effects: the primer combination designed by the present invention has high amplification efficiency and high genome coverage. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a diagram of the sequencing results of the experimental group and the control group provided in this example. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0060] Example 1 Primer amplification experiment Required reagents and consumables: ULSEN® ultra-sensitive herpes zoster virus whole genome capture kit (24 reactions / box), skin lesion samples of clinical cases of herpes zoster in the Phase 3 efficacy clinical trial of herpes zoster live attenuated vaccine, and Illumina DNA Prep library preparation kit.

[0061] 1. Herpes zoster virus DNA extraction: Herpes zoster virus is extracted according to the corresponding requirements and steps in the virus DNA extraction kit. The final volume is recommended to be 30-50μLl, and 1μgDNA is required for amplification detection. If the virus is not tested directly after extraction, it can also be stored at -70℃ for future use, but repeated freezing and thawing should be avoided.

[0062] 2. Primer design and amplification reaction In order to effectively amplify the varicella-zoster virus, at the beginning of primer design, the currently sequenced varicella-zoster virus genome is downloaded and the genome is first aligned. According to the results of each alignment, the representative genome sequence in the varicella-zoster virus genome is screened, and these representative genomes are mixed and aligned. According to the length of the amplification, the position of the primer is set, and the design of each primer will be searched in the results of the mixed alignment, in order to reduce the number of primers to obtain more universal primers, that is, primers that can be paired with varicella-zoster virus genome genes.

[0063] In order to be able to effectively amplify after mixing all primers and verify the uniqueness of the primer pool combination, a primer combination control group will be set up, and the parameters of the GC content of all primers will be basically consistent to reduce the problem of low amplification efficiency caused by large differences in Tm values. At the same time, other parameters including hairpin structure, dimer, etc. are also strictly controlled.

[0064] The experimental group primer design consists of 5 sets of primers (A, B, C, D, E), and the 5 sets of primers have corresponding sample loading systems (ie, EG tube 1, EG tube 2, EG tube 3, EG tube 4, EG tube 5). The control group primer design consists of 3 sets of primers (a, b, c), and the 3 sets of primers have corresponding sample loading systems (ie, CG tube 1, CG tube 2, CG tube 3). After the 8 systems are prepared, the amplification procedure is performed. This experiment selected 3 samples that met the experimental requirements for the experimental group and control group experiments.

[0065] The primer sequence information of the experimental group and the control group used in the experiment is shown in Table 1.

[0066] Table 1 Primer sequence information

[0067] The reaction system configurations of the control group are shown in Tables 2, 3, and 4, and the reaction system configurations of the experimental group are shown in Tables 5, 6, 7, 8, and 9.

[0068] Table 2 CG tube 1

[0069] Table 3 CG tube 2

[0070] Table 4 CG tube 3

[0071] Table 5 EG tube 1

[0072] Table 6 EG tube 2

[0073] Table 7 EG tube 3

[0074] Table 8 EG tube 4

[0075] Table 9 EG tube 5

[0076] After the control group and the experimental group were prepared according to the above system, the solutions were mixed and briefly centrifuged, and then amplified according to the procedures in Table 10.

[0077] Table 10 Primer amplification program

[0078] 3. Purification After the amplification reaction is completed, herpes zoster virus gene fragments of different sizes will be obtained in one tube of PCR reaction. After the amplification program is completed, the amplification products of the five systems in the experimental group are concentrated in a 1.5ml EP tube, and the amplification products of the three systems in the control group are concentrated in a 1.5ml EP tube. After purification and qubit quantification, the subsequent library construction experiment can be carried out directly.

[0079] The specific steps are: 1) Add 250μL AMPure XP beads, *Note: This step is 1,0x-1.5x AMPureXP beads; 2) Oscillate at 1800 rpm for 2 min; incubate for 5 min; 3) Let stand on the magnetic rack for 2 minutes or until clarified, then discard the supernatant; 4) Add 500 μL of freshly prepared 80% ethanol, place on the magnetic stand for 30 seconds, and discard the supernatant; 5) Repeat the above steps; 6) Use a 10 μL pipette tip to remove any remaining liquid; 7) Dry on magnetic rack for 2 minutes; 8) Remove from the magnetic stand; add 40 μL RSB; 9) Oscillate at 1800 rpm for 2 min, then incubate at room temperature for 2 min; 10) Let the liquid stand on a magnetic rack until it becomes clear; 11) Transfer 38 μL of supernatant to a new plate.

[0080] 4. Quantification Instrument: Qubit; Reagents: Qubit dsDNA HS (High Sensitivity) Kit (make sure all reagents are at room temperature before use).

