Composition, reagent and kit for detecting HPV (human papillomavirus) and application of composition, reagent and kit

Abstract

The invention provides a composition, a reagent and a kit for detecting HPV (human papillomavirus) and application of the composition, the reagent and the kit. The composition comprises more than one of a primer probe group aiming at HPV56, a primer probe group aiming at HPV16 and a primer probe group aiming at HPV18; each primer probe group comprises a probe, a forward primer and 1-5 reverse primers, in each primer probe group, the sequence of the probe can be complementary with a part of sequences of amplification products of the forward primer and the reverse primer, and the forward primer and the reverse primer are primers with the specific sequence provided by the invention. The reagent and the kit comprise the composition. The composition, the reagent and the kit have high accuracy when being used for HPV detection, and have good application prospects in the aspect of detecting whether HPV56, HPV16 and HPV18 exist in a cell sample and a urine sample or not.

Description

Technical Field

[0001] The present invention belongs to the technical field of HPV detection, and specifically relates to a composition, reagent, and kit for detecting HPV and their applications. Background Art

[0002] Human papillomavirus (HPV) is a spherical DNA virus that is widely present in nature, with humans as its sole reservoir. It is highly resistant to desiccation and can be stored for long periods of time. To date, 200 HPV genotypes have been identified, at least 40 of which affect the anogenital mucosa. HPV types are classified according to their carcinogenicity risk as "low-risk," "uncertain," or "high-risk." Low-risk HPV types include HPV6, 11, 42, 43, and 44, which primarily cause benign lesions such as genital warts and rarely lead to cervical cancer. High-risk HPV types primarily cause high-grade cervical intraepithelial neoplasia (CIN) and are closely associated with the development of cervical cancer. Current research indicates that HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are high-risk HPV types. HPV16 is the most common type in patients with cervical squamous cell carcinoma (76.6%), followed by HPV18 (7.9%). The infection rates of HPV16 / 18 in cervical adenocarcinoma are 33.65% and 28.86%, respectively. HPV16 / 18 are associated with the majority of cervical precancerous lesions, and other high-risk types such as HPV33, 52, and 58 also play a significant role in cervical lesions.

[0003] Currently, commonly used cervical cancer screening methods include cytology, HPV testing, and visual inspection with acetic acid (VIA). Current HPV testing methods are largely invasive, requiring a gynecological examination, the collection of cervical exfoliated cells, or a colposcopy-based cervical biopsy. The sample is then tested using molecular biological methods. This sampling and testing method presents several challenges. Many women find gynecological examinations uncomfortable, painful, and embarrassing, leading to their reluctance to comply. Furthermore, unmarried women are often unable to undergo testing due to ethical and technical limitations, leading to selection bias in surveys of HPV prevalence and type distribution. This results in most women being unable to participate in screening programs, and millions of high-risk women miss the opportunity to detect precancerous lesions at a curable stage. Self-sampling HPV testing, first proposed by Wright et al., involves self-sampling cervical exfoliated cells transvaginally using a sampling device. This self-sampling procedure is performed for HPV testing. Under this model, the examinee collects the sample at home and mails the specimen, then obtains the report through the Internet or other means. This saves screening time and transportation costs and is easily accepted by people of various cultural and religious backgrounds. At the same time, gynecologists are freed from the traditional cervical cancer screening model of organizing screening populations, collecting information, and obtaining test samples, saving medical resources and greatly improving the coverage of cervical cancer screening. The most common HPV self-sampling methods include three: vaginal self-sampling, anal self-sampling, and urine self-sampling. Among them, urine self-sampling is a good choice for self-sampling screening because it is cheap, non-invasive, and simple to collect. It can be used for women who do not participate in routine screening, reducing sampling time, reducing sampling costs, and expanding the scope of HPV screening. Currently, urine HPV testing has become the research focus of HPV self-sampling at this stage.

[0004] Over the past decade, the use of urine samples for cervical screening biomarkers has been closely evaluated. Evidence suggests that urine as a liquid biopsy is primarily useful for genitourinary cancers, including bladder, cervical, endometrial, and prostate cancers, where DNA fragments are released directly into the urine. Circulating DNA (cfDNA) in urine is an emerging noninvasive biomarker with applications in cancer mutation detection, infectious disease diagnosis, organ transplant monitoring, and prenatal screening. Due to the extensive fragmentation of urine cfDNA, the diagnostic clinical sensitivity of urine cfDNA testing increases with decreasing target length. Maximizing sensitivity by targeting shorter fragments is particularly important because urine cfDNA concentrations are also low, ranging from <1 ng / mL to 200 ng / mL, and the copy number of specific targets is much lower. In a study detecting fetal cfDNA in maternal urine, reducing the length of the PCR amplification product from 65 to 39 nt increased clinical sensitivity from 25% to 75%. Further reduction to 25 nt is required before achieving 100% detection. The results of a 2024 study showed that in the viral testing of 151 HPV patients, the overall consistency of urine (first empty urine) and cervical swab specimens in the Roche Cobas and Qiagen tests was 44.8% and 44%, respectively. The positive rates of human papillomavirus in urine samples were 57% and 70.3%, respectively. The overall consistency of the two urine samples of Roche Cobas and Qiagen for detecting human papillomavirus type 16 / 18 was 84.3%, and the overall consistency of other high-risk human papillomaviruses was 75.60%. At present, the existing Roche Cobas and Qiagen HPV detection kits have low consistency in detecting urine samples and cervical samples, which may be due to the longer length of the amplification product determined by the methodology. Roche CobasHPV uses the Taqman method, which requires a three-segment primer probe with a length of about 100bp; while Qiagen HPV uses branched nucleic acid hybridization signal amplification technology - hybridization capture method. This method refers to the denaturation and decomposition of the target DNA double strand into single strands. The single-stranded DNA combines with the specific RNA probe to form a DNA-RNA hybrid. The DNA-RNA hybrid combines with the specific antibody on the capture microplate and is fixed on the capture microplate. It is then combined with the specific antibody against the DNA-RNA hybrid coupled with alkaline phosphatase, and the enzymatic chemiluminescence amplification signal is used to achieve accurate detection of the target load. The amplification product used in this method is longer and can detect the entire HPV genome (8000bp).

