Primer probe composition, kit and detection method for 23 human papilloma virus genotyping detection

Abstract

The present application relates to the field of molecular biology, in particular to a primer probe composition, kit and detection method for 23 human papilloma virus genotyping detection, wherein the hydrolysis probe and molecular beacon probe are respectively designed in two different regions of HPV gene sequence E6 and L1, and the fluorescence collection point in the amplification stage is set in the high-temperature denaturation stage, so that the detection in the amplification stage and the melting curve stage is independent and does not interfere with each other, thereby realizing the combined use of real-time fluorescent PCR amplification detection technology and melting curve technology; the technology improves the single-well detection target flux of fluorescent PCR analysis technology, reduces the detection cost, and provides a more optimal detection method for HPV multiplex fluorescent PCR genotyping detection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a primer-probe composition, kit, and detection method for genotyping detection of 23 types of human papillomaviruses. Background Technology

[0002] Human papillomavirus (HPV) belongs to the genus Papillomavirus of the family Papovaviridae. HPV primarily infects humans through direct or indirect contact with contaminated objects or sexual transmission. HPV exhibits both host specificity and tissue specificity, infecting only the epithelial cells of human skin and mucous membranes, causing multiple papillomas or warts on the skin and proliferative lesions of the mucous membranes and genital tract. Persistent HPV infection is a major cause of cervical cancer and some genital tumor-like lesions.

[0003] HPV infections of the genital tract and anus are classified into two main categories based on the pathogenicity or carcinogenic risk of each genotype: high-risk and low-risk. Infection with high-risk HPV is a necessary but not sufficient condition for the development of cervical squamous cell carcinoma. Therefore, only a small percentage of women infected with HPV will develop definite cervical lesions or cervical cancer. In women under 30 years of age, although the HPV infection rate is high, the spontaneous clearance rate is also high. Therefore, excessive treatment is not recommended for women with normal cytology but positive HPV test results to avoid negative consequences such as increased costs and potential impact on fertility. In fact, most HPV infections are transient with a very low risk of progression; only a small percentage of infections are persistent. Currently, the factors determining persistent HPV infection are not fully understood, but regardless of age, persistent infection with high-risk HPV for 1 or 2 years is expected to have a significant subsequent risk of developing cervical intraepithelial neoplasia (CIN) grade 3 or cervical cancer. Studies have shown that HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are high-risk HPV types, while HPV types 26, 53, 66, 73, and 82 are intermediate-risk types. HPV type 16 has the strongest association with cervical cancer, followed by HPV type 18. Currently known synergistic factors increasing the chance of persistent HPV infection include smoking, a weakened immune system, and HIV infection. The progression of HPV-related cervical tumors is relatively slow; severe dysplasia progresses to invasive cervical cancer in an average of 3-7 years. Therefore, patients with HPV infection are suitable for infrequent testing. Furthermore, HPV cannot proliferate in vitro, so it cannot be detected using culture methods. Traditional methods mainly rely on morphological and immunological methods for detection, but their specificity and sensitivity are not ideal, and they cannot perform HPV typing. Therefore, molecular biological methods are currently the main technology for HPV typing.

[0004] Current HPV genotyping methods include: 1) In situ hybridization: This method uses nucleic acid probes (DNA or RNA) to perform in situ hybridization with the sample. The advantages of in situ hybridization are that it can locate suspicious cells and has semi-quantitative capabilities; the disadvantages are low sensitivity, high sample quality requirements, high labor intensity, and high cost. Furthermore, each HPV type requires a specific probe. 2) PCR-reverse dot hybridization: This method combines in vitro PCR amplification with reverse dot hybridization. Specific primers are designed based on the genetic characteristics of HPV to amplify the target fragment containing the HPV genotype. The amplified product is then hybridized with a genotyping probe fixed on a membrane strip. The presence or absence of the hybridization signal determines the presence of these HPV genotypes, which can be used for auxiliary diagnosis of clinical HPV infection. The biggest drawback of this method is the susceptibility to contamination during hybridization, which can affect the interpretation of results. 3) Flow cytometry: This technique hybridizes PCR amplification products with cross-linked fluorescently labeled probes on microspheres, and finally detects the fluorescence signal on a multifunctional flow cytometer. Currently, commercially available kits can detect 27 HPV types at once. The disadvantages are that the amplified products are easily contaminated if the container is opened, and the detection time is 6 hours. 4) Melting curve method: Probes labeled with fluorescent and quenching groups are added to the PCR system. During PCR, single-stranded oligonucleotide sequences complementary to the probe sequence are amplified. After amplification, a melting curve analysis process is added to obtain the melting curve and determine the melting point (Tm value) for each type. However, the disadvantage is that the melting point ranges of the melting peaks between different targets are relatively close, making it easy for adjacent melting peaks to interfere with each other, resulting in fusion peaks, etc. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for genotyping detection of 23 types of human papillomaviruses, so as to solve the shortcomings of the existing detection technology, such as long PCR detection time, insufficient number of genotypes, and unclear genotyping.

