Primer and probe combination for detecting human papilloma virus nucleic acid 14 typing based on digital PCR method, kit and application

Abstract

The invention relates to the technical field of gene detection, in particular to a primer and probe combination and a kit for detecting human papilloma virus nucleic acid 14 typing based on a digital PCR (polymerase chain reaction) method and application of the primer and probe combination and the kit. 14 HPV high-risk types including human papilloma viruses HPV52, HPV35, HPV18, HPV66, HPV39, HPV56, HPV68, HPV16, HPV31, HPV58, HPV59, HPV33, HPV45 and HPV51 can be quantitatively detected by utilizing a micro-droplet digital PCR method, compared with the prior art, the quantitative detection of more HPV types can be realized, the specificity is strong, the sensitivity is high, the lowest detection limit is reduced to 0.1 copies / uL, and real absolute quantification is realized.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, and in particular to a primer and probe combination, a kit and an application thereof for detecting human papillomavirus nucleic acid 14 typing based on a digital PCR method. Background Art

[0002] Human papillomavirus (HPV) belongs to the Papillomaviridae family and is a group of circular double-stranded DNA viruses that can infect human skin and mucous membranes. HPV is tissue-specific and causes a variety of papillomas or warts on the human skin and proliferative lesions of the genital epithelium by infecting human skin and mucous membrane epithelial cells. Therefore, accurate and rapid identification of specific HPV types is of great significance for the early diagnosis, treatment and prognosis assessment of the disease.

[0003] At present, the main detection methods for human papillomavirus include NGS-based detection technology, fluorescent PCR detection technology, gene chip or hybridization capture detection technology, but all of them are qualitative detection technologies. Under normal circumstances, most HPV infections are transient infections. Only persistent high-risk infections will greatly increase the probability of cervical cancer. The HPV viral load directly reflects the proliferation of HPV in the body. The higher the viral load, the more difficult it is for the body to clear it, and the higher the probability of persistent infection. The HPV viral load cannot be obtained by qualitative detection alone, which in turn affects the early diagnosis and treatment of the disease. Therefore, it is urgent to develop a quantitative detection technology that can directly reflect the HPV viral load, so as to facilitate real-time monitoring of changes in the HPV viral load in patients and assist doctors in diagnosis.

[0004] Droplet Digital PCR (ddPCR) is a nucleic acid quantitative analysis method based on traditional PCR technology. Its core principle is to distribute the reaction system containing DNA templates into a large number of independent micro-reaction units for amplification and optical detection, thereby achieving absolute quantitative analysis. Unlike traditional quantitative PCR (qPCR), ddPCR does not require a standard curve to determine the starting concentration of the target sequence. Since it does not rely on amplification efficiency and standard curves, it has higher accuracy and repeatability, and can directly give the starting concentration of the target sequence to achieve true absolute quantification.

[0005] Patent CN105018647B discloses a kit and a detection method for accurate quantitative typing detection of HPV16 / 18 based on digital PCR. The kit can be used for droplet processing of sample solutions in the detection of tissue samples with extremely low HPV content, and the HPV type and copy number can be directly judged according to the fluorescence type and the number of fluorescent droplets, which greatly simplifies the operation steps and improves the detection accuracy and reproducibility. However, it can only detect two HPV typings, HPV16 and HPV18, and the detection time takes 3 hours, and the lowest detection sensitivity can only reach 1 copy.

[0006] Patent CN106191317A discloses a primer pair and a kit for detecting HPV16 virus based on digital PCR. The kit can detect HPV virus through the digital PCR method, but can only detect HPV16 typing, and the minimum detection limit is only 1 copy.

[0007] Patent CN118360437A discloses a kit for early screening and monitoring of HPV-related tumors, which is used for the detection of free HPV in human plasma. Through mixed probes, free HPV16 / 18 / 52 / 56 in human plasma can be detected simultaneously in one reaction system, and can be accurately typed, but only four types can be detected, and the minimum detection limit is 250 copies / mL.

[0008] At present, the existing droplet digital PCR technology can detect fewer HPV types, and it is difficult to detect multiple HPV types. This is mainly because the gene sequences of different HPV types are slightly different, especially the differences between high-risk types are more subtle, which increases the difficulty of designing highly specific primers and probes that do not produce cross reactions. The more detection types, the more primers are used, and the more likely it is that the primers will cross-react, affecting the accuracy, specificity and sensitivity of the detection. Summary of the invention

[0009] In order to address the deficiencies in the prior art, a quantitative detection technology is needed that can simultaneously detect multiple HPV typings and directly reflect the HPV viral load. The present invention provides a primer and probe combination, a kit and an application for detecting 14 types of human papillomavirus nucleic acid typing based on a digital PCR method, which can detect 14 types of HPV typing.

