A human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens and its screening method

Abstract

The present invention discloses a human papillomavirus typing primer set for targeted pathogen high-throughput sequencing and a screening method thereof. The primer set of the present invention has the advantages of high specificity, good stability, absence of primer dimers and hairpin structures, low cost, etc., and can be applied to targeted amplification, library construction, targeted pathogen high-throughput sequencing and subsequent bioinformatics analysis of pathogen-infected samples, and can efficiently and accurately detect and classify multiple HPV types.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a human papillomavirus typing primer set for targeted pathogen high-throughput sequencing and a screening method thereof. Background Art

[0002] Human papillomavirus is a virus that is closely related to a variety of human diseases, especially cervical cancer, anal cancer and some skin diseases. At present, there are 231 known types of HPV, which are classified by the WHO International Agency for Research on Cancer into high-risk, medium-risk and low-risk types according to their carcinogenic risks. 14 types of high-risk HPV, such as HPV16 and HPV18, are directly related to the occurrence of cancers such as cervical cancer, while low-risk HPV, such as HPV-6 and HPV-11, can also cause benign lesions such as genital warts. Therefore, accurate typing and detection of HPV are of great significance in clinical diagnosis, vaccine development and public health management.

[0003] At present, the detection and typing of HPV mainly rely on molecular biology technology, and common methods include PCR (polymerase chain reaction) amplification, gene chips, Sanger sequencing, NGS sequencing, etc. These technologies play an important role in the screening and typing of HPV. Although the existing HPV detection and typing methods have improved the accuracy and sensitivity of detection to a certain extent, there are still some limitations. Traditional HPV typing detection usually requires the use of multiple pairs of specific primers to cover various high-risk and low-risk types, which is prone to non-specific amplification, resulting in false positive results and reducing the reliability of the detection. The existing technology is not sensitive enough for the detection of low-abundance HPV types, and it is difficult to accurately detect them in a complex metagenome background. Not only does it increase the cost and complexity of detection, but it also reduces the specificity and accuracy of the detection. The missed detection of low-abundance HPV by the existing technology may lead to misjudgment of clinical diagnosis and affect the early prevention and treatment of the disease.

[0004] The HPV genome is highly diverse, and the gene sequences between different types vary significantly. Therefore, primer design must take into account both specificity and coverage, and be able to specifically amplify the target type without non-specific amplification with non-target sequences. In order to cover multiple HPV types, traditional methods often require the use of a large number of different primers, which not only increases the complexity and cost of the experiment, but may also cause mutual interference between primers. Therefore, it is necessary to design a scheme that can detect multiple HPV types with fewer primers. In pathogen detection, sensitivity is a key indicator. Designing efficient primers to ensure that low-abundance HPV DNA in the sample can be detected is an important goal of primer design. These primers need to be widely used in clinical diagnosis and public health monitoring, including cervical cancer screening, vaccine effectiveness evaluation, and etiology research on other HPV-related diseases. Summary of the invention

[0005] The present application proposes a human papillomavirus typing primer set for targeted pathogen high-throughput sequencing and a screening method thereof. The primer set has the advantages of a small number of primers but can achieve typing of more HPV types, and can solve the problems of poor primer specificity, poor stability, the presence of primer dimers and hairpin structures, and high cost in the prior art.

[0006] A human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens, comprising the following primers:

[0007] Upstream primer 1, the nucleotide sequence is shown in SEQ ID NO.1;

[0008] Upstream primer 2, the nucleotide sequence is shown in SEQ ID NO.2;

[0009] Upstream primer 3, the nucleotide sequence is shown in SEQ ID NO.3;

[0010] Upstream primer 4, the nucleotide sequence is shown in SEQ ID NO.4;

[0011] Upstream primer 5, the nucleotide sequence is shown in SEQ ID NO.5;

[0012] Upstream primer 7, the nucleotide sequence is shown in SEQ ID NO.7;

[0013] Upstream primer 8, the nucleotide sequence is shown in SEQ ID NO.8;

