Primer combination and kit for detecting human papilloma virus nucleic acid 28 typing based on sanger sequencing method

Abstract

The invention relates to the technical field of gene detection, and provides a primer combination and a kit for detecting human papilloma virus nucleic acid 28 types based on a sanger sequencing method.Specific amplification primers are designed for the human papilloma virus nucleic acid 28 types respectively and used for assisting a fluorescent nucleic acid detection kit in detecting objection samples of human papilloma viruses, and the detection sensitivity of the human papilloma virus nucleic acid 28 types is improved. The nucleic acid sequence and subtype of a sample are determined through sanger sequencing, the sensitivity is high, 0.5 copies / ul can be detected, and typing determination of different samples can be effectively assisted. Meanwhile, a packaging box and a tube cover of the kit are optimized, human papilloma virus nucleic acid 28 typing marks are added to the packaging box and the tube cover, and the kit marks and the tube cover marks are in one-to-one correspondence, so that the kit is used for rapidly distinguishing different subtype detection tubes and is also beneficial to split charging and use.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, and in particular to a primer combination and a kit for detecting 28 types of human papillomavirus nucleic acid based on Sanger sequencing. Background Art

[0002] Human papillomavirus (HPV) is a group of DNA viruses that can infect human skin and mucous membranes and is closely related to the occurrence of a variety of malignant tumors, especially cervical cancer. It has been confirmed that persistent infection with high-risk human papillomavirus is a necessary factor for the occurrence of cervical cancer and precancerous lesions, that is, HPV infection is the most critical link in the process of cervical cancer. There are currently more than 200 different types of HPV. HPV typing tests can predict the risk of the subject, determine the screening interval, and distinguish between persistent infection and transient infection so that early treatment can be carried out to reduce the incidence and mortality rate.

[0003] Therefore, accurate and rapid identification of specific HPV types is of great significance for the early diagnosis, treatment and prognosis assessment of the disease.

[0004] Over the years, there have been many methods for detecting HPV, such as cytology, immunohistochemistry detection of HPV antigens, HPV antibody detection, etc. However, since the above detection methods cannot type the HPV virus and have disadvantages such as low sensitivity, poor specificity, and long time consumption, these detection methods have gradually been eliminated.

[0005] At present, the detection of HPV mainly adopts molecular biological methods, such as nucleic acid hybridization, gene chip, polymerase chain reaction, etc. Among them, the most widely used is the fluorescence quantitative PCR method, which is time-saving, highly sensitive, and can be automated, eliminating manual operation, thus becoming the mainstream HPV virus detection method. However, fluorescence PCR usually determines the typing of HPV through fluorescence signals. Due to the sensitivity and specificity of different nucleic acid detection kits, it is impossible to give a definite result for the fluorescent signal of samples with different characteristics, and it is impossible to accurately determine the typing of samples.

[0006] Therefore, the use of DNA sequencing technology is the only standard for determining sample typing. Although the second-generation sequencing (NGS) in DNA sequencing can perform high-throughput sequencing and sequence a large number of DNA fragments at the same time, it is only suitable for large-scale genome sequencing projects. When the sequencing read length is short, the accuracy of the sequencing results is slightly lower than that of the first-generation sequencing, and a higher sequencing depth is required to compensate. As a classic DNA sequencing technology, Sanger sequencing is widely used in clinical molecular biology testing for its high accuracy and good repeatability. However, due to the problem of amplification sensitivity, false negatives may occur when the sample concentration is lower than 0.5 copies / μL. Therefore, the design of specific primers for different HPV types and the development of efficient detection kits are still a major challenge currently faced. Summary of the invention

[0007] In order to solve the above-mentioned problems of detection sensitivity and accuracy, the present invention conducts sequence analysis on human papillomavirus nucleic acid 28 typing, designs specific amplification primers for human papillomavirus nucleic acid 28 typing, and uses them for first-generation sequencing to determine the sample typing, which can effectively assist in solving the typing determination of abnormal samples.