[0005] In summary, the current methods and kits for detecting HPV need to be further improved in order to improve the accuracy of the test results and even increase the convenience of testing. Summary of the Invention

[0006] Based on this, the present invention aims to provide a composition, reagent, and kit for detecting HPV, and their use. The composition, reagent, and kit of the present invention have high accuracy when used for HPV detection and have promising application prospects in detecting the presence of HPV56, HPV16, and HPV18 in cell and urine samples.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] In a first aspect, the present invention provides a composition for detecting HPV, comprising one or more of a primer probe set for HPV56, a primer probe set for HPV16, and a primer probe set for HPV18; each primer probe set comprises one probe, one forward primer, and one to five reverse primers; in each primer probe set, the sequence of the probe is complementary to a portion of the sequence of an amplification product of the forward primer and the reverse primer; wherein:

[0009] The forward primer in the primer probe set for HPV56 is a primer having a sequence as shown in SEQ.ID.NO: 2, and the reverse primer is selected from one or more primers having sequences as shown in SEQ.ID.NO: 3 to 7;

[0010] The forward primer in the primer probe set for HPV16 is a primer having a sequence as shown in SEQ.ID.NO: 9, and the reverse primer is selected from one or more primers having sequences as shown in SEQ.ID.NO: 10 to 14;

[0011] The forward primer in the primer probe set for HPV18 is a primer having a sequence as shown in SEQ.ID.NO: 16, and the reverse primer is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 21.

[0012] In some embodiments of the present invention, the probe is a universal probe.

[0013] In some embodiments of the present invention, in each primer-probe set, part or all of the sequence of the probe is identical to the partial sequence of the 5' end of the forward primer; preferably, the sequence length of the probe is 20 bp to 25 bp.

[0014] In some embodiments of the present invention, the probe is modified with a fluorescent detection group; preferably, the fluorescent detection group includes a fluorescent reporter group and a quencher group or other modification groups that can produce signal changes with the fluorescent reporter group through fluorescence resonance energy transfer.

[0015] In some embodiments of the present invention, the fluorescent detection group is selected from CY5, CY3, FAM, ROX, HEX, VIC, BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, QXL and DDQI.

[0016] In some embodiments of the present invention, the fluorescent detection groups are modified at the 5' end and the 3' end of the probe, respectively.

[0017] In some embodiments of the present invention, the probe in the primer probe set for HPV56 is a probe having a sequence as shown in SEQ.ID.NO: 1.

[0018] In some embodiments of the present invention, the probe in the primer probe set for HPV16 is a probe having a sequence as shown in SEQ.ID.NO:8.

[0019] In some embodiments of the present invention, the probe in the primer probe set for HPV18 is a probe having a sequence as shown in SEQ.ID.NO:15.

[0020] In some embodiments of the present invention, the reverse primer in the primer probe set for HPV56 is selected from one or more primers having sequences as shown in SEQ.ID.NO: 3 to 6; preferably, selected from one or more primers having sequences as shown in SEQ.ID.NO: 3 to 5; more preferably, selected from one or more primers having sequences as shown in SEQ.ID.NO: 3 to 4.

[0021] In some embodiments of the present invention, the reverse primer in the primer probe set for HPV56 is any one of the primers having the sequence shown in SEQ.ID.NO: 3 to 7; preferably, it is the primer having the sequence shown in SEQ.ID.NO: 3.

[0022] In some embodiments of the present invention, the reverse primer in the primer probe set for HPV16 is selected from one or more primers having sequences as shown in SEQ.ID.NO: 10 to 13; preferably, it is selected from one or more primers having sequences as shown in SEQ.ID.NO: 10 to 12; more preferably, it is selected from one or more primers having sequences as shown in SEQ.ID.NO: 10 to 11.

[0023] In some embodiments of the present invention, the reverse primer in the primer probe set for HPV16 is any one of the primers having the sequence shown in SEQ.ID.NO: 10 to 14; preferably, it is the primer having the sequence shown in SEQ.ID.NO: 10.

[0024] In some embodiments of the present invention, the reverse primer in the primer probe set for HPV18 is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 20; preferably, it is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 19; more preferably, it is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 18.

[0025] In some embodiments of the present invention, the reverse primer in the primer probe set for HPV18 is any one of the primers having a sequence as shown in SEQ.ID.NO: 17 to 21; preferably, it is a primer having a sequence as shown in SEQ.ID.NO: 17.

[0026] In a second aspect, the present invention provides a reagent for detecting HPV, wherein the reagent comprises the composition described in the first aspect.

[0027] In some embodiments of the present invention, the reagent includes a probe having a sequence as shown in SEQ.ID.NO: 8, a primer having a sequence as shown in SEQ.ID.NO: 9, a primer having a sequence as shown in SEQ.ID.NO: 10, a probe having a sequence as shown in SEQ.ID.NO: 15, a primer having a sequence as shown in SEQ.ID.NO: 16, and a primer having a sequence as shown in SEQ.ID.NO: 17.

[0028] In some embodiments of the present invention, the reagent includes a probe having a sequence as shown in SEQ.ID.NO: 1, a primer having a sequence as shown in SEQ.ID.NO: 2, a primer having a sequence as shown in SEQ.ID.NO: 3, a probe having a sequence as shown in SEQ.ID.NO: 8, a primer having a sequence as shown in SEQ.ID.NO: 9, a primer having a sequence as shown in SEQ.ID.NO: 10, a probe having a sequence as shown in SEQ.ID.NO: 15, a primer having a sequence as shown in SEQ.ID.NO: 16, and a primer having a sequence as shown in SEQ.ID.NO: 17.

