Primer probe combination for detecting HPV (human papillomavirus) and / or HPV typing, kit and application

Abstract

The invention provides a primer probe combination for detecting human papilloma virus (HPV) and / or HPV typing, and belongs to the technical field of human papilloma virus (HPV) detection. The primer probe combination comprises one or more of HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6, can distinguish different types of HPV, and has the advantages of good specificity, accuracy, low detection limit and the like. According to the method, a digital PCR method (dPCR) is used for detecting a fluorescence signal of each liquid drop, the copy number of a target gene segment in a whole reaction system is calculated according to the Poisson distribution principle, and absolute quantification can be realized without depending on a standard curve.

Description

Technical Field

[0001] The present invention belongs to the technical field of human papillomavirus (HPV) detection, and in particular relates to a primer-probe combination, a kit and an application thereof for detecting HPV and / or HPV typing. Background Art

[0002] HPV-related malignant tumors include: cervical cancer, vaginal cancer, vulvar cancer, oropharyngeal cancer, penile cancer or anal cancer, etc. Cervical cancer is the main representative of this series of malignant tumors. Tumor recurrence and metastasis are the main factors affecting the five-year survival rate, so its recurrence monitoring is the key to reducing the risk of death in patients. Imaging examination and exfoliative cytology diagnosis are commonly used post-treatment follow-up methods, and histopathological diagnosis is the gold standard. However, it is difficult to detect the recurrence of tiny tumors by imaging. Secondly, frequent radiation exposure and high costs will reduce compliance with regular recurrence monitoring. Histopathology relies on methods such as surgery or biopsy to obtain tumor tissue, which is an invasive operation and is not convenient for recurrence identification. High-risk human papillomavirus (HR-HPV), including HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, etc., can identify HPV-related malignant tumors; secondly, circulating tumor DNA (ctDNA) carries the same genetic characteristics as the primary tumor, and HPV ctDNA is present in the blood of patients with HPV-related tumors. Therefore, HPV ctDNA is an ideal marker for HPV-related tumor recurrence. However, HPV ctDNA in patients with HPV-related malignant tumors is diluted by non-tumor-derived DNA and accounts for a low proportion of plasma free DNA. The sensitivity of real-time fluorescence quantitative PCR (QPCR) is not enough for plasma HPV ctDNA detection. The sensitivity and specificity of digital PCR (dPCR) detection are significantly higher than those of QPCR, and can effectively detect HPVDNA and its concentration in tissues, exfoliated cells and blood. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a primer-probe combination, a kit and an application for detecting HPV and / or HPV typing.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] In the present invention, by screening the primer probes HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, and HPV68E6, it was found that the three groups of primer probes HPV16E6, HPV18E6, and HPV45E6 did not perform well in the test. It is not suitable for detecting HPV and / or HPV typing, but the primer probes HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 have good discrimination for detecting HPV and / or HPV typing using the dPCR method, and have strong specificity, high sensitivity and high accuracy when used to detect HPV and / or HPV typing.

[0006] Based on this, the present invention provides a primer-probe combination for detecting HPV and / or HPV typing, including one or more of the primer-probes HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6;

[0007] The nucleotide sequences of the HPV16E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 4 to 6;

[0008] The nucleotide sequences of the HPV18E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 10 to 12;

[0009] The nucleotide sequences of the HPV31E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 13 to 15;

[0010] The nucleotide sequences of the HPV45E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 25 to 27;

[0011] The nucleotide sequences of the HPV33E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 16 to 18;

[0012] The nucleotide sequences of the HPV35E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 19 to 21;

[0013] The nucleotide sequences of the HPV59E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 43 to 45;

[0014] The nucleotide sequences of the HPV68E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 49 to 51;

[0015] The nucleotide sequences of the HPV58E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 40 to 42;

[0016] The nucleotide sequences of the HPV51E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 31 to 33;

[0017] The nucleotide sequences of the HPV52E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 34 to 36;

[0018] The nucleotide sequences of the HPV56E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 37 to 39;

[0019] The nucleotide sequences of the HPV66E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 46 to 48;

[0020] The nucleotide sequences of the HPV39E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 22-24.

[0021] In the present invention, the 5' end of the probe is modified with a fluorescent group. The fluorescent groups modified at the 5' end of the probe nucleotide sequences in HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 are VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5 and ATTO425, respectively.

[0022] The primer-probe combination of the present invention can accurately detect a variety of different high-risk HPV types, and the HPV type is one or more of HPV16, HPV18, HPV31, HPV45, HPV33, HPV35, HPV58, HPV59, HPV68, HPV51, HPV52, HPV56, HPV66 and HPV39.

[0023] In the present invention, as a preferred embodiment, the primer probe combination is divided into primer probe combination 1, primer probe combination 2 or primer probe combination 3; the probe combination 1 is HPV16E7, HPV18E7, HPV31E7 and HPV45E7; the probe combination 2 is HPV33E7, HPV35E7, HPV59E6, HPV68E6 and HPV58E7; the probe combination 3 is HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6.

[0024] The present invention also provides an application of the primer-probe combination mentioned above in the preparation of a product for detecting HPV and / or HPV typing.

[0025] The product of the present invention includes a reagent or a kit. The primer-probe combination of the present invention is used to detect HPV and / or HPV typing with high specificity, high sensitivity and high accuracy.

[0026] The present invention also provides a kit for detecting HPV and / or HPV typing, comprising the above primer-probe combination.

