Primer group for detecting mutation site of human cytomegalovirus UL54 drug-resistant gene and application of primer group

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a primer group for detecting mutation sites of human cytomegalovirus UL54 drug-resistant genes and application of the primer group. The primer group can be used for constructing an HCMV drug-resistant gene library, sequencing HCMV drug-resistant genes and detecting mutation sites of the HCMV drug-resistant genes.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a primer set for detecting the UL54 drug resistance gene mutation site of human cytomegalovirus and its application. Background Technology

[0002] Human cytomegalovirus (HCMV) belongs to the Herpesviridae family, β-herpesvirinae subfamily, cytomegalovirus genus, and human herpesvirus type 5. It is widely distributed in nature, and natural infection is common in the general population.

[0003] HCMV is an opportunistic pathogen in immunocompromised or immunomature individuals and is one of the leading causes of infection or poor prognosis in patients undergoing allogeneic hematopoietic stem cell transplantation. Most infected individuals are asymptomatic, but in children with congenital infection or individuals with suppressed immune function, infection is easily reactivated, leading to severe and life-threatening complications. Furthermore, intrauterine cytomegalovirus infection is also a major cause of birth defects. The high incidence of adverse reactions and treatment failure necessitates reliable and effective diagnosis of emerging antiviral drug resistance. Therefore, drug intervention and treatment of cytomegalovirus are of paramount importance.

[0004] Currently, the main drugs used clinically to treat HCMV infection include ganciclovir (GCV) and its prodrugs valganciclovir, cidofovir (CDV), foscarnet (PFA), and letermovir. UL54 is the target of many antiviral drugs (GCV, CDV, PFA), therefore, drug resistance mutations in UL54 can seriously affect clinical efficacy. Long-term or repeated use of antiviral drugs (especially GCV, CDV, and PFA) can lead to the emergence of drug-resistant HCMV strains. UL54 mutations can alter drug binding sites or enzyme activity, resulting in drug resistance. Immunosuppressed patients (such as organ transplant recipients and HIV-infected individuals) rely on antiviral drugs to control HCMV infection; the emergence of drug-resistant strains may lead to treatment failure or even endanger life. Therefore, monitoring UL54 drug resistance mutations can help guide clinical medication (such as switching to letermovir or other drugs with different mechanisms of action).

[0005] Therefore, there is an urgent need to develop a kit for detecting the UL54 drug resistance gene mutation site of human cytomegalovirus. Summary of the Invention

[0006] The first aspect of the present invention is to provide a primer set.

[0007] A second aspect of the present invention is to provide a reagent kit.

[0008] The object of a third aspect of the present invention is to provide the application of the primer set of the first aspect of the present invention or the reagent kit of the second aspect of the present invention.

[0009] The fourth aspect of this invention aims to provide a method for constructing an HCMV drug resistance gene library.

[0010] The fifth aspect of this invention is to provide an HCMV resistance gene library.

[0011] The sixth aspect of this invention aims to provide a method for sequencing HCMV resistance genes.

[0012] The seventh aspect of this invention aims to provide a method for detecting HCMV drug resistance gene mutation sites.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides a primer set comprising a first primer set and a second primer set: The first primer set includes a combination of nucleotide sequences or continuous fragments as shown in SEQ ID NO:1-24, or a nucleotide sequence having more than 85% sequence identity with it, or a complementary sequence thereof; The second primer set includes a combination of nucleotide sequences or continuous fragments as shown in SEQ ID NO:25-46, or a nucleotide sequence having more than 85% sequence identity with it, or a complementary sequence thereof; The first primer set and the second primer set were used for PCR (preferably multiplex PCR).

[0014] In some embodiments, the primer set is used to detect HCMV resistance gene mutation sites.

[0015] In some embodiments, the primer set is used to detect HCMV resistance gene mutation sites in the sample to be tested.

[0016] In this invention, the HCMV resistance gene is the UL54 gene.

[0017] In this invention, the HCMV resistance gene mutation site is the UL54 gene mutation site.

[0018] In this invention, the HCMV drug resistance gene mutation includes drug resistance mutations caused by base mutations, insertions, or deletions.