[0029] In some embodiments of the present invention, the reagent includes a probe having a sequence as shown in SEQ.ID.NO: 1, a primer having a sequence as shown in SEQ.ID.NO: 2, a primer having a sequence as shown in SEQ.ID.NO: 7, a probe having a sequence as shown in SEQ.ID.NO: 8, a primer having a sequence as shown in SEQ.ID.NO: 9, a primer having a sequence as shown in SEQ.ID.NO: 14, a probe having a sequence as shown in SEQ.ID.NO: 15, a primer having a sequence as shown in SEQ.ID.NO: 16, and a primer having a sequence as shown in SEQ.ID.NO: 21.

[0030] In some embodiments of the present invention, the reagent includes a probe having a sequence as shown in SEQ.ID.NO: 1, a primer having a sequence as shown in SEQ.ID.NO: 2, a primer having a sequence as shown in SEQ.ID.NO: 3, a primer having a sequence as shown in SEQ.ID.NO: 4, a probe having a sequence as shown in SEQ.ID.NO: 8, a primer having a sequence as shown in SEQ.ID.NO: 9, a primer having a sequence as shown in SEQ.ID.NO: 10, a primer having a sequence as shown in SEQ.ID.NO: 11, a probe having a sequence as shown in SEQ.ID.NO: 15, a primer having a sequence as shown in SEQ.ID.NO: 16, a primer having a sequence as shown in SEQ.ID.NO: 17, and a primer having a sequence as shown in SEQ.ID.NO: 18.

[0031] In some embodiments of the present invention, the reagent includes a probe having a sequence as shown in SEQ.ID.NO: 1, a primer having a sequence as shown in SEQ.ID.NO: 2, a primer having a sequence as shown in SEQ.ID.NO: 3, a probe having a sequence as shown in SEQ.ID.NO: 8, a primer having a sequence as shown in SEQ.ID.NO: 9, a primer having a sequence as shown in SEQ.ID.NO: 11, a probe having a sequence as shown in SEQ.ID.NO: 15, a primer having a sequence as shown in SEQ.ID.NO: 16, and a primer having a sequence as shown in SEQ.ID.NO: 17.

[0032] In some embodiments of the present invention, the reagent includes a probe having a sequence as shown in SEQ.ID.NO: 1, a primer having a sequence as shown in SEQ.ID.NO: 2, a primer having a sequence as shown in SEQ.ID.NO: 3, a probe having a sequence as shown in SEQ.ID.NO: 8, a primer having a sequence as shown in SEQ.ID.NO: 9, a primer having a sequence as shown in SEQ.ID.NO: 10, a probe having a sequence as shown in SEQ.ID.NO: 15, a primer having a sequence as shown in SEQ.ID.NO: 16, and a primer having a sequence as shown in SEQ.ID.NO: 18.

[0033] In some embodiments of the present invention, the reagents further include a PCR reaction premix.

[0034] In some embodiments of the present invention, the PCR reaction premix includes Tris-HCl buffer, (NH4)2SO4, MgCl2, DNA polymerase, and dNTPs; preferably, the PCR reaction premix includes 30mmol / L Tris-HCl buffer, 70mmol / L (NH4)2SO4, 3mmol / L MgCl2, 3U hot start Taq enzyme, and 0.5mmol / L dNTPs.

[0035] In some embodiments of the present invention, the pH value of the Tris-HCl buffer is 8.5.

[0036] In some embodiments of the present invention, the dNTPs include dATP, dCTP, dGTP, and dUTP.

[0037] In a third aspect, the present invention provides a kit for detecting HPV, wherein the kit comprises the composition described in the first aspect or the reagent described in the second aspect.

[0038] In a fourth aspect, the present invention provides a method for detecting HPV, comprising performing PCR amplification using the composition described in the first aspect, the reagent described in the second aspect, or the kit described in the third aspect.

[0039] In some embodiments of the invention, the method comprises detecting HPV in a cell sample or a urine sample.

[0040] In some embodiments of the present invention, when detecting HPV in a cell sample, the method further comprises controlling the length of the amplified product of the forward primer and the reverse primer to be between 45 and 300 bp, preferably between 45 and 250 bp, and more preferably between 45 and 200 bp.

[0041] In some embodiments of the present invention, when detecting HPV in a urine sample, the method further comprises controlling the length of the amplification product of the forward primer and the reverse primer to be between 45 and 200 bp, preferably between 45 and 150 bp, and more preferably between 45 and 100 bp.

[0042] In some embodiments of the present invention, the reaction procedure of the PCR amplification is: 95°C for 2 min; 95°C for 15 s, 57°C for 1 min, 45 cycles; 95°C for 105 s, 50°C for 30 s, 1 cycle.

[0043] In some embodiments of the present invention, the PCR amplification reaction system includes: 1X PCR reaction premix, 50-100 nM forward primer, 250-500 nM reverse primer, 150-400 nM probe, 6-10 μL cell sample or urine sample, and a total reaction volume of 15-35 μL.

[0044] In some embodiments of the present invention, the HPV includes one or more of HPV56, HPV16, and HPV18.

[0045] In a fifth aspect, the present invention provides a method for improving the detection rate of HPV in urine samples, the method comprising performing PCR amplification using the composition described in the first aspect, the reagent described in the second aspect, or the kit described in the third aspect, and controlling the length of the amplified product of the forward primer and the reverse primer to be between 45 and 200 bp, preferably between 45 and 150 bp, and more preferably between 45 and 100 bp.

[0046] In some embodiments of the present invention, the reaction procedure of the PCR amplification is: 95°C for 2 min; 95°C for 15 s, 57°C for 1 min, 45 cycles; 95°C for 105 s, 50°C for 30 s, 1 cycle.

[0047] In some embodiments of the present invention, the PCR amplification reaction system includes: 1X PCR reaction premix, 50-100 nM forward primer, 250-500 nM reverse primer, 150-400 nM probe, 6-10 μL cell sample or urine sample, and a total reaction volume of 15-35 μL.

[0048] In some embodiments of the present invention, the HPV includes one or more of HPV56, HPV16, and HPV18.