[0027] In the present invention, the kit also includes an internal reference gene and a PCR reaction mixture. The internal reference gene preferably includes RPP30, and the nucleotide sequences of the probe, upstream primer and downstream primer of RPP30 are shown in SEQ ID NO.52-54; the PCR reaction mixture preferably includes the following components at final concentrations: 1×PCR buffer, 50-400 μM dNTP, 1-2.5 mM MgCl2, 0.1 U-5 U Taq DNA polymerase, 400-750 nM upstream primer and downstream primer, 150-500 nM probe. The present invention uses a PCR reaction mixture for PCR amplification, especially the dosage of the upstream primer, downstream primer and probe provided in the present invention, which improves the specificity, sensitivity and accuracy of detecting HPV and / or HPV typing.

[0028] The present invention also provides a method for detecting HPV and / or HPV typing using a dPCR method, the method being used for non-diagnostic purposes, the method comprising performing PCR amplification using the above primer-probe combination or kit by a dPCR method. As a preferred embodiment, the method comprises the following steps:

[0029] (1) extracting DNA from the sample to be tested;

[0030] (2) using the DNA from step (1) as a template, and performing dPCR amplification using primer and probe combinations;

[0031] (3) Determine whether the sample is HPV positive based on the fluorescence amplification signals of different channels and perform HPV typing.

[0032] In the present invention, the sample type of the sample to be tested includes any one of a tissue sample, an exfoliated cell sample and a plasma sample from a patient with HPV-related lesions.

[0033] The present invention provides an application of a dPCR method in preparing HPV and / or HPV typing products in a detection sample.

[0034] In the present invention, the exfoliated cells include cervical exfoliated cells, penis exfoliated cells, vulva exfoliated cells, oropharyngeal exfoliated cells or anal exfoliated cells. In the present invention, the above sample includes any one of a tissue sample, an exfoliated cell sample and a plasma sample of a patient with HPV-related lesions.

[0035] dPCR is a nucleic acid molecule absolute quantitative technology that divides the PCR reaction system into tens of thousands of independent droplets, that is, tens of thousands of independent reactions. The working principle of the dPCR instrument used in the present invention is to divide the reaction system into tens of thousands of micro-droplets through vibration injection technology, and each micro-droplet can independently complete the PCR amplification reaction.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The present invention provides a primer-probe combination for detecting HPV and / or HPV typing, wherein the primer-probe combination can distinguish different high-risk subtypes of HPV, and has the characteristics of good specificity, accuracy and low detection limit. After amplification, the dPCR method of the present invention produces fluorescence in microdroplets with target nucleic acid molecules, and does not produce fluorescence in microdroplets without target nucleic acid molecules. By detecting the fluorescence signal of each droplet, the copy number of the target gene fragment in the entire reaction system is calculated according to the Poisson distribution principle, and absolute quantification can be achieved without relying on a standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1The results of detecting the corresponding HPV typing by the dPCR method using different primer probes, where 1A in A is the result of detecting HPV16-positive samples by primer probe HPV16E6, and 1E is the result of detecting HPV16-positive samples by primer probe HPV16E7; 2A in B is the result of detecting HPV18-positive samples by primer probe HPV18E6, and 2E is the result of detecting HPV18-positive samples by primer probe HPV18E7; 1A in C is the result of detecting negative samples by primer probe HPV31E7, and 1B is the result of detecting negative samples by primer probe HPV31E7. 31E7 is the result of detecting HPV31 positive samples; 1D in D is the result of detecting HPV45 negative samples by primer probe HPV45E6, and 1F is the result of detecting HPV45 positive samples by primer probe HPV45E7; 1G in E is the result of detecting negative samples by primer probe HPV52E7, and 1H is the result of detecting HPV52 positive samples by primer probe HPV52E7; 2B in F is the result of detecting negative samples by primer probe HPV58E7, and 2D is the result of detecting HPV58 positive samples by primer probe HPV58E7;

[0039] Figure 2 In the figure, 2E in A is the result of detecting negative samples by primer probe HPV51E6, and 2F is the result of detecting positive samples by primer probe HPV51E6; 1A and 1B in B are the results of detecting positive samples by primer probe HPV33E7; 1C and 1D in C are the results of detecting positive samples by primer probe HPV35E7; 1E and 1F in D are the results of detecting positive samples by primer probe HPV39E6; 1G and 1H in E are the results of detecting positive samples by primer probe HPV59E6; 2A and 2C in F are the results of detecting positive samples by primer probe HPV56E6; 1E and 1F in G are the results of detecting positive samples by primer probe HPV66E6; 1G and 1H in H are the results of detecting positive samples by primer probe HPV68E6;

[0040] Figure 3These are the results of detecting HPV45-positive samples with primer probe HPV45E7 in different concentration groups, wherein wells 1A and 1B are the results of detecting HPV45-positive samples with primer probe HPV45E7 in group 1; wells 1C and 1D are the results of detecting HPV45-positive samples with primer probe HPV45E7 in group 2; wells 1E and 1F are the results of detecting HPV45-positive samples with primer probe HPV45E7 in group 3; wells 1G and 1H are the results of detecting HPV45-positive samples with primer probe HPV45E7 in group 4; wells 2A and 2C are the results of detecting HPV45-positive samples with primer probe HPV45E7 in group 5;