[0019] In this invention, the UL54 gene mutation sites include: S290R, D301N, E303D / G, N408D / K, N408S, N410K, F412C, F412L / S, F412V, D413A / E, D413Y, D413N, N495K, K500N, L501I, L501F, T503I, A505V, L516R, K513E / N / R, D515Y, L516P / W, I521T, P522A / S, C524del, V526L, C539G, D542E, L545S / W, Q578H, Q578L, D58 At least one of 8E, D588N, T700A, V715M / A, I726T / V, E756D / Q / N, E756K, L773V, L776M, V781I, V787A, V787E, V787L, L802M, K805Q, A809V, V812L, T813S, T821I, A834P, T838A, G841S, G841A, E951D, L957F, D981del2, and A987G; further comprising at least one of N495K, D588N, Q578H, T700A, L773V, L776M, and K805Q.

[0020] In this invention, the sample to be tested is selected from at least one of the body fluids, tissues, cells, and excretions of the object to be tested.

[0021] In this invention, the sample to be tested is selected from at least one of serum, plasma, whole blood, respiratory tract samples (e.g., sputum, bronchoalveolar lavage fluid), cerebrospinal fluid, and eye samples (e.g., aqueous humor, vitreous humor).

[0022] In this invention, the test subject includes mammals, such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0023] In this invention, the object to be tested includes humans.

[0024] A second aspect of the present invention provides a kit comprising the primer set of the first aspect of the present invention.

[0025] In some embodiments, the kit further includes PCR reaction solution and DNA polymerase.

[0026] In some embodiments, the PCR reaction solution includes: buffer, magnesium ions, and dNTPs.

[0027] In some embodiments, the kit further includes: a substance for breaking DNA, a reagent combination for end repair and adding "A", a reagent combination for adapter ligation, and / or a reagent combination for nucleic acid extraction or purification; further comprising: a reagent combination for end repair and adding "A", a reagent combination for adapter ligation, and a reagent combination for nucleic acid extraction or purification.

[0028] In some embodiments, the kit is used to detect HCMV resistance gene mutation sites.

[0029] In some embodiments, the kit is used to detect HCMV resistance gene mutation sites in the sample to be tested.

[0030] A third aspect of the invention provides the use of the primer set of the first aspect of the invention or the kit of the second aspect of the invention in any one of a1)-a4): a1) Construct an HCMV resistance gene library; a2) Sequencing of HCMV resistance genes; a3) Detect HCMV resistance gene mutation sites; a4) Prepare a product, which is used in any one of a1)-a3).

[0031] In some implementations, the application described in a3) does not involve the diagnosis or treatment of diseases.

[0032] In some implementations, the product is a reagent kit or reagent kit.

[0033] A fourth aspect of the present invention provides a method for constructing an HCMV resistance gene library, comprising the steps of using the primer set of the first aspect of the present invention or the kit of the second aspect of the present invention.

[0034] In some embodiments, the method includes the following steps: Using the nucleic acid (e.g., DNA) of the sample to be tested as a template, multiplex PCR amplification is performed using the first primer set and the second primer set from the primer set of the first aspect of the present invention, respectively.

[0035] In some embodiments, the method further includes the following steps: performing end repair, A addition, and adapter addition on the multiplex PCR amplification products of the first primer set and the second primer set, respectively, and mixing the library obtained by amplification of the first primer set with the library obtained by amplification of the second primer set.

[0036] A fifth aspect of the present invention provides an HCMV resistance gene library, obtained by the method of the fourth aspect of the present invention.

[0037] A sixth aspect of the present invention provides a method for sequencing HCMV resistance genes, wherein the HCMV resistance gene library of the fifth aspect of the present invention is sequenced.

[0038] In some implementations, the sequencing is next-generation sequencing.

[0039] A seventh aspect of the present invention provides a method for detecting HCMV drug resistance gene mutation sites, comprising the steps of the method of the sixth aspect of the present invention.

[0040] In some embodiments, the method includes the following steps: analyzing the sequencing results to detect HCMV drug resistance gene mutation sites.

[0041] In some implementations, bioinformatics software is used to analyze the results of drug resistance site mutations in order to accurately obtain HCMV drug resistance mutant genes, mutation frequencies, and drug resistance.