[0049] The beneficial effects of the present invention are as follows:

[0050] Each set of primer probe groups in the composition, reagent and kit of the present invention is reasonably designed, the primer amplification efficiency is high, sufficient effective quantitative data can be obtained, and the detection results are highly accurate. The method for detecting HPV of the present invention can be used to detect HPV in cell samples or urine samples, and the detection accuracy is high. The method for detecting HPV of the present invention can be a single-plex detection or a multiplex detection. When multiplex detection is performed, there is almost no non-specific binding phenomenon in the primers in the multiple sets of primer probe groups, and the detection results are highly accurate. It is possible to detect multiple types of HPV at one time, thereby improving the detection efficiency and convenience. In addition, the present invention also provides a method for improving the detection rate of HPV in urine samples, which can effectively improve the accuracy of HPV detection in urine samples by using the composition, reagent or kit of the present invention for amplification and controlling the length of the amplified product of the forward primer and the reverse primer to be between 45 and 200 bp. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The middle left figure is the quantitative results of the cell sample detection in Example 1. From left to right in the figure, the quantitative results of quantitative result numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 4 are respectively shown. Figure 1 The middle right picture is based on Figure 1 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV56 copies in the cell sample.

[0052] Figure 2 The middle left figure is the quantitative results of the urine sample test in Example 1. From left to right in the figure, the quantitative results of quantitative result numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 5 are respectively represented. Figure 2 The middle right picture is based on Figure 2 The quantitative results in the middle left panel show the linear relationship between the length of the amplified product and the number of HPV56 copies in the urine sample.

[0053] Figure 3 The middle left figure is the quantitative results of the cell sample detection in Example 2. From left to right in the figure, the quantitative results of quantitative result numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 7 are respectively represented. Figure 3 The middle right picture is based on Figure 3 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV16 copies in the cell sample.

[0054] Figure 4 The middle left figure is the quantitative results of the urine sample test in Example 2. From left to right in the figure, the quantitative results of quantitative result numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 8 are respectively represented. Figure 4 The middle right picture is based on Figure 4 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV16 copies in the urine sample.

[0055] Figure 5 The middle left figure is the quantitative results of the cell sample detection in Example 3. From left to right in the figure, the quantitative results of quantitative result numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 10 are respectively shown. Figure 5 The middle right picture is based on Figure 5 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV18 copies in the cell sample.

[0056] Figure 6 The middle left figure is the quantitative results of the urine sample test in Example 3. From left to right in the figure, the quantitative results numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 11 are respectively shown. Figure 6 The middle right picture is based on Figure 6 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV18 copies in the urine sample.

[0057] Figure 7 The middle left figure is the quantitative result of HPV56 detected by the cell sample in Example 4. From left to right in the figure, the quantitative results numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 12 are respectively shown. Figure 7 The middle right picture is based on Figure 7 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV56 copies in the cell sample.

[0058] Figure 8 The middle left figure is the quantitative result of HPV16 detected by the cell sample in Example 4. From left to right in the figure, the quantitative results of the quantitative results numbered 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 in Table 12 are respectively shown. Figure 8 The middle right picture is based on Figure 8 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV16 copies in the cell sample.

[0059] Figure 9 The middle left figure is the quantitative result of HPV18 detected by the cell sample in Example 4. From left to right in the figure, the quantitative results of the quantitative results numbered 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 in Table 12 are respectively shown. Figure 9The middle right picture is based on Figure 9 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV18 copies in the cell sample.

[0060] Figure 10 The middle left figure is the quantitative result of HPV56 detected by the urine sample in Example 4. From left to right in the figure, the quantitative results numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in Table 13 are respectively represented. Figure 10 The middle right picture is based on Figure 10 The quantitative results in the middle left panel show the linear relationship between the length of the amplified product and the number of HPV56 copies in the urine sample.

[0061] Figure 11 The middle left figure is the quantitative result of HPV16 detected by the urine sample in Example 4. From left to right in the figure, the quantitative results numbered 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 in Table 13 are respectively represented. Figure 11 The middle right picture is based on Figure 11 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV16 copies in the urine sample.

[0062] Figure 12 The middle left figure is the quantitative result of HPV18 detected in the urine sample of Example 4. From left to right in the figure, the quantitative results of the quantitative results numbered 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 in Table 13 are respectively represented. Figure 12 The middle right picture is based on Figure 12 The quantitative results in the middle left figure show the linear relationship between the length of the amplified product and the number of HPV18 copies in the urine sample.

[0063] Figure 13 The middle left figure is the quantitative results of the cell sample detection in Example 1. From left to right in the figure, the quantitative results using primer probe sets P1F4R16, P1F4R17, P1F4R18, P1F4R19, and P1F4R20 are shown. Each primer probe set has 3 samples, and from left to right they are positive sample replicate 1, positive sample replicate 2, and blank control sample.

[0064] Figure 13 The middle right figure is the quantitative results of the cell sample detection in Example 2. From left to right in the figure, the quantitative results using primer probe sets P1F5R21, P1F5R22, P1F5R23, P1F5R24, and P1F5R25 are shown. Each primer probe set has 3 samples, and from left to right they are positive sample replicate 1, positive sample replicate 2, and blank control sample.

[0065] Figure 14 The middle left figure is the quantitative results of the urine sample test in Example 3. From left to right in the figure, the quantitative results using primer probe sets P1F1R3, P1F1R26, P1F1R27, and P1F1R28 are shown. Each primer probe set has 3 samples, and from left to right they are positive sample replicate 1, positive sample replicate 2, and blank control sample.

[0066] Figure 14 The middle right figure is the quantitative results of the urine sample test in Example 4, and from left to right in the figure are positive sample replicate 1, positive sample replicate 2, blank control sample replicate 1, and blank control sample replicate 2.

[0067] Figure 15 It is a sequence relationship diagram of forward primer F2 and reverse primer R29. DETAILED DESCRIPTION

[0068] The following examples further illustrate the technology of the present invention. These examples are for illustration and example only and are not intended to limit the scope of the present invention in any form.