[0010] A primer and probe combination for detecting human papillomavirus nucleic acid 14 typing based on a digital PCR method, comprising primer pairs and probes matching each primer pair, wherein the nucleotide sequences thereof are shown in SEQ ID NOs: 1 to 42; wherein the primer pairs comprise forward primers and reverse primers corresponding to human papillomavirus nucleic acid 14 typing.

[0011] Preferably, the primer and probe combination for human papillomavirus nucleic acid 14 typing is as follows:

[0012] A primer and probe combination for HPV-18, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.1, a specific reverse primer sequence as shown in SEQ ID NO.2, and a probe sequence as shown in SEQ ID NO.3;

[0013] A primer and probe combination for HPV-59, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.4, a specific reverse primer sequence as shown in SEQ ID NO.5, and a probe sequence as shown in SEQ ID NO.6;

[0014] A primer and probe combination for HPV-68, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.7, a specific reverse primer sequence as shown in SEQ ID NO.8, and a probe sequence as shown in SEQ ID NO.9;

[0015] A primer and probe combination for HPV-33, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.10, a specific reverse primer sequence as shown in SEQ ID NO.11, and a probe sequence as shown in SEQ ID NO.12;

[0016] A primer and probe combination for HPV-52, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.13, a specific reverse primer sequence as shown in SEQ ID NO.14, and a probe sequence as shown in SEQ ID NO.15;

[0017] A primer and probe combination for HPV-66, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.16, a specific reverse primer sequence as shown in SEQ ID NO.17, and a probe sequence as shown in SEQ ID NO.18;

[0018] A primer and probe combination for HPV-31, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.19, a specific reverse primer sequence as shown in SEQ ID NO.20, and a probe sequence as shown in SEQ ID NO.21;

[0019] A primer and probe combination for HPV-51, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.22, a specific reverse primer sequence as shown in SEQ ID NO.23, and a probe sequence as shown in SEQ ID NO.24;

[0020] A primer and probe combination for HPV-35, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.25, a specific reverse primer sequence as shown in SEQ ID NO.26, and a probe sequence as shown in SEQ ID NO.27;

[0021] A primer and probe combination for HPV-16, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.28, a specific reverse primer sequence as shown in SEQ ID NO.29, and a probe sequence as shown in SEQ ID NO.30;

[0022] A primer and probe combination for HPV-45, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.31, a specific reverse primer sequence as shown in SEQ ID NO.32, and a probe sequence as shown in SEQ ID NO.33;

[0023] A primer and probe combination for HPV-39, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.34, a specific reverse primer sequence as shown in SEQ ID NO.35, and a probe sequence as shown in SEQ ID NO.36;

[0024] A primer and probe combination for HPV-56, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.37, a specific reverse primer sequence as shown in SEQ ID NO.38, and a probe sequence as shown in SEQ ID NO.39;

[0025] A primer and probe combination for HPV-58, the nucleotide sequence of which includes a specific forward primer sequence as shown in SEQ ID NO.40, a specific reverse primer sequence as shown in SEQ ID NO.41, and a probe sequence as shown in SEQ ID NO.42;

[0026] Furthermore, the human papillomavirus nucleic acid 14 types detected by the digital PCR method are HPV18, HPV59, HPV68, HPV33, HPV52, HPV66, HPV31, HPV51, HPV35, HPV16, HPV45, HPV39, HPV56, and HPV58.

[0027] Furthermore, the primer and probe combination also includes an internal standard primer pair and a probe, and the nucleotide sequences of the internal standard primer pair and the probe are shown in SEQ ID NO.43 to SEQ ID NO.45.

[0028] Furthermore, the 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group; the fluorescent group includes any one of FAM, HEX, ROX, and CY5; the quenching group includes any one of BHQ1 and BHQ2.

[0029] Another object of the present invention is to provide a kit for detecting 14 types of human papillomavirus nucleic acid typing based on the digital PCR method, comprising the primer and probe combination, PCR reaction solution, negative control and positive control; wherein the negative control is DEPC water, and the positive control is a mixture of 14 typing plasmids and an internal standard gene plasmid.