[0008] A primer combination for detecting 28 types of human papillomavirus nucleic acid based on Sanger sequencing method, comprising primer pairs 16-F3 / R3, 18-F2 / R2, 26-F3 / R3, 31-F / R, 33-F / R, 35-F / R, 39-F3 / R3, 45-F / R, 51-F / R, 52-F3 / R3, 53-F3 / R3, 56-F3 / R3, 58-F / R, 59-F / R, 66-F3 / R3, 68-F3 / R3, 73-F3 / R3, 82-R2 / R3, 06-F / R, 11-F2 / R2, 40-F3 / R3, 42-F2 / R2, 43-F3 / R3, 44-F2 / R2, 54-F / R, 61-F / R, 81-F3 / R3, 83-F2 / R2, the base sequences of the primer pairs are shown in SEQ ID NO: 1-56.

[0009] Preferably, the primer combination comprises at least one of the primer pairs shown in claim 1.

[0010] A kit for detecting 28 types of human papillomavirus nucleic acid based on Sanger sequencing method comprises the above primer combination, extraction reagent, detection reagent, human papillomavirus negative control group NC and human papillomavirus positive control group PC.

[0011] Furthermore, the human papillomavirus negative control group NC is a negative sample of normal cervical exfoliated cells; the human papillomavirus positive control group PC is a mixed sample of 28 typing plasmids, and the concentration of each typing plasmid is 100 copies / μl; the extraction reagent is a nucleic acid extraction or purification reagent.

[0012] Furthermore, the detection reagent includes 10x Buffer A buffer and human papillomavirus PCR reaction solution.

[0013] Further, the 10x Buffer A buffer comprises the following components: Tris HCl, KCl, (NH 4 ) 2 SO 4 、TWEEN-20、MgSO 4 and DEPC.H 2 O.

[0014] Preferably, the 10x Buffer A buffer solution is composed of the following components, measured by volume percentage per 10 mL: 20-30% 1M Tris HCl, pH = 9.0, 20-30% 2.5M KCl, 2-5% 2.5M (NH 4 ) 2 SO 4 , 2-5% 100% TWEEN-20, 2-5% 1M MgSO 4 , the remainder is DEPC.H 2 O.

[0015] Furthermore, the human papillomavirus PCR reaction solution includes: the primer combination, 10xBufferA, 10mM-dNTP, 5U / μl-Hotstart E-Taq, DEPC. 2 O.

[0016] Preferably, the human papillomavirus PCR reaction solution is composed of the following components, based on the dosage of 30 μl / reaction: 0.5-0.8 μl of the primer combination for detecting human papillomavirus 28 typing, 5.0 μl of 10xBufferA, 2.5 μl of 10mM-dNTP, 1-2 μl of 5U / μl-Hotstart E-Taq, and the balance of DEPC. 2 O.

[0017] Furthermore, the kit is used to detect the following 28 types of human papillomavirus nucleic acid typing: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, 06, 11, 40, 42, 43, 44, 54, 611, 81, 83.

[0018] Preferably, 28 typing marks are provided on the packaging box and tube cover of the kit.

[0019] The method for using the above kit comprises the following steps:

[0020] (1) Collecting samples and extracting the collected samples;

[0021] (2) Take out the corresponding reaction solution from the kit, thaw it on ice, vortex mix, and centrifuge for 10 seconds;

[0022] (3) Add the calculated amount of human papillomavirus PCR reaction solution to the sample centrifuge tube, vortex mix, centrifuge for 10 seconds and then directly perform PCR amplification reaction:

[0023] (4) detecting the amplified product obtained by the amplification reaction by agarose gel electrophoresis;

[0024] (5) The amplification results were evaluated by gel electrophoresis and further Sanger sequencing was performed.

[0025] Furthermore, the reaction system of the PCR amplification reaction is: 30 μl of human papillomavirus PCR reaction solution and 20 μl of DNA reaction template;

[0026] Furthermore, the reaction procedure of the PCR amplification reaction is: 95°C for 3 min; 95°C for 20 s, 60°C for 30 s, 72°C for 45 s, 45 cycles; 72°C for 3 min.