[0014] Upstream primer 10, the nucleotide sequence is shown in SEQ ID NO.10;

[0015] Upstream primer 11, the nucleotide sequence is shown in SEQ ID NO.11;

[0016] Downstream primer 1, the nucleotide sequence is shown in SEQ ID NO.12;

[0017] Downstream primer 2, the nucleotide sequence is shown in SEQ ID NO.13;

[0018] Downstream primer 3, the nucleotide sequence is shown in SEQ ID NO.14;

[0019] Downstream primer 4, the nucleotide sequence is shown in SEQ ID NO.15;

[0020] Downstream primer 7, the nucleotide sequence is shown in SEQ ID NO.18;

[0021] Downstream primer 8, the nucleotide sequence is shown in SEQ ID NO.19;

[0022] Downstream primer 9, the nucleotide sequence is shown in SEQ ID NO.20;

[0023] Downstream primer 10, the nucleotide sequence is shown in SEQ ID NO.21;

[0024] Downstream primer 11, the nucleotide sequence is shown in SEQ ID NO.22;

[0025] Downstream primer 13, the nucleotide sequence is shown in SEQ ID NO.24;

[0026] Downstream primer 14, the nucleotide sequence is shown in SEQ ID NO.25;

[0027] Downstream primer 15, the nucleotide sequence is shown in SEQ ID NO.26;

[0028] Downstream primer 17, the nucleotide sequence is shown in SEQ ID NO.28;

[0029] Downstream primer 18, the nucleotide sequence is shown in SEQ ID NO.29.

[0030] Optionally, the human papillomavirus includes at least one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV53, HPV73, and HPV82.

[0031] A human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens, comprising the following primers:

[0032] Upstream primer 1, the nucleotide sequence is shown in SEQ ID NO.1;

[0033] Upstream primer 2, the nucleotide sequence is shown in SEQ ID NO.2;

[0034] Upstream primer 3, the nucleotide sequence is shown in SEQ ID NO.3;

[0035] Upstream primer 4, the nucleotide sequence is shown in SEQ ID NO.4;

[0036] Upstream primer 5, the nucleotide sequence is shown in SEQ ID NO.5;

[0037] Upstream primer 6, the nucleotide sequence is shown in SEQ ID NO.6;

[0038] Upstream primer 7, the nucleotide sequence is shown in SEQ ID NO.7;

[0039] Upstream primer 8, the nucleotide sequence is shown in SEQ ID NO.8;

[0040] Upstream primer 9, the nucleotide sequence is shown in SEQ ID NO.9;

[0041] Upstream primer 10, the nucleotide sequence is shown in SEQ ID NO.10;

[0042] Upstream primer 11, the nucleotide sequence is shown in SEQ ID NO.11;

[0043] Downstream primer 1, the nucleotide sequence is shown in SEQ ID NO.12;

[0044] Downstream primer 2, the nucleotide sequence is shown in SEQ ID NO.13;

[0045] Downstream primer 3, the nucleotide sequence is shown in SEQ ID NO.14;

[0046] Downstream primer 4, the nucleotide sequence is shown in SEQ ID NO.15;

[0047] Downstream primer 5, the nucleotide sequence is shown in SEQ ID NO.16;

[0048] Downstream primer 6, the nucleotide sequence is shown in SEQ ID NO.17;

[0049] Downstream primer 7, the nucleotide sequence is shown in SEQ ID NO.18;

[0050] Downstream primer 8, the nucleotide sequence is shown in SEQ ID NO.19;

[0051] Downstream primer 9, the nucleotide sequence is shown in SEQ ID NO.20;

[0052] Downstream primer 10, the nucleotide sequence is shown in SEQ ID NO.21;

[0053] Downstream primer 11, the nucleotide sequence is shown in SEQ ID NO.22;

[0054] Downstream primer 12, the nucleotide sequence is shown in SEQ ID NO.23;

[0055] Downstream primer 13, the nucleotide sequence is shown in SEQ ID NO.24;

[0056] Downstream primer 14, the nucleotide sequence is shown in SEQ ID NO.25;

[0057] Downstream primer 15, the nucleotide sequence is shown in SEQ ID NO.26;

[0058] Downstream primer 16, the nucleotide sequence is shown in SEQ ID NO.27;

[0059] Downstream primer 17, the nucleotide sequence is shown in SEQ ID NO.28;

[0060] Downstream primer 18, the nucleotide sequence is shown in SEQ ID NO.29.