[0030] Furthermore, the PCR reaction solution includes: the primer and probe combination, 2x PCR MIX, DNA template and DEPC.H2O, wherein the 2x PCR MIX is a nucleic acid amplification reaction reagent, including DNA polymerase, dNTPs, Mg 2+ , buffer solution.

[0031] Preferably, based on the dosage of 30 μl / reaction, the PCR reaction solution includes 15 μL PCR MIX, 1.0-2.0 μL 10 μM-forward primer, 1.0-2.0 μL 10 μM-reverse primer, 0.3-0.8 μL probe, 5 μL DNA template, and the balance is DEPC.H2O.

[0032] Further, the PCR reaction solution includes a first PCR reaction solution, a second PCR reaction solution, a third PCR reaction solution and a fourth PCR reaction solution;

[0033] The first PCR reaction solution includes primers and probes corresponding to HPV18, HPV59, HPV68 and HPV33, and the nucleotide sequences of the corresponding forward primers, reverse primers and probes are shown in SEQ ID NOs: 1 to 12 in sequence;

[0034] The second PCR reaction solution includes primers and probe combinations corresponding to HPV52, HPV66, HPV31 and HPV51, and the nucleotide sequences of the corresponding forward primers, reverse primers and probes are shown in SEQ ID NOs: 13 to 24 in sequence;

[0035] The third PCR reaction solution includes primers and probe combinations corresponding to HPV35, HPV16, and HPV45, and the nucleotide sequences of the corresponding forward primers, reverse primers, and probes are shown in SEQ ID NOs: 25 to 33 in sequence;

[0036] The fourth PCR reaction solution includes primers and probe combinations corresponding to HPV39, HPV56, and HPV58, and the nucleotide sequences of the corresponding forward primers, reverse primers, and probes are shown in SEQ ID NOs: 34 to 42 in sequence;

[0037] Wherein, the third PCR reaction solution further includes an internal standard primer pair and a probe having nucleotide sequences as shown in SEQ ID NOs: 43 to 45.

[0038] The present invention also provides the use of the above-mentioned kit in detecting human papillomavirus genotyping using a digital PCR method.

[0039] Furthermore, a method for detecting human papillomavirus genotyping using a digital PCR method comprises the following steps:

[0040] (1) Collecting samples and extracting the collected samples;

[0041] (2) preparing PCR reaction solution;

[0042] (3) transferring the PCR reaction solution into a fully automatic droplet preparation instrument to generate a droplet chip;

[0043] (4) performing PCR amplification reaction on the generated microdroplet chip;

[0044] (5) placing the PCR amplified samples in a biochip reader for reading;

[0045] (6) Obtain the interpretation result based on the number of droplets.

[0046] Furthermore, the reaction procedure of the PCR amplification reaction is: 50°C for 5 min; 95°C for 2 min; 95°C for 15 s, 55°C for 30 s, and 45 cycles.

[0047] The advantages of the present invention are:

[0048] The present invention, through the design of primer probes and the optimization of the reaction system, can detect 14 high-risk HPV types including HPV52, HPV35, HPV18, HPV66, HPV39, HPV56, HPV68, HPV16, HPV31, HPV58, HPV59, HPV33, HPV45 and HPV51 in cervical exfoliated cells, genital urethral secretions and urine by digital PCR method. Compared with the prior art, the present invention can realize the detection of more HPV typing and has strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1It is a scatter plot of the quantitative results of HPV18, 59, 68, and 33 positive samples after quadruple ddPCR in Example 3 of the present invention;

[0050] Figure 2 It is a scatter plot of the quantitative results of HPV52, 66, 31, 51 positive samples after quadruple ddPCR in Example 3 of the present invention;

[0051] Figure 3 It is a scatter plot of the quantitative results of HPV35, internal standard, 16, and 65 positive samples after quadruple ddPCR in Example 3 of the present invention;

[0052] Figure 4 It is a scatter plot of the quantitative results of HPV39, 56, 58 positive samples after quadruple ddPCR in Example 3 of the present invention;

[0053] Figure 5 This is a graph showing the sensitivity detection results of HPV18 at different concentrations in Example 4 of the present invention;

[0054] Figure 6 is a graph showing the sensitivity detection results of HPV68 at different concentrations in Example 4 of the present invention;

[0055] Figure 7 1 is a graph showing repeated sensitivity detection results of HPV18 at concentrations of 100 copies / μL, 10 copies / μL, 1 copy / μL, and 0.1 copies / μL, respectively, in Example 4 of the present invention;

[0056] Figure 8 1 is a graph showing repeated sensitivity detection results of HPV68 at concentrations of 100 copies / μL, 10 copies / μL, 1 copy / μL, and 0.1 copies / μL, respectively, in Example 4 of the present invention;

[0057] Fig. 9 This is a graph showing the sensitivity repeated detection results of HPV18 and HPV68 at a concentration of 0.01 copies / μL in Example 4 of the present invention. DETAILED DESCRIPTION

[0058] 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.