[0041] Figure 4 The results of using primer probe internal reference, HPV16E7, HPV18E7, HPV31E7 and HPV45E7 in tube 1 to detect mixed samples of different concentrations (the samples were prepared by mixing gDNA, HPV16, HPV18, HPV31 and HPV45), among which the target gene concentration of 1E and 1F was 1.25E+05 copies / mL, and the target gene concentration of 1G and 1H was 2.5E+03 copies / mL;

[0042] Figure 5 The results of using primer probe internal reference, HPV33E7, HPV35E7, HPV59E6, HPV68E6 and HPV58E7 in tube 2 to detect mixed samples of different concentrations (prepared by mixing gDNA, HPV33, HPV35, HPV59, HPV68 and HPV58), among which, the target gene concentrations of (2E, 2F) and (1A, 1B) were 1.25E+05 copies / mL, and the target gene concentrations of (2G, 2H) and (1C, 1D) were 2.5E+03 copies / mL and 1.25E+04 copies / mL, respectively;

[0043] Figure 6 Results of using primer probe internal reference, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 in tube 3 to detect mixed samples of different concentrations (prepared by mixing gDNA, HPV51, HPV52, HPV56, HPV66 and HPV39), where the target gene concentrations of (2A, 2B) and (2E, 2F) were 1.25E+05 copies / mL, and the target gene concentrations of (2C, 2D) and (2G, 2H) were 2.5E+03 copies / mL;

[0044] Figure 7Results of using primer-probe internal reference, HPV51E6, HPV35E7, HPV59E6 and HPV58E7 in tube 4 to detect mixed samples of different concentrations (prepared by mixing gDNA, HPV51, HPV35, HPV59 and HPV58), among which (1C, 1D) and (1E, 1F) detected target gene concentrations of 1.25E+05copies / mL and 2.5E+03copies / mL, respectively;

[0045] Figure 8 Results of using primer-probe internal reference, HPV33E7, HPV52E7, HPV56E6 and HPV39E6 in tube 5 to detect mixed samples of different concentrations (gDNA, HPV33, HPV52, HPV56 and HPV39 mixed preparation), among which (2A, 2C) and (2E, 2G) detected target gene concentrations of 1.25E+05copies / mL and 2.5E+03copies / mL, respectively;

[0046] Fig. 9 The results of three repeated tests of HPV16 positive simulation samples by primer-probe combination 1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0047] Fig.10 The results of three repeated tests of HPV18 positive simulation samples by primer-probe combination 1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0048] Fig.11 The results of three repeated tests of HPV31 positive simulation samples by primer-probe combination 1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0049] Fig.12 The results of three repeated tests of HPV45 positive simulation samples by primer-probe combination 1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0050] Fig.13The results of three repeated tests of HPV16 positive simulated samples by primer-probe combination 1-1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulated samples respectively; 2C and 2E are positive and negative controls;

[0051] Fig.14 The results of three repeated tests of HPV18 positive simulation samples by primer-probe combination 1-1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0052] Fig.15 The results of three repeated tests of HPV31 positive simulation samples by primer-probe combination 1-1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0053] Fig.16 The results of three repeated tests of HPV45 positive simulation samples by primer-probe combination 1-1, (1A, 1B, 1C), (1D, 1E, 1F) and (1G, 1H, 2A) are the test results of low, medium and high concentration simulation samples respectively; 2C and 2E are positive and negative controls;

[0054] Fig.17 The dPCR test results of the present invention for high and low concentrations of HPV16 samples;

[0055] Fig.18 The dPCR test results of the present invention for high and low concentrations of HPV18 samples;

[0056] Fig.19 The dPCR test results of the present invention for high and low concentrations of HPV31 samples;

[0057] Fig. 20 The dPCR test results of the present invention for high and low concentrations of HPV45 samples;

[0058] Fig.21 The dPCR test results of the present invention for high and low concentrations of HPV33 samples;

[0059] Fig. 22 The dPCR test results of the present invention for high and low concentrations of HPV35 samples;

[0060] Fig.23 The dPCR test results of the present invention for high and low concentrations of HPV59 samples;

[0061] Fig.24 The dPCR test results of the present invention for high and low concentrations of HPV68 samples;

[0062] Fig.25 The dPCR test results of the present invention for high and low concentrations of HPV58 samples;

[0063] Fig.26 The dPCR test results of the present invention for high and low concentrations of HPV51 samples;

[0064] Fig. 27 The dPCR test results of the present invention for high and low concentrations of HPV52 samples;

[0065] Fig.28 The dPCR test results of the present invention for high and low concentrations of HPV56 samples;

[0066] Fig.29 The dPCR test results of the present invention for high and low concentrations of HPV66 samples;

[0067] Fig.30 The dPCR test results of the present invention for high and low concentrations of HPV39 samples;

[0068] Fig.31 The QPCR test results for high and low concentrations of HPV16 samples;

[0069] Fig.32 The QPCR test results for high and low concentrations of HPV18 samples;

[0070] Fig.33 These are the QPCR test results for high and low concentrations of HPV31 samples;

[0071] Fig.34 These are the QPCR test results for high and low concentrations of HPV45 samples;

[0072] Fig.35 The QPCR test results for high and low concentrations of HPV33 samples;

[0073] Fig.36 The QPCR test results for high and low concentrations of HPV35 samples;

[0074] Fig.37 These are the QPCR test results for high and low concentrations of HPV59 samples;

[0075] Fig.38 The QPCR test results for high and low concentrations of HPV68 samples;