[0061] Optionally, the human papillomavirus comprises HPV2, HPV6, HPV7, HPV11, HPV13, HPV16, HPV17, HPV18, HPV22, HPV23, HPV26, HPV27, HPV31, HPV32, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV44, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV57, HPV58, HPV59, At least one of HPV61, HPV62, HPV64, HPV66, HPV67, HPV68, HPV69, HPV70, HPV72, HPV73, HPV80, HPV81, HPV82, HPV83, HPV84, HPV96, HPV97, HPV106, HPV107, HPV110, HPV114, HPV118, HPV120, HPV122, HPV145, HPV151, HPV159, HPV167, and HPV185.

[0062] Optionally, the kit comprises the above-mentioned human papillomavirus typing primer set for targeted pathogen high-throughput sequencing.

[0063] The present invention also proposes the use of the above-mentioned typing primer set and kit in targeted amplification, library construction, targeted pathogen high-throughput sequencing, and bioinformatics analysis of pathogen infection samples.

[0064] A method for screening a human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens, comprising the following steps:

[0065] Step 1) obtaining multiple genome sequences of each type of human papillomavirus as reference genome sequences;

[0066] Step 2) Find and extract the conserved region sequences of each type through multiple sequence alignment of the reference genome sequence of each type;

[0067] Step 3) designing primer pairs for the conserved region sequences of each type extracted in step 2);

[0068] Step 4) extracting the corresponding amplicon sequence from the primers obtained in step 3), and aligning the amplicon sequence to itself and the nt database, performing coverage and specificity analysis on the amplicon sequence, calculating the coverage and specificity, and screening primers with coverage and specificity percentages greater than 90%, to obtain a first screening primer set;

[0069] The coverage is the ratio of amplicon sequences of a single type to the reference genome sequence of the same type; the specificity is the ratio of amplicon sequences of a single type to the total number of sequences that are only matched to their own type when compared to the nt database;

[0070] Step 5) removing primers with a high risk of generating hairpin structures or primer dimers from the first screening primer set to obtain a second screening primer set;

[0071] Step 6) Performing electrophoresis experiment on the primers in the second screening primer set for the corresponding HPV type, and screening out the primer pairs that can amplify a single band without non-specific amplification through the electrophoresis graph to obtain the third screening primer set;

[0072] Step 7) The amplicon sequences of the third screening primer set are mixed in equal proportions to construct a library and subjected to targeted high-throughput sequencing. The sequencing data are aligned to the amplicon sequence, the number of reads is calculated, and the primer pairing combination with the highest amplicon sequence alignment ratio is selected to form the typing primer set.

[0073] The NT library is a nucleic acid sequence database, which is mainly used to store and manage nucleic acid sequence data, especially for bioinformatics research and data analysis. Those skilled in the art can directly download and use it.

[0074] Optionally, the genome sequence obtained in step 1) is a human papillomavirus genome sequence in which the uncertain base N accounts for less than 1% and is a full-length genome sequence.

[0075] Optionally, in step 2), the sequence similarity of the conserved region is greater than 95% and the length is greater than 300 bp.

[0076] Optionally, in step 3), the length of the designed primers is 18-27 bp, and the length of the amplicon is 100-500 bp.

[0077] Optionally, in step V), the primers are subjected to hairpin structure and primer dimer analysis, and are scored and ranked, the lower the score, the better, and primer combinations with low scores are screened out to obtain a second screening primer set.

[0078] Optionally, if the electrophoresis test result of the primer pair in step 6) is unqualified, return to step 3) to redesign the primer pair.