[0042] In some implementations, the method does not involve the diagnosis or treatment of the disease.

[0043] The beneficial effects of this invention are: This invention provides a primer set that can be used to construct an HCMV resistance gene library, sequence HCMV resistance genes, and detect HCMV resistance gene mutation sites. Furthermore, based on this primer set and the kit or method for NGS detection of HCMV drug resistance gene mutation sites, it solves the technical problems of traditional gene mutation diagnostic methods, such as poor accuracy in low-copy, low-level drug resistance samples and the limitation of detection only of previously reported sites. It can accurately analyze mutation sites and mutation frequencies with high sensitivity and accuracy, which is of great significance for guiding clinical drug treatment. It can ensure sequencing quality even in the case of low-copy, low-frequency mutations, and achieve the goal of accurate identification of HCMV drug resistance gene mutation sites. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the primer set design strategy for Example 1.

[0045] Figure 2 This is a flowchart of the method for detecting drug resistance gene mutation sites of human cytomegalovirus in Example 2.

[0046] Figure 3 This is a schematic diagram illustrating the verification results of the method for detecting drug resistance gene mutation sites of human cytomegalovirus in Example 2.

[0047] Figure 4 This is a schematic diagram showing the results of detecting drug resistance gene mutation sites in clinical samples using the method in Example 2. Detailed Implementation

[0048] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0049] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0050] In this invention, the term "sequence" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of a molecule. Therefore, this term encompasses double-stranded and single-stranded DNA, as well as RNA. The term "nucleic acid" as used herein is a single-stranded or double-stranded covalently linked sequence of nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides can consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized in vitro or isolated from natural sources. Nucleic acids may further comprise modified DNA or RNA, such as methylated DNA or RNA. According to the principle of base complementarity, when the nucleotide sequence is known, the nucleotide information of its complementary sequence can be obtained accordingly.

[0051] In this invention, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide chains or between two regions of the same polynucleotide chain. It is known that an adenine base in a first region of a polynucleotide can form a specific hydrogen bond ("base pair") with a base in a second polynucleotide region antiparallel to the first region (if that base is thymine or uracil). Similarly, it is known that a cytosine base in a first polynucleotide chain can pair with a base in a second polynucleotide chain antiparallel to the first region (if that base is guanine). If, when a first region of a polynucleotide is arranged antiparallel to a second region of the same or a different polynucleotide, at least one nucleotide in the first region can pair with a base in the second region, then the two regions are complementary. Therefore, two complementary polynucleotides do not need to pair at every nucleotide position. "Complementarity" means that the first polynucleotide and the second polynucleotide are 100% or "completely" complementary, and therefore form a base pair at every nucleotide site. "Complementary" also refers to a first polynucleotide that is not 100% complementary (e.g., 90%, 80%, 70%, 60%, or 50% complementary) containing mismatched nucleotides at one or more nucleotide positions. In one embodiment, two complementary polynucleotides are capable of hybridizing with each other under highly stringent hybridization conditions.

[0052] In this invention, "sequence identity" refers to the "sequence identity percentage" or "identity percentage" between two polynucleotides, that is, the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide is present in both the target and reference sequences. Since vacancies are not nucleotides, vacancies present in the target sequence are not counted. Similarly, vacancies present in the reference sequence are not counted because nucleotides from the target sequence are counted but nucleotides from the reference sequence are not. At least 85% sequence identity includes at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the full length of the sequence having sequence identity.

[0053] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the BLAST website of the National Center for Biotechnology Information (NCBI) of the U.S. government. Bl2seq uses the BLASTN algorithm for comparing two sequences. BLASTN is used for comparing nucleic acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, and are part of the EMBOSS suite of bioinformatics programs.

[0054] In this invention, a “fragment” refers to a continuous segment that has sequence identity with at least 55%, at least 65%, at least 75%, at least 85%, at least 95%, or 100% of the full length of the sequence.