[0081] Operation procedure: a) 1 μL dye reagent + 199 μL buffer, vortex mix to get the working solution, protect from light for later use; b) Take 1-20 μL of library to Qubit working solution, the final volume is 200 μL; c) Vortex for 2-3 seconds and place at room temperature away from light for 2 minutes; d) Place the Qubit on the plate and perform the assay.

[0082] Reference document: QUICK REFERENCE - Qubit™ Assays (MAN0017210 vA.0) Example 2 Library Construction 1. DNA fragmentation 1.1 Qubit quantification, add 2-30μL DNA (1-500ng) to the PCR tube; *Note: For multiple samples, the concentration should be greater than 50 or less than 50ng to facilitate subsequent PCR amplification; 1.2 Add pure water to the sample well to make the total volume reach 30 μL; 1.3 Vortex BLT for 10 seconds to check whether the beads are completely suspended; 1.4 Prepare the fragmentation master reaction solution. For each reaction, add 11 μL of BLT and 11 μL of TB1. Vortex the master reaction solution to mix well. 1.5 Take 20 μL of the main reaction solution and add it to the PCR sample well, vortex to mix (a total of 50 μL); 1.6 Place in PCR instrument and run TAG Program (reaction system: 50 μL / well): heat cover at 100°C, incubate at 55°C for 15 min, and store at 10°C.

[0083] 2. Purification 2.1 Add 10 μL TSB to the fragmentation reaction solution, gently pipette and mix 10 times, and resuspend the beads; *Note: TSB (check for precipitation) and TWB are stored at 15-30℃ and restored to room temperature before use; 2.2 Run the program in the PCR instrument: (set the hot cover to 100°C, the system is 60μL); 37°C, 15min; store at 10°C; 2.3 Place the reaction tube in the magnetic rack, wait for the liquid to become clear (about 3 minutes), then aspirate and discard the supernatant; 2.4 Remove the reaction tube from the magnetic rack, slowly add 100 μL TWB, resuspend the magnetic beads, place the reaction tube in the magnetic rack, wait for the liquid to clarify (about 3 minutes), aspirate and discard the supernatant; 2.5 Repeat step 2.4 once; 2.6 Remove the reaction tube from the magnetic rack, slowly add 100 μL TWB and resuspend the magnetic beads, place the reaction tube in the magnetic rack, wait for the liquid to clarify (about 3 minutes), and keep the reaction tube on the magnetic rack until step 3.2 of amplifying the fragmented and labeled DNA is performed.

[0084] 3. Amplify fragmented and labeled DNA 3.1 Prepare the PCR master reaction solution (for each sample): 22μL EPM + 22μL water, vortex to mix, and shake gently; *Note: EPM: thaw on ice, invert to mix, and then centrifuge briefly; DNA Adapter: thaw at room temperature, vortex to mix and then centrifuge briefly for test tubes, and centrifuge briefly before use for well plates; 3.2 Remove the supernatant on the magnetic stand and immediately add 40 μL of PCR master reaction solution to the sample well and mix well; 3.3 For Index Adapter Tubes, add 5 μL i7 adapter and 5 μL i5 adapter respectively and mix by pipetting; 3.4 Set the hot cover to 100℃, the system to 50μL, and run the PCR program. The PCR reaction program is shown in Table 11, and the total DNA starting amount and PCR cycle number are shown in Table 12. Note: The safe stop point can be stored at 4℃ for up to 2 days; or placed on the PCR for no more than 24 hours.

[0085] Table 11 PCR reaction program

[0086] Table 12 Relationship between DNA starting amount and cycle number

[0087] 4. Library purification 4.1 Centrifuge the reaction tube and place it in a magnetic rack to wait for the liquid to clarify (about 5 minutes); 4.2 Pipette and transfer 45 μL of supernatant to a new centrifuge tube; 4.3 Fully oscillate and resuspend SPB; *Note: SPB needs to be restored to room temperature before use and vortexed to mix; 4.4 For 100-500 ng of starting DNA, perform the following steps: a. Add 40 μL H2O to the reaction tube containing the supernatant; b. Add 45 μL SPB to the reaction tube, mix thoroughly, and incubate at room temperature for 5 minutes; c. Place the reaction tube on the magnetic rack and wait for the liquid to become clear (about 5 minutes); d. During the incubation period, vortex the SPB thoroughly and then take 15 μL and add it to a new centrifuge tube; e. Transfer 125 μL of supernatant from the reaction tube on the magnetic stand to a new centrifuge tube (containing 15 μL SPB) and mix thoroughly.