[0027] The advantages of the present invention are:

[0028] The present invention designs specific primers for different HPV typing, and can assist in detecting samples with objectionable human papillomavirus through the Sanger sequencing method, solve the typing determination of a small number of samples with abnormalities, and clarify the nucleic acid sequence and subtype of the samples. Compared with the prior art, the detection result of the kit of the present invention is highly accurate and has an extremely low error rate. For the sequencing of short-fragment DNA, especially sequences less than 1000 bases, the results are reliable and highly sensitive, and 0.5 copies / ul can be detected, which saves more cost and time. At the same time, the present invention optimizes the packaging box and tube cover of the kit, adds 28 typing labels to the packaging box and tube cover, and the kit label corresponds to the tube cover label one by one, which is used to quickly distinguish different subtype detection tubes, and is also conducive to packaging and use, solving the problem that each subtype has a separate detection reagent in the prior art, resulting in too many components in the kit and easy confusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of a detection kit for detecting 28 types of human papillomavirus nucleic acid based on Sanger sequencing method created by the present invention;

[0030] Figure 2 These are different test results of the same sample using different test reagents (a shows HPV18 positive, b shows HPV18 negative);

[0031] Figure 3 This is an image of the PCR amplification results of HPV18; the left wells are DNA marker (the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp (bright band), 250bp, and 100bp), the positive control PC amplification product, and the HPV18 sample (abnormal sample) amplification product;

[0032] Figure 4 This is the result of Sanger sequencing of the amplified product;

[0033] Figure 5 This is an imaging diagram of the PCR amplification results of HPV18 at different sample concentrations; the first well from the left is a DNA marker (the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp (target band), 250bp, and 100bp), and the second to 21st wells from the left are 20 replicate samples (1 is a HPV18 sample concentration gradient of 1 copy / μl; 0.5 is a HPV18 sample concentration gradient of 0.5 copies / μl; 0.1 is a HPV18 sample concentration gradient of 0.1 copies / μl);

[0034] Figure 6It is an imaging diagram of the PCR amplification results of HPV16 at different sample concentrations; among them, the first well from the left is a DNA marker (the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp (target band), 250bp, and 100bp), and the second to 21st wells from the left are 20 replicate samples (1 is the HPV16 sample concentration gradient of 1 copies / μl; 0.5 is the HPV16 sample concentration gradient of 0.5 copies / μl; 0.1 is the HPV16 sample concentration gradient of 0.1 copies / μl). DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the invention to clearly and completely describe the technical solutions in the embodiments of the invention. Obviously, the described embodiments are only part of the embodiments of the invention, not all of them. Based on the embodiments in the invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the invention.

[0036] It should be noted that the various technical terms mentioned in the invention are all technical terms that have long been clearly known in the relevant technical field, so no further explanation will be given. They do not affect and should not constitute the accurate understanding of the technical solution by those skilled in the art.

[0037] Unless otherwise specified, all reagents and materials used in the present invention are purchased from the market.

[0038] Example 1 Primer design and screening

[0039] The human papillomavirus (HPV) genome sequence was searched, and the sequences of 28 HPV types were downloaded from the NCBI database for homology comparison analysis. Three pairs of specific primers were designed for the conserved regions of each type. The designed primer pairs were uploaded to NCBI for homology comparison with other pathogens, and the results showed that the homology with other pathogens was low.

[0040] The optimal primer combination was screened by analyzing the specificity, concentration and sensitivity of the primers for detecting 28 human papillomavirus genotypes including 18 high-risk types HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82 and 10 low-risk types HPV06, 11, 40, 42, 43, 44, 54, 61, 81, 83, including upstream primers and downstream primers, as shown in Table 1.

[0041] Example 2 Preparation of detection kit

[0042] This embodiment constructs a kit for detecting the following 28 types of human papillomavirus nucleic acid typing: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, 06, 11, 40, 42, 43, 44, 54, 61, 81, 83, including the following components:

[0043] Primer combinations, extraction reagents, detection reagents, human papillomavirus negative control group NC and human papillomavirus positive control group PC.

