Primer group for detecting mutation site of human cytomegalovirus UL97 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 a mutation site of a human cytomegalovirus UL97 drug-resistant gene 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] The application belongs to the technical field of gene detection, and particularly relates to a primer set for detecting a human cytomegalovirus UL97 drug-resistant gene mutation site and application thereof. BACKGROUND

[0002] Human cytomegalovirus (HCMV) belongs to the Herpesviridae family, the beta Herpesvirinae subfamily, the Cytomegalovirus genus, and human herpes virus 5, and is widely present in nature and naturally infects the general population.

[0003] HCMV is a conditional pathogen of immunodeficient or immunologically immature individuals and is one of the main causes of infection or poor prognosis in patients undergoing allogeneic hematopoietic stem cell transplantation. Most infected individuals have no obvious symptoms, but in congenitally infected children or immunosuppressed individuals, the infection is easily activated, causing severe lesions and endangering life. In addition, intrauterine infection with cytomegalovirus is one of the main causes of birth defects. The high incidence of adverse reactions and treatment failures requires reliable and effective diagnosis of emerging antiviral drug resistance. Therefore, drug intervention and treatment of cytomegalovirus are particularly important.

[0004] Cytomegalovirus drug resistance mutations are mainly caused by mutations in the UL97 and UL54 genes, and other gene mutations are less common. The UL97 gene encodes a protein kinase that plays a key role in viral replication, especially in the phosphorylation activation of antiviral drugs such as ganciclovir (GCV), which is the main target of drugs such as ganciclovir and valganciclovir. Identification of drug resistance sites can help doctors identify drug-resistant strains in a timely manner and avoid ineffective treatment (such as common mutations M460V / I, H520Q, C592G, etc.). Differentiate between primary drug resistance (drug-resistant strains present before treatment) and secondary drug resistance (mutations during treatment), and optimize individualized drug regimens.

[0005] HCMV UL97 drug resistance site research not only has direct guiding value for clinical treatment, but also promotes basic research and drug development. The development of rapid and sensitive drug resistance detection technology, especially for immunosuppressed patients (such as organ transplant recipients and HIV-infected individuals), can save lives through accurate detection of UL97 mutations. With the increasing prominence of drug resistance, continuous monitoring and research of UL97 mutations is an important part of HCMV management.

[0006] Therefore, there is an urgent need to develop a kit for detecting human cytomegalovirus UL97 drug-resistant gene mutation sites. SUMMARY

[0007] The first aspect of the present application aims to provide a primer set.

[0008] The second aspect of the present application aims to provide a kit.

[0009] The third aspect of the present application aims to provide use of the primer set of the first aspect of the present application or the kit of the second aspect of the present application.

[0010] The fourth aspect of the present application aims to provide a method for constructing a HCMV drug-resistant gene library.

[0011] The fifth aspect of the present application aims to provide a HCMV drug-resistant gene library.

[0012] The sixth aspect of the present application aims to provide a method for sequencing a HCMV drug-resistant gene.

[0013] The seventh aspect of the present application aims to provide a method for detecting a mutation site of a HCMV drug-resistant gene.

[0014] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides a primer set, which comprises 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: 1-16 or a continuous fragment thereof, 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: 17-32 or a continuous fragment thereof, 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 respectively subjected to PCR (preferably multiplex PCR).

[0015] In some embodiments, the primer set is used for detecting a mutation site of a HCMV drug-resistant gene.

[0016] In some embodiments, the primer set is used for detecting a mutation site of a HCMV drug-resistant gene in a sample to be tested.

[0017] In the present application, the HCMV drug-resistant gene is a UL97 gene.

[0018] In the present application, the mutation site of the HCMV drug-resistant gene is a UL97 gene mutation site.

[0019] In the present application, the mutation of the HCMV drug-resistant gene includes a drug-resistant mutation caused by base mutation, insertion or deletion.