[0059] It should be noted that the various installation methods and technical terms mentioned in the invention are technical terms that have long been clearly known in the relevant technical field, so no further explanation is given. In addition, the same reference numerals are used for the same components, but this does not affect and should not constitute an accurate understanding of the technical solution by those skilled in the art.

[0060] Example 1 Design of primers and probes

[0061] Download the whole genome sequences of 14 HPV types from the NCBI database for comparison and analysis, select the conserved regions of the L1 gene of each type to design specific primers and probes, and select the human RNase P gene as the internal standard gene target sequence to design specific primers and probes. Perform BLAST analysis on the designed primers and probes on NCBI to analyze the specificity of the primers and probes to ensure that there is no cross reaction with other sequences.

[0062] Finally, a unique primer and probe combination for 14 types of human papillomavirus was obtained, and the ddPCR method can be used to detect 14 types of HPV typing, including primer pairs and probes matching each primer pair, whose nucleotide sequences are shown in SEQ ID NO: 1 to 42; and also including internal standard primer pairs and probes, whose nucleotide sequences are shown in SEQ ID NO.43 to SEQ ID NO.45, as shown in Table 1 for details:

[0063] Among them, the primer pairs include forward primers and reverse primers corresponding to 14 types of human papillomavirus nucleic acid including HPV18, HPV59, HPV68, HPV33, HPV52, HPV66, HPV31, HPV51, HPV35, HPV16, HPV45, HPV39, HPV56, and HPV58.

[0064] The 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group; the fluorescent group includes any one of FAM, HEX, ROX, and CY5; the quenching group includes any one of BHQ1 and BHQ2.

[0065] Table 1 Nucleotide sequences of primer and probe combinations for HPV genotyping

[0066]

[0067]

[0068]

[0069] Example 2 Preparation of detection kit

[0070] In this embodiment, a kit for detecting the following 14 types of human papillomavirus nucleic acid typing: HPV18, HPV59, HPV68, HPV33, HPV52, HPV66, HPV31, HPV51, HPV35, HPV16, HPV45, HPV39, HPV56, HPV58 was prepared, including the following components:

[0071] Primer and probe combination, PCR reaction solution (the PCR reaction solution in this embodiment includes the first PCR reaction solution, the second PCR reaction solution, the third PCR reaction solution and the fourth PCR reaction solution), negative control (the negative control in this embodiment is DEPC.H2O) and positive control (the positive control in this embodiment is a mixture of 14 typing plasmids and an internal standard gene plasmid).

[0072] The PCR reaction solution includes the primer and probe combination, 2x PCRMIX (the 2x PCRMIX is a nucleic acid amplification reaction reagent (purchased from Shanghai Yisheng Biotechnology Co., Ltd., including DNA polymerase, dNTPs, Mg 2+ , buffer), DNA template and DEPC.H2O, see Table 2 for details.

[0073] The first PCR reaction solution includes primers and probe combinations corresponding to HPV18, HPV59, HPV68 and HPV33, and the nucleotide sequences of the corresponding forward primers, reverse primers and probes are shown in SEQ ID NOs: 1 to 12 in sequence;

[0074] The second PCR reaction solution includes primers and probe combinations corresponding to HPV52, HPV66, HPV31 and HPV51, and the nucleotide sequences of the corresponding forward primers, reverse primers and probes are shown in SEQ ID NOs: 13 to 24 in sequence;

[0075] The third PCR reaction solution includes primer and probe combinations corresponding to HPV35, HPV16, and HPV45, and the nucleotide sequences of the corresponding forward primers, reverse primers, and probes are shown in SEQ ID NOs: 25 to 33, and the nucleotide sequences of the internal standard primer pairs and probes are shown in SEQ ID NOs: 43 to 45.

[0076] The fourth PCR reaction solution includes primer and probe combinations corresponding to HPV39, HPV56, and HPV58, and the nucleotide sequences of the corresponding forward primers, reverse primers, and probes are shown in SEQ ID NOs: 34-42 in sequence.