[0088] 4.5 For starting DNA <100 ng, perform the following steps: a. Add 81 μL SPB to the reaction tube containing the supernatant and mix thoroughly; 4.6 Incubate at room temperature for 5 minutes, place the reaction tube on a magnetic rack, wait for the liquid to clarify (about 5 minutes), aspirate and discard the supernatant; 4.7 Add 200 μL of freshly prepared 80% ethanol to the reaction tube, incubate for 30 seconds, discard the supernatant, and repeat this step once; 4.8 Keep the reaction tube on the magnetic rack, aspirate and discard the residual ethanol in the reaction tube, and let it dry for 5 minutes; 4.9 Remove the reaction tube from the magnetic stand, add 32 μL RSB to resuspend the magnetic beads, and incubate at room temperature for 2 minutes; 4.10 Place the reaction tube on the magnetic rack, wait for the liquid to clarify (about 2 minutes), and transfer 30 μL of the supernatant to a new centrifuge tube.

[0089] 5. Library quality control 5.1 Take 1 μL of the constructed library and run it on Agilent Bioanalyzer 2100 (optional); 5.2 Qubit quantification; molar concentration (nM) = mass concentration (ng / μL) * 10 to the sixth power / (660 * average fragment length of the library).

[0090] 6. MiSeq machine operation 6.1 NaOH denaturation of library The recommended concentration table is shown in Table 13.

[0091] Table 13 Recommended concentrations for use in the machine

[0092] 6.2 Experimental preparation: 1N NaOH, PCR grade water, HT1, pipette and tips, vortex oscillator, handheld centrifuge.

[0093] 6.3 Operation steps: 1. 20μL 1N NaOH + 80μL ultrapure water to get 0.2 N NaOH; 2. Vortex for 3-5 seconds, centrifuge briefly, and repeat once; 3. 5 μL 4 nM or 2 nM library + 5 μL 0.2 N NaOH; 4. Vortex for 3-5 seconds, centrifuge briefly, and repeat once; 5. Denature at room temperature for 5-10 minutes; 6. Add 990 μL of ice-cold HT1; 7. Vortex for 5-10 seconds, centrifuge briefly, and repeat once; 8. Perform the second dilution according to Table 14 or Table 15, depending on the library type and optimized loading concentration.

[0094] Table 14 4nM starting concentration library

[0095] Table 15 2nM starting concentration library

[0096] 9. Place the diluted and denatured library on ice or in a 2-8°C refrigerator, ready for use on the machine.

[0097] 7. Experimental results and analysis The results of the control group and experimental group are as follows Figure 1 As shown, the gene coverage of the three samples in the experimental group were 99.42%, 99.29% and 99.31%, respectively, all above 99%; the gene coverage of the three samples in the control group were 53.73%, 3.68% and 0.58%, respectively. Compared with the experimental group, the coverage of the control group was unstable and significantly lower than that of the experimental group.

[0098] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A primer combination for amplifying the whole genome sequence of varicella-zoster virus, characterized in that: The primer combination includes 5 sets of primers; Preferably, the sequence of the primer combination is shown as SEQ ID No.1-10.

2. A kit, characterized in that: The kit comprises the primer combination according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes conventional PCR amplification reagents, PCR product purification reagents, DNA quantification reagents and DNA library construction reagents; Preferably, the kit further comprises instructions.

4. A method for obtaining the complete genome sequence of varicella-zoster virus, characterized in that: The method comprises using the primer combination described in claim 1 to amplify the whole genome sequence of varicella-zoster virus.

5. The method according to claim 4, characterized in that The amplification includes LCR, NASBA, SDA, TMA, bDNA, PCR methods; Preferably, the amplification is performed by PCR method; Preferably, the amplification is performed under similar amplification conditions.

6. The method according to claim 5, characterized in that Based on a total volume of 50 μL, the PCR amplification system includes: 25 μL of PCR reaction enzyme, 5 μL of primers, 1 ng to 1 μg of DNA sample, and the balance is water; Preferably, the reaction procedure of the PCR amplification is pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 10s, extension at 72°C for 90s, denaturation, annealing and extension for 30 cycles; and finally extension at 72°C for 2min.

7. The method according to claim 5, characterized in that The samples include blood, saliva, cerebrospinal fluid, and blister fluid.

8. A method for detecting varicella-zoster virus in a sample, characterized in that: The method comprises using the primer combination according to claim 1, the kit according to claim 2 or the method according to claim 4 to perform amplification, obtain an amplification product, and use the amplification product for sequencing; Preferably, the sample includes a human sample and an environmental sample, and the human sample is an in vitro human sample; Preferably, the human body sample includes blood, saliva, cerebrospinal fluid, and blister fluid.

9. A library for detecting varicella-zoster virus, characterized in that: The library comprises the primer combination of claim 1.

10. Any of the following applications, characterized in that: The applications include: 1) Use of the primer combination of claim 1 in preparing a product for amplifying or detecting the whole genome sequence of varicella-zoster virus; 2) Use of the primer combination of claim 1 or the kit of claim 2 in amplifying the whole genome sequence of varicella-zoster virus or in sequencing varicella-zoster virus.

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