[0079] Optionally, the types that can be distinguished by the amplicons of the screened primer set must include 14 high-risk types HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 66 / 68 and 3 medium-risk types HPV53 / 73 / 82 that are highly correlated with cervical cancer as announced by the World Health Organization.

[0080] Optionally, the amplicon sequence of the screened primer set can distinguish a total of 59 types of HPV2 / 6 / 7 / 11 / 13 / 16 / 17 / 18 / 22 / 23 / 26 / 27 / 31 / 32 / 33 / 34 / 35 / 39 / 40 / 42 / 44 / 45 / 51 / 52 / 53 / 55 / 56 / 57 / 58 / 59 / 61 / 62 / 64 / 66 / 67 / 68 / 69 / 70 / 72 / 73 / 80 / 81 / 82 / 83 / 84 / 96 / 97 / 106 / 107 / 110 / 114 / 118 / 120 / 122 / 145 / 151 / 159 / 167 / 185.

[0081] Compared with the prior art, the advantages of the present invention are:

[0082] The primer set of the present invention has the advantages of high specificity, good stability, absence of primer dimers and hairpin structures, low cost, etc. It can be applied to targeted amplification, library construction, targeted pathogen high-throughput sequencing and subsequent bioinformatics analysis of pathogen-infected samples, and can efficiently and accurately detect and separate multiple HPV types.

[0083] The primer screening method of the present invention has the following advantages: by utilizing the high throughput and high sensitivity of metagenomic sequencing technology, primers that can cover multiple HPV types are designed, the number of primers is reduced, and the detection efficiency is improved. The present invention uses bioinformatics analysis tools to compare and analyze the HPV genome sequence, and screens out highly conserved and specific sequence regions as the basis for primer design. This can not only ensure the specificity of the primers, but also cover more HPV types. Using multiple PCR technology, multiple HPV types are amplified simultaneously in one reaction system, which improves the detection throughput and efficiency and reduces the experimental operation steps and costs. The present invention verifies the specificity, sensitivity and stability of the primers through dry experiments and wet experiments to ensure the effectiveness of the designed primers in practical applications. It can be flexibly targeted at different application scenarios (such as clinical diagnosis, public health monitoring, etc.) to ensure its reliability and operability in different detection environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0085] Figure 1is a flow chart of the method of the present invention;

[0086] Figure 2 This is the electrophoresis verification test result diagram of primer pairing PCR products amplifying HPV types 6, 11, 16, 18, 31, 33, 39, 40, 42, 45, 51, and 52;

[0087] Figure 3 This is the electrophoresis verification test result diagram of primer pairing PCR products amplifying HPV types 53, 54, 55, 56, and 57;

[0088] Figure 4 This is the electrophoresis verification test result diagram of primer pairing PCR products amplifying HPV types 58, 61, 62, and 66;

[0089] Figure 5 This is the electrophoresis verification test result diagram of primer pairing PCR products amplifying HPV types 67, 81, and 82;

[0090] Figure 6 This is the electrophoresis verification test result diagram of primer pairing PCR products amplifying HPV types 16, 58, 34, and 54;

[0091] Figure 7 This is the electrophoresis verification test result diagram of primer pairing PCR products amplifying HPV types 35, 40, 43, 44, 52, 54, 58, 59, 61, 68, 69, 71, and 82;

[0092] Figure 8 This is the electrophoresis verification test result of primer pairing PCR products that amplified HPV types 26, 73, 82, 68, and 43. DETAILED DESCRIPTION

[0093] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail. The experimental methods described in the following examples are all conventional methods unless otherwise specified: if no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0094] Example 1

[0095] The screening method of the primer set in the present invention is as follows Figure 1 Shown

[0096] 1. Screen and download reference genome sequences

[0097] The primer set to be designed in this embodiment is required to be able to be used to distinguish the following HPV2 / 6 / 7 / 11 / 13 / 16 / 17 / 18 / 22 / 23 / 26 / 27 / 31 / 32 / 33 / 34 / 35 / 39 / 40 / 42 / 44 / 45 / 51 / 52 / 53 / 55 / 56 / 57 / 58 / 59 / 61 / 62 / 64 / 66 / 67 / 68 / 69 / 70 / 72 / 73 / 80 / 81 / 82 / 83 / 84 / 96 / 97 / 106 / 107 / 110 / 114 / 118 / 120 / 122 / 145 / 151 / 159 / 167 / 185, a total of 59 types of viruses.