[0020] In the present application, the UL97 gene mutation sites include at least one of F324Y, K359E, K359Q, L405P, T409M, H411Y / N, M460I / V, M460T, V466G, C480F, C518Y, H520Q, P521L, A591V, C592G, A594V, A594T, A594E / G, A594P / S, L595S / F, L595W, E596G / Y, K599T, C603W, D605E, C607Y / F, C603R, C603S, I610T, A613V; further include at least one of H411Y, M460V, H520Q, C592G, A594V, L595F, C603W.

[0021] In the present application, the sample to be tested is selected from at least one of body fluid, tissue, cell, excretion of the subject to be tested.

[0022] In the present application, the sample to be tested is selected from at least one of serum, plasma, whole blood, respiratory tract sample (e.g. sputum, bronchoalveolar lavage fluid), cerebrospinal fluid, ocular sample (e.g. aqueous humor, vitreous).

[0023] In the present application, the subject to be tested includes mammals, such as humans, non-human primates (e.g. chimpanzees, apes), rodents (e.g. rats, mice, guinea pigs), pets (e.g. cats, dogs), livestock (e.g. horses, cows, sheep, pigs, rabbits).

[0024] In the present application, the subject to be tested includes humans.

[0025] In a second aspect of the present application, a kit is provided, which includes the primer set of the first aspect of the present application.

[0026] In some embodiments, the kit further includes a PCR reaction solution and a DNA polymerase.

[0027] In some embodiments, the PCR reaction solution includes a buffer, magnesium ions and dNTPs.

[0028] In some embodiments, the kit further includes a substance for breaking DNA, a reagent combination for end repair and "A" addition, a reagent combination for linker ligation, and / or a reagent combination for nucleic acid extraction or purification; further including a reagent combination for end repair and "A" addition, a reagent combination for linker ligation, and a reagent combination for nucleic acid extraction or purification.

[0029] In some embodiments, the kit is used for detecting HCMV drug-resistant gene mutation sites.

[0030] In some embodiments, the kit is used for detecting HCMV drug resistance gene mutation sites in a sample to be tested.

[0031] In a third aspect of the present application, there is provided use of the primer set of the first aspect of the present application or the kit of the second aspect of the present application in any one of a1) to a4): a1) constructing a HCMV drug resistance gene library; a2) sequencing the HCMV drug resistance gene; a3) detecting HCMV drug resistance gene mutation sites; a4) preparing a product for any one of a1) to a3).

[0032] In some embodiments, the use of a3) does not involve diagnosis or treatment of a disease.

[0033] In some embodiments, the product is a kit or a reagent set.

[0034] In a fourth aspect of the present application, there is provided a method for constructing a HCMV drug resistance gene library, comprising the step of using the primer set of the first aspect of the present application or the kit of the second aspect of the present application.

[0035] In some embodiments, the method comprises the following steps: using the first primer set and the second primer set of the primer set of the first aspect of the present application to perform multiplex PCR amplification, respectively, using the nucleic acid (e.g. DNA) of the sample to be tested as a template.

[0036] In some embodiments, the method further comprises the following steps: performing end repair, A-tailing and adaptor 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 and the library amplified by the second primer set.

[0037] In a fifth aspect of the present application, there is provided a HCMV drug resistance gene library, which is obtained by the method of the fourth aspect of the present application.

[0038] In a sixth aspect of the present application, there is provided a method for sequencing the HCMV drug resistance gene, which is sequencing the HCMV drug resistance gene library of the fifth aspect of the present application.

[0039] In some embodiments, the sequencing is next-generation sequencing.

[0040] In a seventh aspect of the present application, there is provided a method for detecting HCMV drug resistance gene mutation sites, comprising the steps of the method of the sixth aspect of the present application.

[0041] In some embodiments, the method comprises the step of analyzing the sequencing results to detect the HCMV drug-resistant gene mutation sites.