[0069] Example 1

[0070] A method for detecting HPV56, the probes and primers used are shown in Table 1, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0071] Table 1 Probes and primers for detecting HPV56

[0072]

[0073] Table 2 PCR amplification reaction system

[0074] Reagent name concentration PCR reaction premix (DNA, 2×) 1X Forward primer 100nM Reverse primer 500nM probe 400nM Cell sample or urine sample 10 μL Total reaction volume 35μL

[0075] In Table 2, the components of the PCR reaction premix include 30mmol / L Tris-HCl buffer (pH=8.5), 70mmol / L (NH4)2SO4, 3mmol / L MgCl2, 3U LionTaq hot start enzyme, 0.5mmol / L dNTPs

[0076] (dATP:dCTP:dGTP:dUTP=1:1:1:1).

[0077] Table 3 PCR amplification reaction program

[0078]

[0079] The primers and probes shown in Table 1 were divided into five groups: P1F1R1, P1F1R2, P1F1R3, P1F1R4, and P1F1R5. Single-plex assays were performed on cell samples containing only HPV type 56. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was prepared using ddH2O instead of the cell sample in Table 2. The two replicates of the same positive sample were prepared using two aliquots (10 μL each) from the same cell sample and the PCR amplification reaction system shown in Table 2. The positive samples used for each of the five primer-probe groups were the same cell sample (i.e., each positive sample contained equivalent HPV56 concentrations). The three reaction systems were added to separate PCR tubes, sealed, and gently mixed. The samples were then briefly centrifuged and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, centrifuged briefly, and transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results are shown in Table 4 and Figure 1 As shown in Table 4 and Figure 1 It can be seen that the detection of HPV56 in cell samples using the primer probe sets P1F1R1, P1F1R2, P1F1R3, P1F1R4, and P1F1R5 of the present invention has high detection accuracy.

[0080] Table 4 Results of HPV56 detection in cell samples

[0081]

[0082] The primers and probes shown in Table 1 were divided into five groups: P1F1R1, P1F1R2, P1F1R3, P1F1R4, and P1F1R5. Single-plex detection was performed on urine samples containing only HPV56. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was prepared using ddH2O instead of the urine sample in Table 2. The two replicates of the same positive sample were prepared using two aliquots (10 μL each) of the same urine sample and the PCR amplification reaction system shown in Table 2. The positive sample used for each of the five primer-probe groups was the same urine sample (i.e., each positive sample contained a comparable HPV56 concentration). The three reaction systems were added to separate PCR tubes, sealed, and gently mixed. The samples were then briefly centrifuged and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, centrifuged briefly, and transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results are shown in Table 5 and Figure 2 As shown in Table 5 and Figure 2 It can be seen that the detection of HPV56 in urine samples using the primer probe set P1F1R1, P1F1R2, P1F1R3, P1F1R4, and P1F1R5 of the present invention has a high detection accuracy, and the quantitative concentration (copy number) of HPV56 in urine samples tends to decrease with the increase of the length of the amplified product and shows a good linear relationship.

[0083] Table 5 Results of HPV56 detection in urine samples

[0084]

[0085] Example 2

[0086] A method for detecting HPV16, the probes and primers used are shown in Table 6, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0087] Table 6 Probes and primers for detecting HPV16

[0088]

[0089] The primers and probes shown in Table 6 were divided into five groups: P2F2R6, P2F2R7, P2F2R8, P2F2R9, and P2F2R10. Single-plex detection was performed on cell samples containing only HPV type 16. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was prepared using ddH2O instead of the cell sample in Table 2. The two replicates of the same positive sample were prepared by taking two aliquots (10 μL each) from the same cell sample and preparing the PCR amplification reaction system shown in Table 2. The positive samples used for the five primer-probe groups were the same cell sample (i.e., each positive sample contained a comparable HPV16 concentration). Each reaction system was added to a sealed PCR tube, gently mixed, and then briefly centrifuged and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, centrifuged briefly, and then transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results are shown in Table 7 and Figure 3 As shown in Table 7 and Figure 3 It can be seen that the detection of HPV16 in cell samples using the primer probe sets P2F2R6, P2F2R7, P2F2R8, P2F2R9, and P2F2R10 of the present invention has high detection accuracy.

[0090] Table 7 Results of HPV16 detection in cell samples

[0091]

[0092] The primers and probes shown in Table 6 were divided into five groups: P2F2R6, P2F2R7, P2F2R8, P2F2R9, and P2F2R10. Single-plex detection was performed on urine samples containing only HPV type 16. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was prepared using ddH2O instead of the urine sample in Table 2. The two replicates of the same positive sample were prepared using two aliquots (10 μL each) of the same urine sample and the PCR amplification reaction system shown in Table 2. The positive sample used for each of the five primer-probe groups was the same urine sample (i.e., each positive sample contained a comparable HPV16 concentration). The three reaction systems were added to separate PCR tubes, sealed, and gently mixed. The samples were then briefly centrifuged and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, centrifuged briefly, and transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results are shown in Table 8 and Figure 4 As shown in Table 8 and Figure 4 It can be seen that the detection of HPV16 in urine samples using the primer probe set P2F2R6, P2F2R7, P2F2R8, P2F2R9, and P2F2R10 of the present invention has a high detection accuracy, and the quantitative concentration (copy number) of HPV16 in urine samples tends to decrease with the increase of the length of the amplified product and shows a good linear relationship.

[0093] Table 8 Results of HPV16 detection in urine samples

[0094]

[0095]

[0096] Example 3

[0097] A method for detecting HPV18, the probes and primers used are shown in Table 9, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0098] Table 9 Probes and primers for detecting HPV18

[0099]

[0100] The primers and probes shown in Table 9 were divided into five groups: P3F3R11, P3F3R12, P3F3R13, P3F3R14, and P3F3R15. Single-plex detection was performed on cell samples containing only HPV type 18. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was a reaction system prepared using ddH2O instead of the cell sample in Table 2. The two replicates of the same positive sample were two reaction systems prepared by taking two aliquots (10 μL each) from the same cell sample and preparing them according to the PCR amplification reaction system shown in Table 2. The positive samples used for the five primer-probe groups were the same cell sample (i.e., the HPV18 concentration in each positive sample was equivalent). Each reaction system was added to a PCR tube, sealed, and gently mixed. The sample was then briefly centrifuged and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, centrifuged briefly, and transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results are shown in Tables 10 and Figure 5 As shown in Table 10 and Figure 5 It can be seen that the detection of HPV18 in cell samples using the primer probe sets P3F3R11, P3F3R12, P3F3R13, P3F3R14, and P3F3R15 of the present invention has high detection accuracy.