[0076] Fig.39These are the QPCR test results for high and low concentrations of HPV58 samples;

[0077] Fig.40 These are the QPCR test results for high and low concentrations of HPV51 samples;

[0078] Fig.41 These are the QPCR test results for high and low concentrations of HPV52 samples;

[0079] Fig.42 These are the QPCR test results for high and low concentrations of HPV56 samples;

[0080] Fig.43 The QPCR test results for high and low concentrations of HPV66 samples;

[0081] Fig.44 These are the QPCR test results for high and low concentrations of HPV39 samples. DETAILED DESCRIPTION

[0082] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0083] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0084] Example 1

[0085] (1) Design and screening of primers and probes for HPV typing detection using dPCR

[0086] The E6 and E7 gene sequences of all variants of HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39 were downloaded from NCBI, and the conserved regions were screened using MAGE7. Primers and probes were designed in the conserved regions to ensure that all variants of HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39 were covered. The system contains one upstream primer, one downstream primer, and one probe for HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39, and one upstream primer, one downstream primer, and one probe for the RPP30 internal reference gene. The information of primers and probes used for detecting HPV and / or HPV typing is shown in Table 1.

[0087] Table 1 Primer and probe information

[0088]

[0089]

[0090] (2) Sample processing

[0091] Blood samples from patients with cervical cancer were collected, and the concentration of extracted DNA was tested using a Qubit fluorometer; the purity of ctDNA was tested using an Agilent 2200 / 4200 Tapestation, and the main fragment distribution was 170bp to 200bp, confirming that the extraction was qualified. Human papillomavirus nucleic acid detection and genotyping kit (dPCR method) was used for detection, and the DNA loading amount was required to be greater than 20ng and the volume was ≤14μL.

[0092] (3) PCR reaction system configuration

[0093] The components and their contents described in Table 2 were configured to obtain a PCR reaction system.

[0094] Table 2 PCR reaction system

[0095]

[0096]

[0097] Among them, the upstream primers in Table 2 are the upstream primers of HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, HPV68E6, and RPP30 in Table 1, and the downstream primers are HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7 The downstream primers of HPV5E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, HPV68E6, and RPP30, and the probes are the probes of HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV45E6, HPV45E7, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, HPV68E6, and RPP30 in Table 1, respectively. The specific sequences correspond to Table 1.

[0098] (4) dPCR detection

[0099] The prepared PCR reaction system was placed on the dPCR analyzer, and 1.0E+06 copies / mL of positive plasmid samples and nuclease-free water were amplified according to the PCR reaction amplification program. The amplification program was 60°C for 5 minutes of droplet tiling, 95°C for 5 minutes of enzyme activation, 95°C for 10 seconds, 60°C for 30 seconds, 45 cycles, and the droplet recognition channel was selected as CY5.5. The combination with obvious distinction between positive and negative droplets, concentrated positive droplet clusters, and less Rain was selected. If it did not meet the requirements, the upstream or downstream primers needed to be optimized.

[0100] The dPCR instrument can divide the reaction system into about 20,000 droplets and perform independent PCR amplification reactions on each droplet unit.

[0101] During the process, while extending the DNA chain, the Taq enzyme can dissociate the specific fluorescent probe bound to the template through the activity of its own 5'>3' exonuclease, so that the fluorescent reporter group produces fluorescence under a specific light source. After the PCR amplification cycle, the microdroplets with target nucleic acid molecules will produce fluorescence, and the microdroplets without target nucleic acid molecules will not produce fluorescence, and then the fluorescence signal of each droplet unit is detected. The LED light source emits fluorescence of a specified band through a filter to illuminate the microdroplets in the special flat container. The fluorescence signal generated by the microdroplets passes through the paired filter and enters the camera sensor to form an image. The software distinguishes negative droplets from positive droplets by the fluorescent gray value of each droplet, and uses the droplet recognition algorithm to analyze each sample image. The mutant and wild-type copy numbers of the target gene fragment in the reaction system are calculated by Poisson distribution to achieve the purpose of quantitative detection. Among them, the formula of Poisson distribution is shown in Formula 1.

[0102] Negative droplets ÷ total droplets = e －λ ...Formula 1.

[0103] where λ is the average copy number contained in a single droplet.

[0104] Figure 1 and Figure 2 The results showed that the positive and negative droplets screened out by primer probes HPV16E6, HPV16E7, HPV18E6, HPV18E7, HPV31E7, HPV33E7, HPV35E7, HPV39E6, HPV51E6, HPV52E7, HPV56E6, HPV58E7, HPV59E6, HPV66E6, and HPV68E6 were clearly distinguished, and the positive droplet clusters were concentrated. Compared with the primer probe HPV16E6, HPV16E7 had better discrimination. Compared with the primer probe HPV18E6, HPV18E7 had better discrimination. However, the positive and negative droplets could not be distinguished using the primer probe HPV45E6. The screening using the primer probe HPV45E7 could distinguish the positive and negative droplets, but the positive droplet clusters were not concentrated.