[0042] In some embodiments, the bioinformatics software is used to analyze the drug resistance site mutation results to accurately obtain the HCMV drug-resistant mutation gene, mutation frequency and drug resistance.

[0043] In some embodiments, the method does not involve the diagnosis and treatment of diseases.

[0044] The beneficial effects of the present application are: The present application provides a primer set which can be used to construct an HCMV drug-resistant gene library, sequence the HCMV drug-resistant gene, and detect the HCMV drug-resistant gene mutation sites. Further, the primer set and the kit or method for detecting the HCMV drug-resistant gene mutation sites based on NGS solve the technical problems of the conventional gene mutation diagnosis method, such as poor accuracy for low copy and low level drug resistance samples, and can only detect the sites that have been reported, and the application has limitations, which can accurately analyze the mutation sites and mutation frequency, has high sensitivity and accuracy, has important significance for guiding clinical drug treatment, and can ensure the sequencing quality in the case of low copy and low frequency mutation, and achieve the purpose of accurately identifying the HCMV drug-resistant gene mutation sites. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 It is a schematic diagram of the design strategy of the primer set of Example 1.

[0046] Figure 2 It is a flowchart of the method for detecting the HCMV drug-resistant gene mutation sites of Example 2.

[0047] Figure 3 It is a schematic diagram of the verification results of the method for detecting the HCMV drug-resistant gene mutation sites of Example 2.

[0048] Figure 4 It is a schematic diagram of the results of the method for detecting the drug-resistant gene mutation sites of clinical samples of Example 2. DETAILED DESCRIPTION

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.

[0050] The expression "about" as used herein is as understood by one of ordinary skill in the art and varies according to the context in which it is used. If one of ordinary skill in the art uses the term according to its day-to-day meaning, "about" will mean a specified value plus or minus 10% of that value.

[0051] In the present application, the term "sequence" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded and single-stranded DNA, as well as RNA. The term "nucleic acid" as used herein is a single- or double-stranded covalently linked sequence of nucleotides, in which the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. A polynucleotide can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be manufactured synthetically or isolated from natural sources. Nucleic acids can further comprise modified DNA or RNA, such as DNA or RNA that has been methylated. The nucleotide sequence of a known nucleic acid can be used to derive the nucleotide information of its complementary sequence according to the principle of base complementarity.

[0052] In the present application, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand. An adenine base of a known first region of a polynucleotide is capable of forming a specific hydrogen bond ("base pair") with a base of a second region of a polynucleotide that is antiparallel to the first region, if the base is a thymine or a uracil. Similarly, a cytosine base of a first polynucleotide strand is capable of base pairing with a base of a second polynucleotide strand that is antiparallel to the first region, if the base is a guanine. Two regions are complementary if at least one nucleotide of the first region is capable of base pairing with a base of the second region when the first region of a polynucleotide is arranged in an antiparallel fashion with the second region of the same or a different polynucleotide. Thus, two complementary polynucleotides do not need to base pair at every nucleotide position. "Complementary" refers to a first polynucleotide that is 100% or "perfectly" complementary to a second polynucleotide and thus forms base pairs at every nucleotide position. "Complementary" also refers to a first polynucleotide that contains mismatched nucleotides at one or more nucleotide positions that are not 100% complementary (e.g., 90%, or 80% or 70%, or 60%, or 50% complementary). In one embodiment, two complementary polynucleotides are capable of hybridizing to each other under high stringency hybridization conditions.

[0053] In the present invention, "sequence identity" refers to the "percent sequence identity" or "percent identity" between two polynucleotides, i.e., the percentage of identical matches between the sequences within a comparison window that accounts for additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A match is any position where the same nucleotide occurs in both the target sequence and the reference sequence. Gaps are not counted as nucleotides. Likewise, gaps that occur in the reference sequence are not counted, as are nucleotides from the reference sequence, since the target sequence nucleotides are counted. At least 85% sequence identity includes a contiguous segment of 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.