[0044] In this embodiment, the extraction reagent is a nucleic acid extraction reagent (Shanghai Fengxiebei 20230142). The human papillomavirus negative control group NC is NC1, a normal cervical exfoliated cell negative sample from the sampling box of Jiangsu Jianyou Medical Technology Co., Ltd., and NC2, a normal cervical exfoliated cell negative sample from the sampling box of Chaozhou Cape Biochemical Co., Ltd.

[0045] The primer combinations include: 16-F3 / R3, 18-F2 / R2, 26-F3 / R3, 31-F / R, 33-F / R, 35-F / R, 39-F3 / R3, 45-F / R, 51-F / R, 52-F3 / R3, 53-F3 / R3, 56-F3 / R3, 58-F / R, 59-F / R, 66-F3 / R3, 68-F3 / R3, 73-F3 / R 3, 82-R2 / R3, 06-F / R, 11-F2 / R2, 40-F3 / R3, 42-F2 / R2, 43-F3 / R3, 44-F2 / R2, 54-F / R, 61-F / R, 81-F3 / R3, 83-F2 / R2 primer pairs, the upstream and downstream primers in the primer pairs are the upstream and downstream primers designed and screened in Example 1, and their base sequences are shown in SEQ ID NO: 1-56 in sequence, as shown in Table 1.

[0046] The detection reagents include 10xBuffer A buffer and human papillomavirus PCR reaction solution.

[0047] The 10x Buffer A buffer is shown in Table 2 and is composed of the following components: 20-30% 1M (pH = 9.0) Tris HCl, 20-30% 2.5M KCl, 2-5% 2.5M (NH 4 ) 2 SO 4 , 2-5% 100% TWEEN-20, 2-5% 1M MgSO 4 , the remainder is DEPC.H 2 O.

[0048] The human papillomavirus PCR reaction solution includes the above-mentioned detection of human papillomavirus 28 typing as shown in Table 3, 0.5-0.8 μl of upstream primer, 0.5-0.8 μl of downstream primer, 5.0 μl of 10xBufferA, 2.5 μl of 10mM-dNTP, 1-2 μl of 5U / μl-Hotstart E-Taq, and the balance of DEPC. 2 O, and the reaction solution was prepared to 30 μl / reaction.

[0049] At the same time, if Figure 1 As shown, this embodiment optimizes the packaging box and tube cover of the test kit, adds 28 types of typing labels to the packaging box and tube cover, and the reagent kit label corresponds to the tube cover label one by one, which is used to quickly distinguish different subtype detection tubes.

[0050] Table 1 Primers and nucleotide sequences for HPV genotyping

[0051]

[0052]

[0053]

[0054] Table 2 10x buffer A formula

[0055] Reagent name Original concentration Input volume (μL / 10mL) TrisHCl 1M (pH = 9.0) 20-30％ <![CDATA[(NH 4 ) 2 SO 4 ]]> 2.5M 2-5％ KCl 2.5M 20-30％ TWEEN-20 100％ 2-5％ <![CDATA[MgSO 4 ]]> 1M 2-5％ <![CDATA[DEPC.H 2 The]]> — 30～40％

[0056] Table 3 Human papillomavirus PCR reaction solution

[0057] Material Name Dosage 10xBufferA 5.0μl dNTP(10mM) 2.5μl Hotstart E-Taq (5U / μl) 1~2μl Upstream primer F 0.5~0.8μl Downstream primer R 0.5~0.8μl <![CDATA[DEPC.H 2 The]]> All 30 μl

[0058] Example 3 Method of using the kit

[0059] The sample types applicable to the detection kit of the present invention include but are not limited to: cervical exfoliated cells collected by cervical brushes and cervical swabs, or secretions and epithelial cells collected from vaginal swabs, rectal swabs, body surface swabs, genital urinary tract secretions and urine, and vaginal and rectal mixed swabs.

[0060] The collection, transportation and storage of the test samples of the present invention comply with the "Operational Procedures for the Collection, Transportation and Storage of Clinical Laboratory Specimens". It is recommended that the samples be sent for inspection immediately after sampling. The samples should be stored at room temperature for no more than 2 days, at 2-8°C for no more than 7 days, at -20+5°C for no more than 12 months, and at below -70°C for no more than 24 months.