[0105] In this embodiment, the dosage of the upstream primer, downstream primer and probe of HPV45E7 with poor discrimination is adjusted, that is, the concentrations of the upstream primer, downstream primer and probe in Table 2 are divided into 5 groups, in which the upstream primer of HPV45E7 in Group 1 is 500 nM, the downstream primer is 500 nM and the probe is 250 nM; in Group 2, the upstream primer of HPV45E7 is 500 nM, the downstream primer is 500 nM and the probe is 400 nM; in Group 3, the upstream primer of HPV45E7 is 500 nM, the downstream primer is 500 nM and the probe is 400 nM; in Group 4, the upstream primer of HPV45E7 is 500 nM, the downstream primer is 500 nM and the probe is 400 nM; in Group 5, the upstream primer of HPV45E7 is 500 nM, the downstream primer is 500 nM and the probe is 400 nM; in Group 6, the upstream primer of HPV45E7 is 500 nM, the downstream primer is 500 nM and the probe is 400 nM. The upstream primer of HPV45E7 in group 4 was 750 nM, the downstream primer was 750 nM, and the probe was 400 nM; the upstream primer of HPV45E7 in group 4 was 500 nM, the downstream primer was 500 nM, and the probe was 500 nM; the upstream primer of HPV45E7 in group 5 was 750 nM, the downstream primer was 750 nM, and the probe was 500 nM. Then, the primer and probe HPV45E7 were detected using the method of steps (1) to (4). The results are shown in FIG. Figure 3 .

[0106] According to the conditions that the positive and negative droplets are clearly distinguished, the positive droplets are concentrated, and the Rain is small, Figure 3 The results show that when the upstream primer of HPV45E7 is 500nM, the downstream primer is 500nM and the probe is 500nM, the discrimination is good when the primer probe HPV45E7 is used for detection.

[0107] The primer and probe information of each HPV typing used in the present invention is shown in Table 3.

[0108] Table 3 Primer and probe information for different HPV typing

[0109]

[0110]

[0111] The sequences of the primers and probes in Table 3 are shown in Table 1.

[0112] Example 2

[0113] A primer-probe combination for detecting HPV and / or HPV typing based on a dPCR method, wherein the primer-probe combination is HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6, with RPP30 as an internal reference, wherein the specific sequence of the primer-probe combination and RPP30 is shown in Table 1, wherein the HPV1 The fluorescent markers modified at the 5' end of the probe sequences of HPV6E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 were VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5 and ATTO425, respectively. The fluorescent marker modified at the 5' end of the probe sequence of RPP30 was FAM.

[0114] Example 3

[0115] A kit for dPCR amplification detection of HPV and / or HPV typing, the kit comprising independently packaged internal reference RPP30, primer probe combination and PCR reaction mixture, the primer probe combination is the primer probe combination described in Example 2, each primer is packaged separately, wherein the volume concentration of the primer probe combination is: 10μM upstream primer 1.1μL, 10μM downstream primer 1.1μL, 10μM probe 0.55μL. The PCR reaction mixture is 10×PCR buffer 2.2μL, 10mMdNTP 0.5μL, 25mMMgCl21.5μL, 10U / μLTaq DNA polymerase 0.1μL, template input 14μL and water 0.95μL. The amounts of upstream primers, downstream primers and probes in the RPP30 are shown in Table 3.

[0116] Example 4

[0117] The method for dPCR amplification and detection of HPV and / or HPV typing in this embodiment comprises the following steps:

[0118] (1) Collection and extraction of exfoliated cell samples

[0119] Collect cervical exfoliated cell specimens positive for HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39, transfer them to preservation solution, and store them at room temperature. After DNA extraction, store them in a -80℃ ultra-low temperature refrigerator.

[0120] (2) dPCR amplification

[0121] The PCR reaction system was prepared according to the amounts of the upstream primer, downstream primer and probe in the PCR reaction mixture and the primer-probe combination described in Example 3, and PCR amplification was performed using the primer-probe combination using a dPCR instrument. The PCR amplification procedure is shown in Example 1, and the primer-probe sequences are shown in Table 1.

[0122] The test results are expressed as nucleic acid extract concentration copies / mL:

[0123] Note: 1. Calculation method of copy number: copy number = instrument measurement result (copies / μL) × generated droplet volume (nL, read in the instrument) × number of effective droplets (read in the instrument measurement result) / 1000.

[0124] 2. Actual sample volume (μL) = template sample volume × generated droplet volume (nL, read in the instrument) × number of effective droplets (read in the instrument measurement results) / (1000×22μL).

[0125] 3. Concentration of nucleic acid extract (copies / mL) = number of copies × 1000 / actual sample volume.

[0126] Example 5

[0127] According to the similarity and interference degree between the primers, the target gene concentrations were set to 1.25E+05 copies / mL and 2.5E+03 copies / mL, respectively, of which the HPV68 and HPV58 target gene concentrations were 1.25E+05 copies / mL and 1.25E+04 copies / mL, and five combinations were divided into different HPV typing groups for multiple verification. The method described in Example 4 was used to screen out combinations with obvious distinction between positive and negative droplets, concentrated positive droplet clusters, and less Rain. The grouping of the five combinations is shown in Table 4.

[0128] Table 4 Grouping of the five combinations

[0129]

[0130] Figures 4 to 8 The results showed that there was a stratification phenomenon of target gene positive droplets in tubes 4 and 5, and some target gene positive droplets were diffuse, such as HPV51, HPV59 and HPV35 in tube 4, and internal reference gene and HPV56 in tube 5. Therefore, tubes 4 and 5 were eliminated, and the primer-probe combination of tubes 1, 2 and 3 was used for subsequent verification.