[0054] The percent sequence identity can be calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software readily available for online use and download. Suitable software programs are available from various sources for alignment of nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information BLAST website of the United States government. Bl2seq uses the BLASTN algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs.

[0055] In the present invention, "fragment" refers to a contiguous segment having at least 55%, at least 65%, at least 75%, at least 85%, at least 95%, or 100% sequence identity to the full length of the sequence.

[0056] In the present invention, the term "nucleic acid" refers generally to a monomer or polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs or variants. A nucleic acid molecule can include one or more unmodified or modified nucleotides. A nucleic acid can have any three-dimensional structure and can perform any function, known or unknown. 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 fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (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. A nucleic acid can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs, such as peptide nucleic acid (PNA), morpholino, and locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), 2'-fluoro, 2'-OMe, and phosphorothioated DNA. A nucleic acid can include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. In some examples, a nucleic acid is DNA or RNA or a derivative thereof. A nucleic acid can be single-stranded or double-stranded. A nucleic acid can be circular.

[0057] In the present invention, the term "RNA" stands for "Ribonucleic Acid" is one of the four biological macromolecules contained in the cells of living organisms. RNA is a macromolecular polymer composed of nucleotides. A nucleotide is composed of a base, a ribose, and a phosphate. There are four bases: adenine (A), guanine (G), uracil (C), and cytosine (C).

[0058] In the present invention, the term "DNA" stands for "Deoxyribonucleic Acid" is one of the four biological macromolecules contained in the cells of living organisms. DNA carries the genetic information necessary for the synthesis of RNA and proteins, and is essential for the development and normal operation of living organisms. DNA is a macromolecular polymer composed of deoxyribonucleotides. A deoxyribonucleotide is composed of a base, a deoxyribose, and a phosphate. There are four bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

[0059] In the present disclosure, "sequencing" refers to determining the sequence (e.g., identity and order of monomeric units) of a biomolecule, e.g., a nucleic acid, such as DNA or RNA. In some embodiments, sequencing can be performed by a genetic analyzer.

[0060] The present application is further described in detail by the following specific examples.

[0061] It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application.

[0062] The experimental methods in the following examples, unless otherwise specified, are generally performed according to conventional methods or according to the conditions recommended by the manufacturers. The materials, reagents, etc. used in the present examples, unless otherwise specified, are reagents and materials obtained from commercial channels. The reagents used are indicated by the manufacturers, and similar products of other manufacturers have substitutability.

[0063] The reagents and instruments involved in the following examples are shown in Tables 1 and 2.

[0064] Table 1: Reagent names and manufacturers

[0065] Table 2: Instrument names and manufacturers

[0066] Example 1: A primer set for detecting mutation sites of human cytomegalovirus UL97 drug-resistant gene The inventors obtained a primer set targeting the full-length of HCMV UL97 gene through statistical analysis of a large number of NCBI public sources and actual sequencing data, and a high-performance primer combination verified by experiments. It has the following characteristics: 1. Obtained through big data training, ensuring that each pair of primers has good HCMV genome conservation, is not easy to be off-target, and has 3' end specificity relative to the human genome (reference European GRCh38.p13 and yellow GCA_000004845.2). There are at least 10 base differences between the human genome and the human genome, and the HCMV sequence has better sensitivity in the human nucleic acid background; 2. The design Tm value and amplification length difference between each pair of primers are very small, and are optimized through experiments. By adjusting the primer concentration ratio, the actual amplification efficiency is close, avoiding the preference problem caused by multiplex PCR amplification, and ensuring that each sequence is well and uniformly amplified; 3. The primer design region avoids the hot spot interval of drug resistance mutations, maximizes the reduction of background sequencing data interference data analysis problems caused by unamplified primers.