[0077] Table 2 PCR reaction solution components

[0078]

[0079]

[0080] Example 3 Digital PCR Detection

[0081] The test kit is suitable for samples such as cervical exfoliated cells, genital and urinary tract secretions and urine collected by cervical brush and cervical swab.

[0082] The kit used in this example can simultaneously perform rapid typing and quantitative detection of 14 types of human papillomavirus in a quadruple fluorescence channel.

[0083] The present embodiment uses a digital PCR method to detect human papillomavirus genotyping, comprising the following steps:

[0084] (1) Sample processing

[0085] Using the nucleic acid extraction or purification reagent produced by Shanghai Nuclear Biology, take 200 μL of sample (the sample in this example is a positive sample mixed with 14 HPV typing plasmids) and 20 μL of proteinase K and add them to the pre-packaged nucleic acid extraction reagent for extraction. The extraction procedure refers to the reagent manual.

[0086] (2) Preparation of PCR reaction solution

[0087] Four tubes of reaction solution (corresponding to the first PCR reaction solution, the second PCR reaction solution, the third PCR reaction solution and the fourth PCR reaction solution in Example 2) need to be prepared, with a total volume of 30 μL per tube of reaction solution. The specific components are shown in Table 2.

[0088] (3) Preparation of microdroplets

[0089] Transfer 30uL of the PCR reaction solution from step (2) into the Loader T200 fully automatic droplet preparation instrument to generate a droplet chip;

[0090] (4) PCR amplification

[0091] The generated microdroplet chip was manually transferred to a Cycler T200 PCR amplification instrument for PCR amplification reaction. The reaction program of the PCR amplification reaction is shown in Table 3.

[0092] (5) Detection of droplets

[0093] After the PCR reaction is completed, the chip is placed in the Imager T200 biochip reader for reading, and a qualitative analysis of the reading results is performed (in the read droplets, those with fluorescent signals are positive, and those without fluorescent signals are negative);

[0094] (6) Analyze data and display results

[0095] The ddPCR data were analyzed using the “Biochip Data Analysis Green” software, and the samples were quantitatively analyzed. Based on the number of positive and negative droplets, the software automatically reported the number of viral copies (copies / L) in the sample.

[0096] The results are as follows Figure 1-4 As shown, each subtype of human papillomavirus positive in the read droplet will produce a fluorescent signal in the corresponding fluorescent channel. Figure 1 The scatter plot of the quantitative results of HPV18, 59, 68, and 33 positive samples in four fluorescence channels, FAM, HEX, ROX, and CY5, is shown. Figure 2 The scatter plot of the quantitative results of HPV52, 66, 31, and 51 positive samples in four fluorescence channels: FAM, HEX, ROX, and CY5 is shown. Figure 3 The scatter plots of the quantitative results of HPV35, 16, 65, and internal standard positive samples in four fluorescence channels, FAM, HEX, ROX, and CY5, are shown. Figure 4 The scatter plots of the quantitative results of HPV39, 56, and 58 positive samples in four fluorescence channels, FAM, HEX, ROX, and CY5, are shown.

[0097] The results of quadruple droplet digital PCR tests show that the primer pairs do not affect each other and can still achieve specific amplification well, and each subtype can be detected.

[0098] Table 3 Amplification reaction program

[0099]

[0100] Example 4

[0101] The detection method and steps of this embodiment are the same as those of embodiment 3. The mixed samples of HPV18 and HPV68 were detected using the first PCR reaction solution in embodiment 2. The mixed samples were diluted into gradient concentrations of 100 copies / μL, 10 copies / μL, 1 copy / μL, 0.1 copies / μL and 0.01 copies / μL, and the detection was repeated 3 times for each gradient concentration. The detection results are shown in FIG. Figure 5-9 As shown by Figure 5 and Figure 6 The sensitivity test results of HPV18 and HPV68 at different concentrations are shown. The detection rates of HPV18 and HPV68 at 100 copies / μL, 10 copies / μL, 1 copy / μL, and 0.1 copies / μL are all 100%, and the detection rate at 0.01 copies / μL is 33%. Figure 7 and Figure 8The displayed HPV18 and HPV68 repeated detection results at 100 copies / μL, 10 copies / μL, 1 copy / μL, and 0.1 copies / μL respectively. Fig. 9 From the repeated detection results of HPV18 and HPV68 at 0.01 copies / μL, it can be seen that the repeated detection results are the same. The detection rates of HPV18 and HPV68 at 100 copies / μL, 10 copies / μL, 1 copies / μL, and 0.1 copies / μL are all 100%. Therefore, the minimum detection limit of HPV18 and HPV68 typing in this kit is 0.1 copies / μL.