[0131] Example 6

[0132] A primer-probe combination for detecting HPV and / or HPV typing based on a dPCR method, wherein the primer-probe combination is primer-probe combination 1, primer-probe combination 2 or primer-probe combination 3; the primer-probe combination 1 is HPV16E7, HPV18E7, HPV31E7 and HPV45E7; the primer-probe combination 2 is HPV33E7, HPV35E7, HPV59E6, HPV68E6 and HPV58E7; the primer-probe combination 3 is HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6, with RPP30 as an internal reference, wherein the primer-probe combination 1 is HPV16E7, HPV18E7, HPV31E7 and HPV45E7; the primer-probe combination 2 is HPV33E7, HPV35E7, HPV59E6, HPV68E6 and HPV58E7; the primer-probe combination 3 is HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6, with RPP30 as an internal reference, wherein the primer-probe combination The specific sequence of the needle combination and RPP30 is shown in Table 1, wherein the 5' end of the probe sequence of HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 is sequentially modified with fluorescent labels VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5 and ATTO425. The 5' end of the probe sequence of RPP30 is modified with a fluorescent label FAM.

[0133] Example 7

[0134] A kit for dPCR amplification detection of HPV and / or HPV typing, the kit comprising independently packaged internal reference RPP30, primer probe combination and PCR reaction mixture, the primer probe combination is the primer probe combination described in Example 6, each primer is packaged separately, wherein the volume concentration of the primer probe combination is: 10μM upstream primer 1.1μL, 10μM downstream primer 1.1μL, 10μM probe 0.55μL. The PCR reaction mixture is 10×PCR buffer 2.2μL, 10mMdNTP 0.5μL, 25mMMgCl21.5μL, 10U / μLTaq DNA polymerase 0.1μL, template input 14μL and water 0.95μL. The amounts of upstream primers, downstream primers and probes in the RPP30 are shown in Table 3.

[0135] Example 8

[0136] The method for dPCR amplification and detection of HPV and / or HPV typing in this embodiment comprises the following steps:

[0137] (1) Collection of exfoliated cells and DNA extraction

[0138] Collect cervical exfoliated cells from HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, 39 positive individuals and transfer them to preservation solution. Complete DNA extraction of samples.

[0139] (2) dPCR amplification

[0140] A PCR reaction system is prepared according to the PCR reaction mixture described in Example 7 and the upstream primer, downstream primer and probe in the primer-probe combination described in Example 6, and PCR amplification is performed using the primer-probe combination described in Example 6 using a dPCR instrument, wherein the PCR amplification procedure is as described in Example 1. After PCR amplification, if the microdroplets produce fluorescence, it indicates that the sample to be tested is positive, and if the microdroplets do not produce fluorescence, it indicates that the sample to be tested is negative.

[0141] Example 9

[0142] The fixed-value quality control products purchased from Jingliang were used to verify the accuracy of primer-probe combination 1, primer-probe combination 2 and primer-probe combination 3 described in Example 6 using the method of Example 8. The results are shown in Tables 5 to 7.

[0143] Table 5 Accuracy test results of primer-probe combination 1

[0144]

[0145] Table 6 Accuracy test results of primer probe combination 2

[0146]

[0147]

[0148] Table 7 Accuracy test results of primer probe combination 3

[0149]

[0150] The results in Tables 5 to 7 show that the quality control products of the measured values ​​of primer-probe combination 1 to primer-probe combination 3 all meet the quality control product standards, and the detection accuracy of the present invention is high.

[0151] Example 10

[0152] HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, 39 quality control products with a concentration of 1.0E+06 copies / mL and 20 ng of fragmented genomic DNA were used to verify the specificity of primer probe combination 1, primer probe combination 2 and primer probe combination 3 described in Example 6 using the method of Example 8.

[0153] Table 8 Specificity detection results of primer-probe combinations

[0154] sample Primer probe combination 1 Primer probe combination 2 Primer probe combination 3 HPV16 + - - HPV18 + - - HPV31 + - - HPV45 + - - HPV33 - + - HPV35 - + - HPV59 - + - HPV68 - + - HPV58 - + - HPV51 - - + HPV52 - - + HPV56 - - + HPV66 - - + HPV39 - - + 20ng fragmented genomic DNA - - -

[0155] Note: “+” indicates detected, “-” indicates not detected.

[0156] The results in Table 8 show that there is no cross-reaction between the types and genomic DNA, indicating that the primer-probe combination of the present invention has strong specificity.

[0157] Embodiment 11

[0158] The detection limit was tested by the method of Example 8, and the mixed sample of HPV16, 18, 31, and 45 was diluted to 5 copies / reaction, and the test was repeated 15 times. The results are shown in Table 9. The mixed sample of HPV33, 35, and 59, and the mixed sample of HPV68 and 58 were diluted to 5 copies / reaction, and the test was repeated 15 times. The results are shown in Table 10. The mixed sample of HPV51, 52, and 56 was diluted to 5 copies / reaction, and the sample of HPV66 and 39 was diluted to 5 copies / reaction and the test was repeated 15 times. The results are shown in Table 11.

[0159] Table 9 Detection limit test results of primer probe combination 1

[0160]

[0161] Table 10 Detection limit test results of primer probe combination 2

[0162]

[0163] Table 11 Detection limit test results of primer probe combination 2

[0164]

[0165]

[0166] The results in Tables 9 to 11 show that the detection rate of 5 copies / reaction samples of each type is 100%, and the deviation between the detection concentration (copies / reaction) and the reference concentration (copies / reaction) is less than ±50%. The detection limit of HPV16 / 18 / 31 / 45 / 33 / 35 / 59 / 68 / 58 / 51 / 52 / 56 / 66 / 39 can reach 5 copies / reaction.