[0098] First, we screened and downloaded as many high-quality, complete HPV genome sequences of each type as possible from public databases such as NCBI. During screening, we ensured that the proportion of uncertain bases N in the genome sequence was less than 1% and that it was a full-length genome sequence. We ensured the high quality and integrity of the selected genome sequence to provide reliable reference genome data.

[0099] 2. Extract conserved region sequences through multiple sequence alignment

[0100] Using multiple sequence alignment software such as ClustalW and MAFFT, we compared the genome sequences of each type and found the conserved region sequences with a similarity greater than 95% and a length greater than 300bp. The conserved region sequence is an important basis for designing primers, which can ensure the universality and specificity of primers in different samples.

[0101] 3. Primer design

[0102] Based on the conserved region sequence, use professional primer design software such as Primer3 and primer-blast to design specific primers. The designed primer length is required to be 18-27bp and the amplicon length is 100-500bp.

[0103] 4. Primer evaluation and screening

[0104] A) Analysis of coverage and specificity of amplicon sequences: extract the corresponding amplicon sequences from the designed primers, and align them to the nt database, calculate coverage and specificity, where coverage is the percentage of amplicon sequences of a single type aligned to the reference genome sequence of the type. Specificity is the percentage of amplicon sequences of a single type aligned to the nt database that only align to its own type in the total number of alignments, and then screen out primers with coverage and specificity greater than 90% to obtain the first screening primer set.

[0105] B) Hairpin structure and primer dimer analysis: The hairpin structure and primer dimer analysis was performed on the first screening primer set. The lower the score, the better. According to the score ranking, the primer combination with a low score was screened out to obtain the second screening primer set.

[0106] C) Wet experiment verification: The primers that can amplify a single band without non-specific amplification are screened through the electrophoresis diagram to obtain the third screening primer set. If it is unqualified, return to the primer design step and redesign it. The electrophoresis verification test results of the PCR products of HPV types amplified by some primer pairs are shown in the figure below. Figure 2-8 As shown in Table 1 (in the same primer pairing, as long as one sample can be amplified, the primer pair is considered to be in compliance), the corresponding codes in the electrophoresis diagram are shown in Table 1. The reagents used in the wet experiment, their sources, and the experimental steps are as follows:

[0107] 1. Reagents

[0108]

[0109] 2. Experimental steps

[0110] A. Take a tube of quality control powder, add 300μl Nuclear-free water, and shake to fully dissolve it.

[0111] B. Preparation of extraction reagents: Before use, the reaction reagent tank can be gently inverted and mixed three times, then placed in a plate centrifuge for instant centrifugation, and the aluminum foil film on the 96-well reagent tank can be torn off.

[0112] C. Sample addition: Add 300uL of sample and 20uL of proteinase K to the 1st and 7th wells of the 96-well reagent reservoir.

[0113] D. Turn on the power of the extractor, open the instrument door, insert the reaction reagent slot into the slot so that the reaction reagent slot cannot move forward, backward, left or right, and insert the magnetic rod sleeve into the magnetic rod sleeve mounting bracket.

[0114] E. Close the instrument door, click the start button on the main interface, select the corresponding program DR-4801-KZ, and the machine automatically completes the extraction process. After the program is completed, transfer the eluted nucleic acid in the elution tank to a 1.5ml EP tube.