[0067] Finally, two independently existing primer sets for multiplex PCR amplification were obtained: the first primer set and the second primer set; the sequences are shown in Table 3, and the primer design strategy is as shown in Figure 1

[0068] Table 3 First primer set sequence, second primer set sequence

[0069] Example 2 A method for detecting human cytomegalovirus UL97 drug-resistant gene mutation sites A method for detecting human cytomegalovirus UL97 drug-resistant gene mutation sites, the detection process is as shown in Figure 2 1. Multiplex PCR amplification 1) Take the reaction solution in the CMV UL97 drug-resistant site detection kit, the first primer set in Example 1, and the second primer set from the refrigerator, thaw and vortex at room temperature; 2) Take the polymerase from the refrigerator, do not vortex; 3) Use a small centrifuge to centrifuge all the reagents for 30s, and centrifuge the liquid on the tube cap to the bottom of the tube; 4) Calculate the amount of reaction solution, polymerase, and multiplex PCR primer mix required according to the number of reactions, and mix well into a new EP tube, vortex to mix well, and centrifuge for 30s using a small centrifuge. Get the first primer set multiplex PCR amplification reaction system mixture and the second primer set multiplex PCR amplification reaction system mixture, the amount of each multiplex PCR amplification reaction system component is calculated according to Table 4; Table 4 Multiplex PCR amplification reaction system

[0070] 5) Dispense the multiplex PCR amplification reaction system mixture into 8-row PCR tubes; 6) Add the sample nucleic acid to the appropriate wells of the 8-row PCR tube according to the amount in Table 4, then seal with an 8-row PCR tube cap; vortex to mix well, then centrifuge for 30s using a small centrifuge to collect the liquid to the bottom of the tube; 7) Place the 8-row PCR tube in a general PCR instrument and cover it with a heat cover, program the PCR instrument according to the program in Table 5, and start running.

[0071] Table 5 Multiplex PCR amplification reaction program

[0072] 2. End repair, A addition Use the "Metagenomic DNA Library Construction Kit (Reversible Terminal Termination Sequencing Method)" to perform the following operations:​​ 1) Place the fragmentation buffer, the buffer for filling the gap on ice to thaw, and vortex to mix; 2) Take the fragmentation enzyme from the refrigerator, and do not vortex; 3) Use a small centrifuge to centrifuge all the reagents above for 30 seconds, and centrifuge the liquid on the tube cap to the bottom of the tube; 4) Calculate the amount of fragmentation buffer, fragmentation enzyme, and buffer for filling the gap according to the number of reactions, and mix them in a new EP tube, vortex to mix, and centrifuge for 30 seconds using a small centrifuge; the amount of each component required for the end repair and gap filling plus A reaction system is calculated according to Table 6; Table 6 End repair, gap filling plus A reaction system

[0073] 5) Dispense the mixture into 8-row PCR tubes; 6) After adding the multiplex PCR amplification product (5 μL) to the appropriate wells of the 8-row PCR tube, seal the 8-row PCR tube with the 8-row PCR tube cap, vortex to mix, and centrifuge for 30 seconds using a small centrifuge to collect the liquid at the bottom of the tube; 7) Place the 8-row PCR tube in a general PCR instrument, and cover it with a thermal cap; program the PCR instrument according to the program in Table 7, and start running.

[0074] Table 7 End repair, gap filling plus A reaction program

[0075] 3. Adapter ligation 1) Take the adapter from the refrigerator to thaw at room temperature; 2) Take 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-row PCR tube obtained in step 2 above, seal the 8-row PCR tube with the 8-row PCR tube cap, vortex to mix, and centrifuge for 30 seconds using a small centrifuge to collect the liquid at the bottom of the tube; 4) Place the 8-row PCR tube in a general PCR instrument, and cover it with a thermal cap; program the PCR instrument according to the program in Table 8, and start running.