[0167] Example 12

[0168] The consistency evaluation was performed with reference to the method of Example 8 and the commercial kit. The comparison kit is: High-risk human papillomavirus nucleic acid (typing) detection kit (National Medical Device Registration Standard: 20163401295), the detection principle is fluorescent quantitative PCR, the cycle threshold ≤39 is positive, and 15 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68) can be identified. 402 clinical specimens (some specimens have multiple infections of different types of HPV) were included, and the same sample was tested using the above two methods. The results are shown in Table 12.

[0169] Table 12 Comparison results of the consistency between the detection method of the present invention and the HPV QPCR detection method

[0170] HPV type Number of QPCR positive cases Number of dPCR positive cases Kappa value 95% CI HPV16 55 53 0.872 0.801～0.943 HPV18 19 18 0.915 0.819～1.000 HPV31 25 23 0.867 0.763～0.971 HPV33 31 29 0.956 0.858～1.000 HPV35 16 14 0.862 0.729～0.995 HPV39 24 25 0.935 0.862～1.000 HPV45 13 19 0.805 0.654～0.956 HPV51 26 26 0.918 0.838～0.998 HPV52 58 52 0.853 0.779～0.927 HPV56 / 66 64 59 0.837 0.763～0.911 HPV58 54 54 0.829 0.747～0.911 HPV59 / 68 47 45 0.877 0.803～0.951

[0171] The results in Table 12 show that the two detection methods have excellent consistency in all 14 types.

[0172] Example 13

[0173] Detection of HPV types in plasma ctDNA using different HPV16, 18, 31, and 45 gene primer-probe combinations

[0174] The control group was: a primer-probe combination 1-1 for detecting HPV and / or HPV typing based on the dPCR method, wherein the primer-probe combination was primer-probe combination 1-1, and primer-probe combination 1-1 was HPV16E7-1, HPV18E7-1, HPV31E7-1, and HPV45E7-1. The specific primer sequences were shown in Table 13, and the fluorescent markers modified at the 5' ends of the probe sequences of HPV16E7-1, HPV18E7-1, HPV31E7-1, and HPV45E7-1 were VIC, ROX, CY5, and ATTO425, respectively.

[0175] The test group is: a primer-probe combination 1 for detecting HPV and / or HPV typing based on the dPCR method, wherein the primer-probe combination 1 is HPV16E7, HPV18E7, HPV31E7 and HPV45E7. The specific primer sequences are shown in Table 1, wherein the 5' ends of the probe sequences of HPV16E7, HPV18E7, HPV31E7 and HPV45E7 are sequentially modified with fluorescent markers such as VIC, ROX, CY5 and ATTO425.

[0176] Table 13 Sequence information of primer-probe combination 1-1

[0177]

[0178]

[0179] The method for detecting HPV or HPV typing in a plasma ctDNA sample using the primer-probe combination 1-1 or primer-probe combination 1 comprises the following steps:

[0180] (1) Simulated sample preparation and extraction

[0181] HPV16, 18, 31, and 45, the fixed-value quality control products purchased from Jingliang, were used to break the samples of about 170bp fragments by ultrasound, and diluted with normal human plasma to prepare high, medium, and low concentration samples, respectively, to simulate plasma ctDNA samples. ctDNA extraction was completed.

[0182] Among them, the high, medium and low concentrations of HPV16 were 4.03E+05, 4.03E+04 and 4.03E+02 copies / mL respectively; the high, medium and low concentrations of HPV18 were 4.90E+05, 4.90E+04 and 4.90E+02 copies / mL respectively; the high, medium and low concentrations of HPV31 were 5.38E+05, 5.38E+04 and 5.38E+02 copies / mL respectively; the high, medium and low concentrations of HPV45 were 4.87E+05, 4.87E+04 and 4.87E+02 copies / mL respectively.

[0183] (2) dPCR amplification

[0184] A PCR reaction system was prepared, wherein the PCR reaction system was 10 μM upstream primer 1.1 μL, 10 μM downstream primer 1.1 μL, 10 μM probe 0.55 μL, 10×PCR buffer 2.2 μL, 10 mM dNTP 0.5 μL, 25 mM MgCl2 1.5 μL, 10 U / μL Taq DNA polymerase 0.1 μL, template input 14 μL and water 0.95 μL. PCR amplification was performed using a dPCR instrument with primer probe combination 1-1 or primer probe combination 1, wherein the PCR amplification procedure was as described in Example 1, and the fluorescence detection channels were VIC, CY5, ROX and ATT0425. The detection results are shown in Tables 14 to 15.

[0185] Table 14 Detection results of primer-probe combination 1 for simulated plasma

[0186]

[0187] Table 15 Detection results of primer-probe combination 1-1 for simulated plasma

[0188]

[0189] Figures 9 to 16The results of Table 14 and Table 15 show that the primer probe sequence used in primer probe combination 1 can well detect free ctDNA

[0190] The detection value of primer-probe combination 1-1 was low, and low-concentration samples were not detected, which is not suitable for plasma ctDNA detection.

[0191] Embodiment 14

[0192] This example uses the dPCR amplification method for detecting HPV and / or HPV typing of the present invention and a high-risk human papillomavirus nucleic acid (typing) detection kit (National Medical Device Registration No.: 20163401295) for comparative detection.