[0055] In this invention, the term "nucleic acid" generally refers to a monomeric or polymeric form of a nucleotide of any length, which is a deoxyribonucleotide or ribonucleotide, or an analogue or variant thereof. A nucleic acid molecule may include one or more unmodified or modified nucleotides. Nucleic acids can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of nucleic acids: ribonucleic acid (RNA), deoxyribonucleic acid (DNA), coding or non-coding regions of a gene or gene segment, loci defined by linkage analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, complementary deoxyribonucleic acid (cDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acids can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs, such as peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), ethylene glycol nucleic acids (GNA), threonine nucleic acids (TNA), 2'-fluoro, 2'-OMe, and phosphorylated DNA. Nucleic acids can include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. In some instances, nucleic acids are DNA or RNA, or derivatives thereof. Nucleic acids can be single-stranded or double-stranded. Nucleic acids can be circular.

[0056] In this invention, the term "RNA," short for "ribonucleic acid," is one of the four major biological macromolecules found in biological cells: nucleic acids. RNA is a large polymer composed of nucleotides. Nucleotides are composed of bases, ribose, and phosphate. There are four types of bases: adenine (A), guanine (G), uracil (C), and cytosine (C).

[0057] In this invention, the term "DNA," short for "deoxyribonucleic acid," is one of the four biological macromolecules found in living cells—a type of nucleic acid. DNA carries the genetic information necessary for the synthesis of RNA and proteins and is an essential biological macromolecule for the development and normal functioning of organisms. DNA is a large polymer composed of deoxynucleotides. Deoxynucleotides consist of a base, deoxyribose, and a phosphate group. There are four types of bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

[0058] In this disclosure, "sequencing" refers to the process of determining the sequence (e.g., identity and order of monomeric units) of a biomolecule, such as a nucleic acid, like DNA or RNA. In some embodiments, sequencing can be performed using a gene analyzer.

[0059] The present invention will be further described in detail below through specific embodiments.

[0060] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0061] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0062] The reagents and instruments involved in the following examples are shown in Tables 1 and 2.

[0063] Table 1. Reagent Names and Manufacturers

[0064] Table 2 Instrument Names and Manufacturers

[0065] Through statistical analysis of extensive publicly available NCBI data and actual sequencing data, and by experimentally validating high-performance primer combinations, the inventors obtained a primer set that targets and enriches the full length of the HCMV UL54 gene. This set has the following characteristics: 1. The primers were trained with large datasets to ensure that each primer pair has good HCMV genome conservation and is not prone to off-target effects. In addition, they have 3' end specificity relative to the human genome (referencing European GRCh38.p13 and Asian GCA_000004845.2), with at least 10 bases significantly different from the human genome, and can have good sensitivity to HCMV sequences in human nucleic acid background. 2. The design Tm value and amplification length of each primer pair are very similar and have been optimized experimentally. By adjusting the primer concentration ratio, the actual amplification efficiency is made close, avoiding the bias problem that occurs during multiplex PCR amplification and ensuring that each sequence segment is amplified well and uniformly. 3. The primer design region avoids hotspots of drug resistance mutations, minimizing the interference of unamplified primers with background sequencing data on data analysis.

[0066] Ultimately, two independent primer sets were obtained for multiplex PCR amplification: primer set 1 and primer set 2; their sequences are shown in Table 3, and the primer design strategy is as follows: Figure 1 As shown.

[0067] Table 3. Sequences of the first primer set and the second primer set.

[0068]

[0069] Example 2: A method for detecting the UL54 drug resistance gene mutation site of human cytomegalovirus. A method for detecting the UL54 drug resistance gene mutation site of human cytomegalovirus, the detection procedure is as follows: Figure 2 As shown, the specific steps include the following: 1. Multiplex PCR amplification 1) Remove the reaction solution, the first primer set, and the second primer set from the CMV UL54 resistance site detection kit from the refrigerator, thaw them at room temperature, and vortex to mix. 2) Remove the polymerase from the refrigerator; do not vortex. 3) Using a small centrifuge, centrifuge all the above reagents for 30 seconds, and centrifuge the liquid on the tube cap to the bottom of the tube; 4) Calculate the required amounts of reaction solution, polymerase, and multiplex PCR primer mix according to the number of reactions, and aspirate and mix them into a new EP tube. Vortex to mix, and centrifuge for 30 seconds to obtain the mixture of the first primer set multiplex PCR amplification reaction system and the second primer set multiplex PCR amplification reaction system. The required amounts of components for each multiplex PCR amplification reaction system are calculated according to Table 4. Table 4 Multiplex PCR amplification reaction system