[0115]

[0116]

[0117] Table 1 Electrophoresis and sequencing comparison results of PCR products of HPV types amplified by some primer pairs

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] D) Targeted pathogen sequencing data analysis: The amplicon sequences of the third screening primer set were mixed in equal proportions to construct a library and then subjected to tNGS. The sequencing data results were analyzed, and the sequencing data comparison results of the HPV types amplified by the amplicons of some primer pairs were shown in Table 1. It was verified that the designed HPV-Fa, HPV-Fb, HPV-Ra, and HPV-Rb were not effective. Finally, the primer pairing combination with the highest amplicon sequence comparison ratio was selected as the final primer set. The primer sequences of the final primer set are shown in Table 2. This final primer set can be used to distinguish all 59 types.

[0124] Table 2 Primer ID and sequence of the final primer set

[0125]

[0126] In addition, according to Table 1, it can be seen that the primer combination of HPV-F1, HPV-F2, HPV-F3, HPV-F4, HPV-F5, HPV-F7, HPV-F8, HPV-F10, HPV-F11, HPV-R1, HPV-R2, HPV-R3, HPV-R4, HPV-R7, HPV-R8, HPV-R9, HPV-R10, HPV-R11, HPV-R13, HPV-R14, HPV-R15, HPV-R17, and HPV-R18 can effectively detect 14 high-risk types HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 66 / 68 and 3 medium-risk types HPV53 / 73 / 82.

[0127] 5. Primer application

[0128] The final selected primer set is used for targeted amplification, library construction, targeted pathogen high-throughput sequencing and subsequent bioinformatics analysis of pathogen infection samples to ensure efficient and accurate detection and typing of multiple HPV types. This primer set has the characteristics of a small number of primers, high detection efficiency, wide coverage, strong specificity, high sensitivity, wide application, simple operation and low cost. It is suitable for clinical diagnosis and public health monitoring, and has broad application prospects, clinical value and market value.

[0129] Practical application cases: HPV-F1, HPV-F2, HPV-F3, HPV-F4, HPV-F5, HPV-F7, HPV-F8, HPV-F10, HPV-F11, HPV-R1, HPV-R2, HPV-R3, HPV-R4, HPV-R7, HPV-R8, HPV-R9, HPV-R10, HPV-R11, HPV-R13, HPV-R14, HP V-R15, HPV-R17, HPV-R18 primer combination, using the wet experimental method in the above embodiment to detect the human papillomavirus genotyping test room quality control product (lyophilized) of Guangzhou Kaipu Pharmaceutical Technology Co., Ltd., which contains 14 high-risk types of HPV HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 66 / 68 and 3 medium-risk types HPV53 / 73 / 82. The human papillomavirus typing primer group was verified, and after targeted amplification, library construction, targeted pathogen high-throughput sequencing, and finally bioinformatics analysis was performed through the sequencing results. The analysis results are shown in Table 3, and it can be seen that all high- and medium-risk types can be effectively detected.

[0130] Table 3 Results of pathogen targeted sequencing analysis

[0131]

[0132] In addition, it should be noted that the above are only preferred embodiments of the present invention and are not limited to the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions recorded in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens, characterized in that: The primers included are: Upstream primer 1, the nucleotide sequence is shown in SEQ ID NO.1; Upstream primer 2, the nucleotide sequence is shown in SEQ ID NO.2; Upstream primer 3, the nucleotide sequence is shown in SEQ ID NO.3; Upstream primer 4, the nucleotide sequence is shown in SEQ ID NO.4; Upstream primer 5, the nucleotide sequence is shown in SEQ ID NO.5; Upstream primer 7, the nucleotide sequence is shown in SEQ ID NO.7; Upstream primer 8, the nucleotide sequence is shown in SEQ ID NO.8; Upstream primer 10, the nucleotide sequence is shown in SEQ ID NO.10; Upstream primer 11, the nucleotide sequence is shown in SEQ ID NO.11; Downstream primer 1, the nucleotide sequence is shown in SEQ ID NO.12; Downstream primer 2, the nucleotide sequence is shown in SEQ ID NO.13; Downstream primer 3, the nucleotide sequence is shown in SEQ ID NO.14; Downstream primer 4, the nucleotide sequence is shown in SEQ ID NO.15; Downstream primer 7, the nucleotide sequence is shown in SEQ ID NO.18; Downstream primer 8, the nucleotide sequence is shown in SEQ ID NO.19; Downstream primer 9, the nucleotide sequence is shown in SEQ ID NO.20; Downstream primer 10, the nucleotide sequence is shown in SEQ ID NO.21; Downstream primer 11, the nucleotide sequence is shown in SEQ ID NO.22; Downstream primer 13, the nucleotide sequence is shown in SEQ ID NO.24; Downstream primer 14, the nucleotide sequence is shown in SEQ ID NO.25; Downstream primer 15, the nucleotide sequence is shown in SEQ ID NO.26; Downstream primer 17, the nucleotide sequence is shown in SEQ ID NO.28; Downstream primer 18, the nucleotide sequence is shown in SEQ ID NO.