[0193] (1) Simulated sample preparation and extraction

[0194] HPV16, 18, 31, 45, 33, 35, 58, 59, 68, 51, 52, 56, 66, and 39 were purchased from Jingliang, and the samples were broken into fragments of about 170 bp by ultrasound and diluted with normal human plasma to simulate clinical samples. ctDNA extraction was completed.

[0195] (2) dPCR amplification

[0196] A PCR reaction system was prepared according to the PCR reaction mixture described in Example 7 and the upstream primer, downstream primer and probe in the primer-probe combination described in Example 6, and PCR amplification was performed using the primer-probe combination described in Example 6 using a dPCR instrument, wherein the PCR amplification procedure is as described in Example 1. The fluorescence detection channels are FAM, VIC, CY5, ROX and ATT0425.

[0197] Fluorescence quantitative PCR amplification: Take 4 kinds of human papillomavirus PCR reaction solutions and mix them with enzyme mixture, mix them thoroughly to form PCR mixture, centrifuge them for standby use. The PCR amplification program is 50℃ for 2 minutes UNG enzyme reaction, 94℃ for 5 minutes enzyme activation, 94℃ for 15 seconds, 57℃ for 30 seconds, and 45 cycles. The fluorescence detection channels are FAM, HEX, CY5, ROX and CY5.

[0198] Table 16 dPCR test results of simulated plasma

[0199]

[0200] Table 17 QPCR test results of simulated plasma

[0201]

[0202]

[0203] Note: NoCt in the table means not detected.

[0204] Figures 17 to 44 The results in Tables 16 and 17 show that the detection rate of dPCR in two low-concentration simulated plasma ctDNA samples of each target is 100%, while in QPCR detection, there are cases where the detection is not detected or is lower than the positive judgment value (Ct>39 is judged as negative), such as HPV68 is not detected in both concentrations, and one of the two replicates of HPV16, HPV31, HPV45, and HPV66 (5.00E+02 copies / mL) is not detected. This shows that for the detection of plasma free ctDNA samples, the present invention has a more obvious advantage in detecting HPV and its typing using the dPCR method.

[0205] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting HPV and / or HPV typing, characterized in that: comprising one or more of the primer probes HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6; The nucleotide sequences of the HPV16E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 4 to 6; The nucleotide sequences of the HPV18E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 10 to 12; The nucleotide sequences of the HPV31E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 13 to 15; The nucleotide sequences of the HPV45E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 25 to 27; The nucleotide sequences of the HPV33E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 16 to 18; The nucleotide sequences of the HPV35E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 19 to 21; The nucleotide sequences of the HPV59E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 43 to 45; The nucleotide sequences of the HPV68E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 49 to 51; The nucleotide sequences of the HPV58E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 40 to 42; The nucleotide sequences of the HPV51E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 31 to 33; The nucleotide sequences of the HPV52E7 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 34 to 36; The nucleotide sequences of the HPV56E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 37 to 39; The nucleotide sequences of the HPV66E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 46 to 48; The nucleotide sequences of the HPV39E6 probe, upstream primer and downstream primer are shown in SEQ ID NOs. 22-24.

2. The primer-probe combination according to claim 1, characterized in that: The 5' end of the probe is modified with a fluorescent group.

3. The primer-probe combination according to claim 2, characterized in that: The fluorescent groups modified at the 5' end of the probe nucleotide sequences in the HPV16E7, HPV18E7, HPV31E7, HPV45E7, HPV33E7, HPV35E7, HPV59E6, HPV68E6, HPV58E7, HPV51E6, HPV52E7, HPV56E6, HPV66E6 and HPV39E6 are VIC, ROX, CY5, ATTO425, VIC, ROX, CY5, CY5, ATTO425, VIC, ROX, CY5, CY5 and ATTO425 in sequence; the HPV subtype is one or more of HPV16, HPV18, HPV31, HPV45, HPV33, HPV35, HPV58, HPV59, HPV68, HPV51, HPV52, HPV56, HPV66 and HPV39.

4. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of a product for detecting HPV and / or HPV typing.

5. A kit for detecting HPV and / or HPV typing, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that The kit also includes an internal reference gene and a PCR reaction mixture; the internal reference gene includes RPP30, and the nucleotide sequences of the probe, upstream primer and downstream primer of the RPP30 are shown in SEQ ID NOs.52 to 54; the PCR reaction mixture includes the following components in final concentrations: 1×PCR buffer, 50 to 400 μM dNTP, 1 to 2.5 mM MgCl2, 0.1 U to 5 U Taq DNA polymerase, 400 to 750 nM upstream primer and downstream primer, and 150 to 500 nM probe.

7. A method for detecting HPV and / or HPV typing using a dPCR method, characterized in that: The method is used for non-diagnostic purposes, and comprises performing PCR amplification by a dPCR method using the primer-probe combination according to any one of claims 1 to 3 or the kit according to claim 5 or 6.

8. The method according to claim 7, characterized in that The following steps are involved: (1) extracting DNA from the sample to be tested; (2) using the DNA from step (1) as a template, and performing dPCR amplification using primer and probe combinations; (3) Determine whether the sample is HPV positive based on the fluorescence amplification signals of different channels and perform HPV typing.

9. Use of a dPCR method in preparing a product for detecting HPV and / or HPV typing in a sample.

10. The use according to claim 9, characterized in that: The sample includes any one of a tissue sample, an exfoliated cell sample and a plasma sample from a patient with HPV-related lesions.

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