[0061] The method for using the kit in this embodiment comprises the following steps:

[0062] 1. Sampling:

[0063] The samples in this example are cervical exfoliated cells collected by cervical brush and cervical swab

[0064] 2. Sampling:

[0065] The collected samples are extracted using HPV nucleic acid extraction reagent and sample release agent to form a homogenized liquid sample for testing.

[0066] 3. Add sample:

[0067] Take out the corresponding reaction solution from the kit, thaw on ice, vortex mix, and centrifuge for 10 seconds; calculate the number of PCR reaction wells required for each reaction system (the calculation formula is: number of wells n = number of samples (N) + 2), add the calculated amount of human papillomavirus PCR reaction solution to the sample centrifuge tube, vortex mix, centrifuge for 10 seconds, and perform amplification reaction according to the amplification reaction system and amplification reaction program shown in Tables 4 and 5;

[0068] 4. Sanger sequencing:

[0069] 5 μl of amplified product was added with 1 μl of 6x glycerol gel loading buffer I containing bromophenol blue and EDTA (purchased from Sangon Biotech (Shanghai) Co., Ltd., Lot: J427KA9254), mixed thoroughly, and added to the prepared 1.2% (w / w) agarose gel comb for electrophoresis at 150V for 20min. The imaging analysis was performed using a gel imaging analyzer (Denojee, Tanon 1600), and samples with clear bands and correct sizes were selected for direct Sanger sequencing (Sangon Biotech (Shanghai) Co., Ltd.) to obtain the corresponding sample sequences.

[0070] Table 4 Amplification reaction system

[0071] Human papillomavirus PCR reaction solution 30μl DNA reaction template 20μl

[0072] Table 5 Amplification reaction program

[0073]

[0074] Example 4 Sample Detection Test

[0075] The actual sample (unknown sample) was tested using the kit for detecting human papillomavirus nucleic acid 28 typing based on the Sanger sequencing method constructed in Example 2 of the present invention.

[0076] The unknown samples were amplified using different HPV fluorescence detection kits to obtain Figure 2 HPV18 positive ( Figure 2 a) and negative ( Figure 2b) The fluorescence amplification curves of the two different results make it impossible to determine the subtype of the unknown sample, and Sanger sequencing is needed for auxiliary proof;

[0077] According to the method of Example 3 of the present invention, the unknown sample is amplified using a kit for typing human papillomavirus nucleic acid 28, and the amplified product is subjected to electrophoresis test. The PCR amplification result is imaged as shown in FIG. Figure 3 As shown, a band of the same size as the positive control was obtained, and then the sample sequence was obtained by Sanger sequencing. The result is as follows Figure 4 As shown, the sample subtype was confirmed to be HPV18 through comparison.

[0078] The kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing constructed in Example 2 of the present invention was used, but the Sanger sequencing method of the present invention was not used to perform sensitivity testing on actual samples (sample results of which were known to be HPV18 typing and HPV16 typing).

[0079] The results are as follows Figure 5 and Figure 6 As shown by Figure 5 It can be seen that when the HPV18 sample concentration gradient is 1 and 0.5 copies / μl, 20 samples have amplification bands 100% detected; but when the HPV18 sample concentration gradient is 0.1 copies / μl, only 18 samples have amplification bands, and the detection rate is 90%, which is lower than 95%. When the sample concentration is lower than 0.5 copies / μl, false negatives may occur. Figure 6 It can be seen that when the HPV16 sample concentration gradient was 1 and 0.5 copies / μl, all 20 samples had amplification bands with 100% detection; but when the HPV16 sample concentration gradient was 0.1 copies / μl, only 10 samples had amplification bands, with a detection rate of 50%. Therefore, the sensitivity of this reagent is limited to 0.5 copies / μl, and false negatives may occur when the sample is lower than 0.5 copies / μl.