29.

2. The human papillomavirus typing primer set for targeted pathogen high-throughput sequencing according to claim 1, characterized in that: The human papillomavirus includes at least one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV53, HPV73, and HPV82.

3. A human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens, characterized in that: The primers included are: Upstream primer 1, the nucleotide sequence is shown in SEQ ID NO.1; Upstream primer 2, the nucleotide sequence is shown in SEQ ID NO.2; Upstream primer 3, the nucleotide sequence is shown in SEQ ID NO.3; Upstream primer 4, the nucleotide sequence is shown in SEQ ID NO.4; Upstream primer 5, the nucleotide sequence is shown in SEQ ID NO.5; Upstream primer 6, the nucleotide sequence is shown in SEQ ID NO.6; Upstream primer 7, the nucleotide sequence is shown in SEQ ID NO.7; Upstream primer 8, the nucleotide sequence is shown in SEQ ID NO.8; Upstream primer 9, the nucleotide sequence is shown in SEQ ID NO.9; Upstream primer 10, the nucleotide sequence is shown in SEQ ID NO.10; Upstream primer 11, the nucleotide sequence is shown in SEQ ID NO.11; Downstream primer 1, the nucleotide sequence is shown in SEQ ID NO.12; Downstream primer 2, the nucleotide sequence is shown in SEQ ID NO.13; Downstream primer 3, the nucleotide sequence is shown in SEQ ID NO.14; Downstream primer 4, the nucleotide sequence is shown in SEQ ID NO.15; Downstream primer 5, the nucleotide sequence is shown in SEQ ID NO.16; Downstream primer 6, the nucleotide sequence is shown in SEQ ID NO.17; Downstream primer 7, the nucleotide sequence is shown in SEQ ID NO.18; Downstream primer 8, the nucleotide sequence is shown in SEQ ID NO.19; Downstream primer 9, the nucleotide sequence is shown in SEQ ID NO.20; Downstream primer 10, the nucleotide sequence is shown in SEQ ID NO.21; Downstream primer 11, the nucleotide sequence is shown in SEQ ID NO.22; Downstream primer 12, the nucleotide sequence is shown in SEQ ID NO.23; Downstream primer 13, the nucleotide sequence is shown in SEQ ID NO.24; Downstream primer 14, the nucleotide sequence is shown in SEQ ID NO.25; Downstream primer 15, the nucleotide sequence is shown in SEQ ID NO.26; Downstream primer 16, the nucleotide sequence is shown in SEQ ID NO.27; Downstream primer 17, the nucleotide sequence is shown in SEQ ID NO.28; Downstream primer 18, the nucleotide sequence is shown in SEQ ID NO.

29.

4. The human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens according to claim 3, characterized in that: The human papillomavirus comprises HPV2, HPV6, HPV7, HPV11, HPV13, HPV16, HPV17, HPV18, HPV22, HPV23, HPV26, HPV27, HPV31, HPV32, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV44, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV57, HPV58, HPV59, HPV At least one of HPV120, HPV122, HPV145, HPV151, HPV159, HPV167, and HPV185.

5. A human papillomavirus typing kit for targeted pathogen high-throughput sequencing, characterized in that: The kit comprises the human papillomavirus typing primer set for high-throughput sequencing of targeted pathogens as described in claim 1 or 3.

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