[0101] Table 10 Results of HPV18 detection in cell samples

[0102]

[0103]

[0104] The primers and probes shown in Table 9 were divided into five groups: P3F3R11, P3F3R12, P3F3R13, P3F3R14, and P3F3R15. Single-plex detection was performed on urine samples containing only HPV type 18. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was a reaction system prepared using ddH2O instead of the urine sample in Table 2. The two replicates of the same positive sample were two reaction systems prepared by taking two aliquots (10 μL each) from the same urine sample and preparing them according to the PCR amplification reaction system shown in Table 2. The positive sample used in each of the five primer-probe groups was the same urine sample (i.e., the HPV18 concentration in each positive sample was equivalent). The three reaction systems were added to PCR tubes, sealed, and the samples were gently mixed. The tubes were then briefly centrifuged and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, centrifuged briefly, and then transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results are shown in Tables 11 and Figure 6 As shown in Table 11 and Figure 6 It can be seen that the detection of HPV18 in urine samples using the primer probe set P3F3R11, P3F3R12, P3F3R13, P3F3R14, and P3F3R15 of the present invention has a high detection accuracy, and the quantitative concentration (copy number) of HPV18 in urine samples tends to decrease with the increase of the length of the amplified product and shows a good linear relationship.

[0105] Table 11 Results of HPV18 detection in urine samples

[0106]

[0107] Example 4

[0108] A method for simultaneously detecting HPV56, HPV16, and HPV18, wherein the probe and primers for HPV56 are shown in Table 1, the probe and primers for HPV16 are shown in Table 6, the probe and primers for HPV18 are shown in Table 9, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0109] The primers and probes shown in Tables 1, 6, and 9 were divided into 5 groups. The first group of primer probes consisted of P1F1R1, P2F2R6, and P3F3R11; the second group of primer probes consisted of P1F1R2, P2F2R7, and P3F3R12; the third group of primer probes consisted of P1F1R3, P2F2R8, and P3F3R13; the fourth group of primer probes consisted of P1F1R4, P2F2R9, and P3F3R14; and the fifth group of primer probes consisted of P1F1R5, P2F2R10, and P3F3R15. The results were for the detection of HPV types including HPV56, HPV16, and HPV18. The cell samples were triple tested, and each set of primer probe groups was used to detect one blank control sample and two replicates of the same positive sample; wherein, the blank control sample was a reaction system prepared by using ddH2O instead of the cell sample in Table 2, and the two replicates of the same positive sample were two reaction systems prepared by taking two small portions (10 μL each) from the same cell sample and preparing them according to the PCR amplification reaction system shown in Table 2; the positive samples used by the five sets of primer probe groups were the same cell samples (i.e., the concentrations of HPV56, HPV16, and HPV18 contained in each positive sample were equivalent). Each reaction system was added to the PCR tube, the cap was sealed, and the sample was gently mixed, then centrifuged briefly and allowed to stand at room temperature for 5 minutes. The PCR tube was then placed in a handheld centrifuge, and after a brief centrifugation, it was transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation; PCR amplification was then performed according to the reaction procedure shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation, and the results are shown in Tables 12 and 12. Figures 7-9 As shown in Table 12 and Figures 7-9 It can be seen that the triple detection of HPV56, HPV16 and HPV18 in cell samples using the five primer probe sets of the present invention has high detection accuracy.

[0110] Table 12 Results of triple HPV testing in cell samples

[0111]

[0112]

[0113] The primers and probes shown in Tables 1, 6, and 9 were divided into 5 groups. The first group of primer probes consisted of P1F1R1, P2F2R6, and P3F3R11; the second group of primer probes consisted of P1F1R2, P2F2R7, and P3F3R12; the third group of primer probes consisted of P1F1R3, P2F2R8, and P3F3R13; the fourth group of primer probes consisted of P1F1R4, P2F2R9, and P3F3R14; and the fifth group of primer probes consisted of P1F1R5, P2F2R10, and P3F3R15. The results were for the detection of HPV types including HPV56, HPV16, and HPV18. The urine samples were triple tested, and each set of primer probe groups was used to detect one blank control sample and two replicates of the same positive sample; wherein, the blank control sample was a reaction system prepared by using ddH2O instead of the urine sample in Table 2, and the two replicates of the same positive sample were two reaction systems prepared by taking two small portions (10 μL each) from the same urine sample and preparing them according to the PCR amplification reaction system shown in Table 2; the positive sample used by the five sets of primer probe groups was the same urine sample (i.e., the concentrations of HPV56, HPV16, and HPV18 contained in each positive sample were equivalent). The three reaction systems were added to the PCR tubes, the caps were sealed, and the samples were gently mixed, then centrifuged briefly and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, and after a brief centrifugation, they were transferred to the consumables of the D600 digital PCR instrument of MacBiotech Co., Ltd.; PCR amplification was then performed according to the reaction procedure shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBiotech Co., Ltd. The results are shown in Tables 13 and 14. Figures 10-12 As shown in Table 13 and Figures 10-12 It can be seen that the triple detection of HPV56, HPV16 and HPV18 in urine samples using the five sets of primer probe sets of the present invention has a high detection accuracy, and the quantitative concentration (copy number) of HPV56, HPV16 and HPV18 in urine samples shows a trend of decreasing with increasing length of the amplification product and exhibits a good linear relationship.