[0102] 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.

[0103] 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 and probe combination for detecting human papillomavirus nucleic acid 14 typing based on digital PCR method, characterized in that: It comprises primer pairs and probes matching each primer pair, and the nucleotide sequences thereof are shown in SEQ ID NOs: 1 to 42; wherein the primer pairs comprise forward primers and reverse primers corresponding to 14 types of human papillomavirus nucleic acid.

2. The primer and probe combination for detecting human papillomavirus nucleic acid 14 typing based on the digital PCR method according to claim 1, characterized in that: The digital PCR method detects 14 types of human papillomavirus nucleic acid, including HPV18, HPV59, HPV68, HPV33, HPV52, HPV66, HPV31, HPV51, HPV35, HPV16, HPV45, HPV39, HPV56, and HPV58.

3. The primer and probe combination for detecting human papillomavirus nucleic acid 14 typing based on the digital PCR method according to claim 1, characterized in that: The primer and probe combination further includes an internal standard primer pair and a probe, and the nucleotide sequences of the internal standard primer pair and the probe are shown in SEQ ID NO.43 to SEQ ID NO.

45.

4. The primer and probe combination for detecting human papillomavirus nucleic acid 14 typing based on the digital PCR method according to claim 1, characterized in that: The 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group; the fluorescent group includes any one of FAM, HEX, ROX, and CY5; the quenching group includes any one of BHQ1 and BHQ2.

5. A kit for detecting human papillomavirus nucleic acid 14 typing by digital PCR method, characterized in that: It comprises the primer and probe combination according to any one of claims 1 to 4, a PCR reaction solution, a negative control and a positive control; wherein the negative control is DEPC water, and the positive control is a mixture of 14 typing plasmids and an internal standard gene plasmid.

6. The kit according to claim 5, characterized in that The PCR reaction solution includes the primer and probe combination, 2x PCR MIX, DNA template and DEPC.H2O, wherein the 2x PCR MIX is a nucleic acid amplification reaction reagent, including DNA polymerase, dNTPs, Mg 2+ , buffer solution.

7. The kit according to claim 5, characterized in that The PCR reaction solution includes a first PCR reaction solution, a second PCR reaction solution, a third PCR reaction solution and a fourth PCR reaction solution; The first PCR reaction solution includes primers and probe combinations corresponding to HPV18, HPV59, HPV68 and HPV33, and the nucleotide sequences of the corresponding forward primers, reverse primers and probes are shown in SEQ ID NOs: 1 to 12 in sequence; The second PCR reaction solution includes primers and probes corresponding to HPV52, HPV66, HPV31 and HPV51, and the nucleotide sequences of the corresponding forward primers, reverse primers and probes are shown in SEQ ID NOs: 13 to 24 in sequence; The third PCR reaction solution includes primers and probes corresponding to HPV35, HPV16, and HPV45, and the nucleotide sequences of the corresponding forward primers, reverse primers, and probes are shown in SEQ ID NOs: 25 to 33 in sequence; The fourth PCR reaction solution includes primers and probe combinations corresponding to HPV39, HPV56, and HPV58, and the nucleotide sequences of the corresponding forward primers, reverse primers, and probes are shown in SEQ ID NOs: 34 to 42 in sequence; Wherein, the third PCR reaction solution further includes an internal standard primer pair and a probe having nucleotide sequences as shown in SEQ ID NOs: 43 to 45.

8. Use of the kit according to claim 5 in detecting human papillomavirus genotyping using a digital PCR method.

9. The method for detecting human papillomavirus genotyping using digital PCR according to claim 8, characterized in that: The following steps are involved: (1) Collecting samples and extracting the collected samples; (2) preparing PCR reaction solution; (3) transferring the PCR reaction solution into a fully automatic droplet preparation instrument to generate a droplet chip; (4) performing PCR amplification reaction on the generated microdroplet chip; (5) placing the PCR amplified samples in a biochip reader for reading; (6) Obtain the interpretation result based on the number of droplets.

10. The method for detecting human papillomavirus genotyping using digital PCR method according to claim 9, characterized in that: The reaction program of the PCR amplification reaction is: 50°C for 5 min; 95°C for 2 min; 95°C for 15 s, 55°C for 30 s, and 45 cycles.

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