[0080] In summary, from the test results of Example 4, it can be seen that due to the sensitivity and specificity of different nucleic acid detection kits, the HPV typing is judged by the fluorescence signal. When different results are judged, the test results of the sample are controversial; and for the different sample fluorescence signals, no definite results can be given, and the Sanger sequencing method is needed to assist in proving the sample gene sequence, clarify the sample typing, and realize the accurate identification of HPV typing. The detection kit of the present application has a high sensitivity of 0.5 copies / μl, which can effectively amplify most of the different samples and realize accurate detection of HPV subtypes through the Sanger sequencing method.

[0081] For those skilled in the art, the invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the invention is defined by the appended claims rather than the above description, so it is intended to include all changes within the meaning and scope of the equivalent elements of the claims. Any reference numerals in the claims should not be regarded as limiting the claims involved.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any minor modifications, equivalent substitutions and improvements made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the technical solution of the present invention.

Claims

1. A primer combination for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing, characterized in that: Including primer pairs 16-F3 / R3, 18-F2 / R2, 26-F3 / R3, 31-F / R, 33-F / R, 35-F / R, 39-F3 / R3, 45-F / R, 51-F / R, 52-F3 / R3, 53-F3 / R3, 56-F3 / R3, 58-F / R, 59-F / R, 66-F3 / R3, 68-F3 / R3, 73-F3 / R3, 82-R2 / R3, 06-F / R, 11-F2 / R2, 40-F3 / R3, 42-F2 / R2, 43-F3 / R3, 44-F2 / R2, 54-F / R, 61-F / R, 81-F3 / R3, 83-F2 / R2, the base sequences of the primer pairs are shown in SEQ ID NOs: 1 to 56.

2. The primer combination for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing method according to claim 1, characterized in that: The primer combination comprises at least one of the primer pairs described in claim 1.

3. A kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing, characterized in that: The method comprises the primer combination as claimed in claim 1, an extraction reagent, a detection reagent, a human papillomavirus negative control group NC and a human papillomavirus positive control group PC; Among them, the human papillomavirus negative control group NC is a negative sample of normal cervical exfoliated cells, the human papillomavirus positive control group PC is a mixed sample of 28 typing plasmids, each typing plasmid concentration is 100 copies / μl, and the extraction reagent is a nucleic acid extraction or purification reagent.

4. A kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing according to claim 3, characterized in that: The detection reagent comprises human papillomavirus PCR reaction solution and 10xBufferA buffer solution.

5. A kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing according to claim 4, characterized in that: The 10xBuffer A buffer solution includes the following components: Tris HCl (pH=9.0), KCl, (NH4)2SO4, TWEEN-20, MgSO4 and DEPC.H2O.

6. A kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing according to claim 4, characterized in that: The human papillomavirus PCR reaction solution comprises: the primer combination as claimed in claim 1, 10xBufferA, 10mM-dNTPl, 5U / μl-Hotstart E-Taq, and DEPC.H2O.

7. A kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing according to claim 3, characterized in that: The kit is used to detect the following 28 types of human papillomavirus nucleic acid typing: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, 06, 11, 40, 42, 43, 44, 54, 61, 81, 83.

8. A method for using the kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing as described in any one of claims 3 to 7, characterized in that: The following steps are involved: (1) Collecting samples and extracting the collected samples; (2) Take out the corresponding human papillomavirus PCR reaction solution from the kit, thaw it on ice, vortex to mix, and centrifuge for 10 seconds; (3) Add the human papillomavirus PCR reaction solution to the sample centrifuge tube, vortex mix, centrifuge for 10 seconds, and then directly perform the PCR amplification reaction; (4) detecting the amplification product obtained by the amplification reaction by agarose gel electrophoresis; (5) The amplification results were evaluated by gel electrophoresis and further Sanger sequencing was performed.

9. The method for using the kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing according to claim 8, characterized in that: The reaction system of the PCR amplification reaction is: 30 μl of human papillomavirus PCR reaction solution and 20 μl of DNA reaction template.

10. The method for using the kit for detecting human papillomavirus nucleic acid 28 typing based on Sanger sequencing according to claim 8, characterized in that: The reaction program of the PCR amplification reaction is: 95°C for 3 min; 95°C for 20 s, 60°C for 30 s, 72°C for 45 s, 45 cycles; 72°C for 3 min.

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