[0114] Table 13 Results of triple HPV testing in urine samples

[0115]

[0116]

[0117] Comparative Example 1

[0118] A method for detecting HPV56, the probes and primers used are shown in Table 14, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0119] Table 14 Probes and primers for detecting HPV56

[0120]

[0121] The primers and probes shown in Table 14 were divided into 5 groups, the 5 primer probe groups were P1F4R16, P1F4R17, P1F4R18, P1F4R19, and P1F4R20, and single-plex detection was performed on cell samples containing only HPV56 type HPV. Each primer probe group was used to detect 1 blank control sample and 2 replicates of the same positive sample; wherein, the blank control sample was a reaction system prepared using ddH2O instead of the cell sample in Table 2, and the 2 replicates of the same positive sample were two reaction systems prepared by taking two aliquots (10 μL each) from the same cell sample and preparing them according to the PCR amplification reaction system shown in Table 2; the positive samples used in the 5 primer probe groups were the same cell sample (i.e., the HPV56 concentration contained in each positive sample was equivalent), and were the same cell sample as the cell sample used in Example 1. The 3 reaction systems were added to the PCR tubes, the caps were sealed, and the samples were gently mixed, then briefly centrifuged and placed at room temperature for 5 minutes. The PCR tube was then placed in a handheld centrifuge, centrifuged briefly, and then transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results were as follows: Figure 13 As shown in the left picture. Figure 13 As can be seen from the left figure, when the five primer-probe sets shown in Table 14 were used for detection, the number of effective droplets in the positive sample detection was small and the differentiation between positive and negative droplets was not obvious, which led to inaccurate quantitative results. It is speculated that this may be due to unreasonable primer design (low Tm value) and insufficient primer amplification efficiency, resulting in the inability to obtain sufficient effective quantitative data.

[0122] Comparative Example 2

[0123] A method for detecting HPV56, the probes and primers used are shown in Table 15, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0124] Table 15 Probes and primers for detecting HPV56

[0125]

[0126]

[0127] The primers and probes shown in Table 15 were divided into 5 groups, and the 5 primer probe groups were P1F5R21, P1F5R22, P1F5R23, P1F5R24, and P1F5R25, respectively. Single-plex detection was performed on cell samples containing only HPV56 type HPV, and each primer probe group was used to detect 1 blank control sample and 2 replicates of the same positive sample; wherein, the blank control sample was a reaction system prepared using ddH2O instead of the cell sample in Table 2, and the 2 replicates of the same positive sample were two reaction systems prepared by taking two aliquots (10 μL each) from the same cell sample and preparing them according to the PCR amplification reaction system shown in Table 2; the positive samples used in the 5 primer probe groups were the same cell sample (i.e., the HPV56 concentration contained in each positive sample was equivalent), and were the same cell sample as the cell sample used in Example 1. The 3 reaction systems were added to the PCR tubes, the caps were sealed, and the samples were gently mixed, then briefly centrifuged and placed at room temperature for 5 minutes. The PCR tube was then placed in a handheld centrifuge, centrifuged briefly, and then transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results were as follows: Figure 13 As shown in the figure on the right. Figure 13 As can be seen from the right figure, the positive droplets (the red dots in the upper part of the figure) in the test results of each group of positive samples all appear to be in a raining state, and the intensity of some positive droplets and negative droplets (the gray dots in the lower part of the figure) is very different (that is, the difference between the positive signal value and the background signal value is very small), which can easily lead to inaccurate test results due to the inability to accurately judge the boundary between positive droplets and negative droplets; in addition, positive droplets appeared in the test results of the blank control samples containing the primer probe groups P1F5R24 and P1F5R25, indicating that non-specific amplification between primers occurred. Using these two sets of primer probe groups for amplification will result in false positives and will cause the quantitative data to be inflated.

[0128] Comparative Example 3

[0129] A method for detecting HPV56, the probes and primers used are shown in Table 16, the PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0130] Table 16 Probes and primers for detecting HPV56

[0131]

[0132] The primers and probes shown in Table 16 were divided into four groups, namely P1F1R3, P1F1R26, P1F1R27, and P1F1R28. Single-plex detection was performed on urine samples containing only HPV56 type HPV. Each primer-probe group was used to test one blank control sample and two replicates of the same positive sample. The blank control sample was a reaction system prepared using ddH2O instead of the urine sample in Table 2. The two replicates of the same positive sample were two reaction systems prepared by taking two aliquots (10 μL each) from the same urine sample and preparing them according to the PCR amplification reaction system shown in Table 2. The positive sample used in the four primer-probe groups was the same urine sample (i.e., the HPV56 concentration contained in each positive sample was equivalent). The three reaction systems were added to PCR tubes, sealed, and the samples were gently mixed. After a brief centrifugation, they were allowed to stand at room temperature for 5 minutes. The PCR tube was then placed in a handheld centrifuge, centrifuged briefly, and then transferred to the consumables of the D600 digital PCR instrument of MacBio Corporation. PCR amplification was then performed according to the reaction program shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of MacBio Corporation. The results were as follows: Figure 14 As shown in the left picture. Figure 14 As can be seen from the left figure, as the length of the amplified product increases, the number of positive droplets decreases. When the amplified product is 350bp, the positive and negative backgrounds cannot be distinguished. Therefore, when detecting the presence of HPV in urine samples, the length of the amplified product of the primer cannot be too high.

[0133] Comparative Example 4

[0134] A method for detecting HPV16, the probes and primers used are shown in Table 17 (the sequence relationship diagram of the forward primer F2 and the reverse primer R29 is shown in Figure 15 The PCR amplification reaction system is shown in Table 2, and the PCR amplification reaction procedure is shown in Table 3.