[0070] 5) Aliquot the mixture of the multiplex PCR amplification reaction system into 8-tube PCR tubes; 6) Add the sample nucleic acid to the appropriate wells of the 8-tube PCR tube according to the amount in Table 4, and seal the tube with the cap; vortex the 8-tube PCR tube to mix, centrifuge for 30 seconds, and collect the liquid at the bottom of the tube. 7) Place the 8-tube PCR array in a standard PCR instrument, cover it with the heat-sealing cap, program the PCR instrument according to the program in Table 5, and start the operation.

[0071] Table 5 Multiplex PCR amplification reaction procedure

[0072] 2. End-stage repair, adding A Perform the following operations using the "Metagenomic DNA Library Construction Kit (Reversible Termination Sequencing Method)": 1) Thaw the fragmentation completion buffer and balance buffer on ice and vortex to mix. 2) Remove the fragmented finishing enzyme from the refrigerator, and do not vortex. 3) Using a small centrifuge, centrifuge all the above reagents for 30 seconds, and centrifuge the liquid on the tube cap to the bottom of the tube; 4) Calculate the required amounts of fragmentation completion buffer, fragmentation completion enzyme, and balancing buffer based on the number of reactions, and aspirate and mix them into a new EP tube. Vortex to mix, and centrifuge for 30 seconds using a small centrifuge. The amount of components required for each end repair, completion, and A reaction system is calculated according to Table 6. Table 6 End-of-phase repair, leveling, and A-reaction system

[0073] 5) Aliquot the mixture into 8-tube PCR tubes; 6) After adding the above multiplex PCR amplification product (5 μL) to the appropriate well of the 8-tube PCR tube, seal it with the 8-tube PCR cap, vortex the 8-tube PCR tube to mix well, centrifuge for 30 seconds, and collect the liquid to the bottom of the tube. 7) Place the 8-tube PCR array in a standard PCR instrument and cover it with the heat-sealing cap; program the PCR instrument according to the procedure in Table 7 and start it.

[0074] Table 7 End-of-phase repair, leveling, and A-reaction procedure

[0075] 3. Connector connection 1) Remove the connector from the refrigerator and allow it to thaw at room temperature; 2) Remove the ligase from the refrigerator, and do not vortex. 3) Add 5 μl of ligase and 30 μl of adapter to each well of the 8-tube PCR tube obtained in step 2 above, seal with the 8-tube PCR tube cap, vortex the 8-tube PCR tube to mix well, centrifuge for 30 seconds, and collect the liquid to the bottom of the tube. 4) Place the 8-tube PCR array in a standard PCR instrument and cover it with the heat-sealing cap; program the PCR instrument according to the procedure in Table 8 and start it.

[0076] Table 8 Connector Connection Response Procedure

[0077] 4. Magnetic bead purification Perform the following procedures using a nucleic acid extraction or purification kit (magnetic bead method): 1) Remove the purified magnetic beads from the refrigerator, allow them to equilibrate at room temperature for 30 minutes, and then vortex to mix. 2) Take the purified cleaning solution out of the refrigerator and add the appropriate volume of anhydrous ethanol according to the instructions to prepare the purified cleaning solution; 3) Remove the purified eluent from the refrigerator; 4) Add 40 μl of purified magnetic beads to the PCR tube containing the ligation product obtained in step 3, vortex to mix, and incubate at room temperature for 10 min. 5) Centrifuge the PCR tubes in a small centrifuge for 30 seconds, place them on a magnetic rack for 2 minutes to absorb the supernatant, and carefully discard the supernatant after it becomes clear. 6) Keep the PCR tube on the magnetic rack, add 200 μl of purification and washing buffer to the PCR tube, and carefully aspirate the supernatant; 7) Repeat step 6), briefly centrifuge the PCR tubes using a small centrifuge, place them on a magnetic rack, remove all residual liquid using a pipette, and allow them to air dry at room temperature for about 2 minutes. 8) Add 40 μl of purification elution buffer to the PCR tube, vortex to mix, and incubate at room temperature for 10 min; 9) Place the PCR tube on a magnetic rack for 1 minute to allow it to absorb. 10) After the supernatant has clarified, transfer all the supernatant to a new PCR tube and perform library quantification.