[0076] Table 8 Adapter ligation reaction program

[0077] 4. Magnetic bead purification Use the nucleic acid extraction or purification kit (magnetic bead method) to perform the following operations: 1) Take the purification magnetic beads from the refrigerator, equilibrate at room temperature for 30 minutes, and vortex to mix; 2) Take the purified wash solution from the refrigerator, add the corresponding volume of anhydrous ethanol according to the instructions, and prepare the purified wash solution; 3) Take the purified elution solution 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 tube in a small centrifuge for 30 s, place it on a magnetic stand for 2 min, and carefully discard the supernatant after the supernatant is clear; 6) Keep the PCR tube on the magnetic stand, add 200 μl of purified wash solution to the PCR tube, and carefully discard the supernatant; 7) Repeat step 6), centrifuge the PCR tube briefly in a small centrifuge, place it on a magnetic stand, and use a pipette to remove all residual liquid, and air dry for about 2 min at room temperature; 8) Add 40 μl of purified elution solution to the PCR tube, vortex to mix, and incubate at room temperature for 10 min; 9) Place the PCR tube on the magnetic stand and adsorb for 1 min; 10) After the supernatant is clear, transfer all the supernatant to a new PCR tube and perform library quantification.

[0078] 5. Library quantification and pooling Refer to the "NGS Library Quantification Kit Instructions" to quantify the library concentration, and mix the library obtained by amplifying the first primer group with the library obtained by amplifying the second primer group according to the pooling rules.

[0079] 6. High-throughput sequencing and bioinformatics analysis According to the operation instructions of the Illumina sequencing reagent kit, the constructed library is subjected to single-end 50bp sequencing on the Illumina NextSeq550Dx (Illumina) instrument. The bioinformatics analysis process is used to analyze the data obtained, and the HCMV drug resistance detection result is obtained.

[0080] Effect example 1. Take 10 HCMV positive plasma samples, respectively, and the DNA of human cells (control, NC), according to the method of example 2, detect, and analyze the primer coverage and sequencing quality according to the average of 10 samples. The results show that NC is negative, which indicates that the primer group of example 1 has good coverage to the full length of UL97 gene, all above 99%; the average depth reaches 5238, through bioinformatics statistical analysis, the data amount of single sample reaches 4 M, which can meet the requirements of on-machine analysis (). Figure 3 ).

[0081] 2. Three clinical plasma samples with positive HCMV were selected, and the method of Example 2 was used for clinical sample verification. The coverage of each drug-resistant site was analyzed, and the specific results of one sample are shown in Table 1: each drug-resistant site of the UL97 gene can be 100% covered (three samples can be 100% covered), which further proves that the primer group of Example 1 has high accuracy. Figure 4

[0082] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the scope of protection of the present application.​

Claims

1. Primer set, which includes first primer set and second primer set: The first primer set includes a combination of nucleotide sequences or continuous fragments as shown in SEQ ID NO:1-16, 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:17-32, 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.

2. A kit comprising the primer set of claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes: PCR reaction solution and DNA polymerase; Preferably, the kit comprises: 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.

4. The use of the primer set according to claim 1 or the kit according to any one of claims 2-3 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).

5. A method for constructing an HCMV resistance gene library, comprising the steps of using the primer set of claim 1 or the kit of any one of claims 2-3.

6. The method according to claim 5, characterized in that, The method includes the following steps: Using the nucleic acid of the sample to be tested as a template, multiplex PCR amplification was performed using the first primer set and the second primer set described in claim 1, respectively; Preferably, 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.

7. An HCMV resistance gene library, obtained by the method described in any one of claims 5-6.

8. A method for sequencing HCMV resistance genes, wherein the HCMV resistance gene library of claim 7 is sequenced.

9. A method for detecting HCMV resistance gene mutation sites, comprising the steps of the method of claim 8; Preferably, the method includes the following steps: analyzing the sequencing results to detect HCMV drug resistance gene mutation sites.

10. The application, method, or library according to any one of claims 4-9, characterized in that, The HCMV resistance gene is the UL97 gene; Preferably, the HCMV resistance gene mutation site is the UL97 gene mutation site.

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