[0135] Table 17 Probes and primers for detecting HPV16

[0136]

[0137] The primers and probes shown in Table 17 were used to perform single-plex detection on urine samples containing only HPV16 type HPV, and two blank control samples and two replicates of the same positive sample were detected respectively; wherein, the two blank control samples were two reaction systems prepared by using ddH2O instead of the urine sample in Table 2, and the two replicates of the same positive sample were two reaction systems prepared by taking two small portions (10 μL per small portion) from the same urine sample (the urine sample used here is the same urine sample as the urine sample used in Example 2) and preparing them according to the PCR amplification reaction system shown in Table 2. The four reaction systems were added to the PCR tubes respectively, the caps were sealed, and the samples were gently mixed, then centrifuged briefly and allowed to stand at room temperature for 5 minutes. The PCR tubes were then placed in a handheld centrifuge, and after a brief centrifugation, they were transferred to the consumables of the D600 digital PCR instrument of Mike Biotech Co., Ltd.; then PCR amplification was performed according to the reaction procedure shown in Table 3. Finally, the results were interpreted by the copy number concentration using the analysis software of the D600 digital PCR instrument of Mike Biotech Co., Ltd., and the results are as follows. Figure 14 As shown in the figure on the right. Figure 15 It can be seen that in order to obtain a shorter amplification product, the designed forward primer and reverse primer will have complementary sequences; combined with Figure 14 As can be seen in the figure on the right, the presence of complementary sequences between primers can lead to false positives in blank control samples due to primer dimerization, resulting in inaccurate quantitative results. Therefore, improving the accuracy of test results by shortening the amplification product is subject to certain limitations. Combined with Comparative Example 3, it can be seen that when detecting the presence of HPV in urine samples, it is necessary to select appropriate primers and control the length of the primer amplification product within a certain range.

Claims

1. A composition for detecting HPV, characterized in that: The composition comprises one or more of a primer probe set targeting HPV56, a primer probe set targeting HPV16, and a primer probe set targeting HPV18; each primer probe set comprises one probe, one forward primer, and one to five reverse primers; in each primer probe set, the sequence of the probe is complementary to a portion of the sequence of the amplification product of the forward primer and the reverse primer; wherein: The forward primer in the primer probe set for HPV56 is a primer having a sequence as shown in SEQ.ID.NO: 2, and the reverse primer is selected from one or more primers having sequences as shown in SEQ.ID.NO: 3 to 7; The forward primer in the primer probe set for HPV16 is a primer having a sequence as shown in SEQ.ID.NO: 9, and the reverse primer is selected from one or more primers having sequences as shown in SEQ.ID.NO: 10 to 14; The forward primer in the primer probe set for HPV18 is a primer having a sequence as shown in SEQ.ID.NO: 16, and the reverse primer is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 21.

2. The composition according to claim 1, characterized in that The probe is a universal probe; preferably, in each primer probe set, part or all of the sequence of the probe is identical to the partial sequence at the 5' end of the forward primer; and / or The probe is modified with a fluorescent detection group; preferably, the fluorescent detection group includes a fluorescent reporter group and a quencher group or other modification groups that can generate signal changes with the fluorescent reporter group through fluorescence resonance energy transfer.

3. The composition according to claim 1 or 2, characterized in that The probe in the primer probe set for HPV56 is a probe having a sequence as shown in SEQ.ID.NO: 1; and / or The probe in the primer probe set for HPV16 is a probe having a sequence as shown in SEQ.ID.NO: 8; and / or The probe in the primer probe set for HPV18 has a sequence shown as SEQ.ID.NO:

15.

4. The composition according to any one of claims 1 to 3, characterized in that The reverse primer in the primer probe set for HPV56 is any one of the primers having a sequence as shown in SEQ.ID.NO: 3 to 7; preferably, it is a primer having a sequence as shown in SEQ.ID.NO: 3; and / or The reverse primer in the primer probe set for HPV16 is any one of the primers having a sequence as shown in SEQ.ID.NO: 10 to 14; preferably, it is a primer having a sequence as shown in SEQ.ID.NO: 10; and / or The reverse primer in the primer probe set for HPV18 is any one of the primers having the sequence shown as SEQ.ID.NO: 17 to 21; preferably, it is the primer having the sequence shown as SEQ.ID.NO:

17.

5. The composition according to any one of claims 1 to 3, characterized in that The reverse primer in the primer probe set for HPV56 is selected from at least one primer having a sequence as shown in SEQ.ID.NO: 3 to 6; preferably, selected from at least one primer having a sequence as shown in SEQ.ID.NO: 3 to 5; more preferably, selected from at least one primer having a sequence as shown in SEQ.ID.NO: 3 to 4; and / or The reverse primer in the primer probe set for HPV16 is selected from one or more primers having sequences as shown in SEQ.ID.NO: 10 to 13; preferably, one or more primers having sequences as shown in SEQ.ID.NO: 10 to 12; more preferably, one or more primers having sequences as shown in SEQ.ID.NO: 10 to 11; and / or The reverse primer in the primer probe set for HPV18 is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 20; preferably, it is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 19; more preferably, it is selected from one or more primers having sequences as shown in SEQ.ID.NO: 17 to 18.

6. A reagent for detecting HPV, characterized in that: The reagent comprises the composition according to any one of claims 1 to 5; preferably, the reagent further comprises a PCR reaction premix; more preferably, the PCR reaction premix comprises Tris-HCl buffer, (NH4)2SO4, MgCl2, DNA polymerase, and dNTPs.

7. A kit for detecting HPV, characterized in that: The kit comprises the composition according to any one of claims 1 to 5 or the reagent according to claim 6.

8. A method for detecting HPV for non-disease diagnosis purposes, characterized in that: The method comprises performing PCR amplification using the composition of any one of claims 1 to 5, the reagent of claim 6, or the kit of claim 7; preferably, the method comprises detecting HPV in a cell sample or a urine sample; more preferably, when detecting HPV in a urine sample, the method further comprises controlling the length of the amplification product of the forward primer and the reverse primer to be between 45 and 200 bp, preferably between 45 and 150 bp, and more preferably between 45 and 100 bp.

9. A method for improving the detection rate of HPV in urine samples, characterized in that: The method comprises performing PCR amplification using the composition of any one of claims 1 to 5, the reagent of claim 6, or the kit of claim 7, and controlling the length of the amplified product of the forward primer and the reverse primer to be between 45 and 200 bp, preferably between 45 and 150 bp, and more preferably between 45 and 100 bp.

10. The method according to claim 8 or 9, characterized in that The reaction program of the PCR amplification is: 95°C for 2 min; 95°C for 15 s, 57°C for 1 min, 45 cycles; 95°C for 105 s, 50°C for 30 s, 1 cycle; and / or The HPV includes one or more of HPV56, HPV16, and HPV18.

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