[0078] 5. Library Quantitative Analysis and Pooling The library concentration was quantified according to the instructions of the NGS Library Quantification Kit. The library amplified by the first primer set and the library amplified by the second primer set were mixed into one tube according to the pooling rule.

[0079] 6. High-throughput sequencing and bioinformatics analysis Following the instructions for the Illumina sequencing kit, the constructed library was sequenced at 50 bp at one end using an Illumina NextSeq 550Dx (Illumina) instrument. The data was then analyzed using a bioinformatics analysis workflow to obtain HCMV resistance detection results.

[0080] Effect Example 1. DNA was collected from 10 HCMV-positive plasma samples and human cell DNA (control, NC). The samples were analyzed according to the method in Example 2. The primer coverage and sequencing quality were analyzed by combining the mean values ​​of the 10 samples. The results showed that NC was negative, indicating that the primer set in Example 1 had good coverage of the full-length UL54 gene, exceeding 99%; the average depth reached 4522, and bioinformatics statistical analysis showed that the data volume of a single sample reached 4 MB, which meets the requirements for sequencing analysis. Figure 3 ).

[0081] 2. Three HCMV-positive clinical plasma samples were selected and clinically validated using the method described in Example 2. The coverage of each resistance site was analyzed. Specific results for one sample are shown below. Figure 4 As shown, all drug resistance sites of the UL54 gene were 100% covered (all three samples were 100% covered), further demonstrating that the primer set in Example 1 has high accuracy.

[0082] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1.A primer set comprising a first primer set and a second primer set: the first primer set comprises a combination of nucleotide sequences as shown in SEQ ID NO: 1-24, or a nucleotide sequence having more than 85% sequence identity thereto, or a complementary sequence thereof; the second primer set comprises a combination of nucleotide sequences as shown in SEQ ID NO: 25-46, or a nucleotide sequence having more than 85% sequence identity thereto, or a complementary sequence thereof; the first primer set and the second primer set are used for PCR respectively. 2.A kit comprising the primer set of claim 1. 3.The kit of claim 2, wherein: the kit further comprises a PCR reaction solution and a DNA polymerase; preferably, the kit comprises a substance for breaking DNA, a reagent combination for end repair and A-tailing, a reagent combination for adapter ligation, and / or a reagent combination for nucleic acid extraction or purification. 4.Use of the primer set of claim 1 or the kit of any one of claims 2-3 in any one of a1)-a4): a1) constructing a HCMV drug-resistant gene library; a2) sequencing a HCMV drug-resistant gene; a3) detecting a HCMV drug-resistant gene mutation site; a4) preparing a product for any one of a1)-a3). 5.A method of constructing a HCMV drug-resistant gene library, comprising the step of using the primer set of claim 1 or the kit of any one of claims 2-3. 6.The method of claim 5, wherein: the method comprises the following steps: using the first primer set and the second primer set of claim 1 to perform multiplex PCR amplification respectively with nucleic acid of a sample to be tested as a template; preferably, the method further comprises the following steps: performing end repair, A-tailing and adapter ligation on the multiplex PCR amplification product of the first primer set and the multiplex PCR amplification product of the second primer set respectively, and mixing the library amplified by the first primer set with the library amplified by the second primer set. 7.A HCMV drug-resistant gene library obtained by the method of any one of claims 5-6. 8.A method of sequencing a HCMV drug-resistant gene, which sequences the HCMV drug-resistant gene library of claim 7. 9.A method of detecting a HCMV drug-resistant gene mutation site, comprising the steps of the method of claim 8; preferably, the method comprises the following steps: analyzing the sequencing results to detect the HCMV drug-resistant gene mutation site. 10.The use, method or library of any one of claims 4-9, wherein: the HCMV drug-resistant gene is a UL54 gene; preferably, the HCMV drug-resistant gene mutation site is a UL54 gene mutation site.

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