[0006] The present invention also provides a primer-probe composition for genotyping detection of 23 types of human papillomaviruses.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A primer-probe composition for genotyping detection of 23 human papillomaviruses (HPVs), comprising a primer set and a probe set; the primer set comprising: primer 1: nucleotide sequence as shown in SEQ ID NO. 1; primer 2: nucleotide sequence as shown in SEQ ID NO. 2; primer 3: nucleotide sequence as shown in SEQ ID NO. 4; primer 4: nucleotide sequence as shown in SEQ ID NO. 5; primer 5: nucleotide sequence as shown in SEQ ID NO. 7; primer 6: nucleotide sequence as shown in SEQ ID NO. 8; primer 7: nucleotide sequence as shown in SEQ ID NO. 10; primer 8: nucleotide sequence as shown in SEQ ID NO. 11; primer 9: nucleotide sequence as shown in SEQ ID NO. 13; primer 10: nucleotide sequence as shown in SEQ ID NO. 14; primer 11: nucleotide sequence as shown in SEQ ID NO. 16; primer 12: nucleotide sequence as shown in SEQ ID NO. 17; primer 13: nucleotide sequence as shown in SEQ ID NO. 14; primer 12: nucleotide sequence as shown in SEQ ID NO. 17; and primer 13: nucleotide sequence as shown in SEQ ID NO. 14. Primer 14: Primer 15: Primer 16: Primer 17: Primer 18: Primer 19: Primer 20: Primer 21: Primer 22: Primer 23: Primer 24: Primer 25: Primer 26: Primer 27: Primer 28: Primer 29: Primer 20: Primer 21: Primer 22: Primer 23: Primer 24: Primer 25: Primer 26: Primer 27: Primer 28: Primer 29: Primer 20: Primer 29: Primer 20: Primer 21: Primer 22: Primer 23: Primer 24: Primer 25: Primer 26: Primer 27: Primer 28: Primer 29: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 29: Primer 20: Primer 21: Primer 22: Primer 23: Primer 24: Primer 25: Primer 26: Primer 27: Primer 28: Primer 29 ... Primer 29: Primer 30: Primer 31: Primer 32: Primer 33: Primer 34: Primer 35: Primer 36: Primer 35: Primer 36: Primer 37: Primer 38: Primer 39: Primer 30: Primer 31: Primer 32: Primer 33: Primer 34: Primer 35: Primer 36: Primer 37: Primer 38: Primer 39 ...Primer 36: nucleotide sequence as shown in SEQ ID NO. 48; primer 37: nucleotide sequence as shown in SEQ ID NO. 50; primer 38: nucleotide sequence as shown in SEQ ID NO. 51; primer 39: nucleotide sequence as shown in SEQ ID NO. 52; primer 40: nucleotide sequence as shown in SEQ ID NO. 53; primer 41: nucleotide sequence as shown in SEQ ID NO. 55; primer 42: nucleotide sequence as shown in SEQ ID NO. 56; primer 43: nucleotide sequence as shown in SEQ ID NO. 57; primer 44: nucleotide sequence as shown in SEQ ID NO. 58; primer 45: nucleotide sequence as shown in SEQ ID NO. 60; primer 46: nucleotide sequence as shown in SEQ ID NO. 61.

[0009] The probe set includes: probe A: the nucleotide sequence shown in SEQ ID NO. 3; probe B: the nucleotide sequence shown in SEQ ID NO. 6; probe C: the nucleotide sequence shown in SEQ ID NO. 9; probe D: the nucleotide sequence shown in SEQ ID NO. 12; probe E: the nucleotide sequence shown in SEQ ID NO. 15; probe F: the nucleotide sequence shown in SEQ ID NO. 18; probe G: the nucleotide sequence shown in SEQ ID NO. 21; probe H: the nucleotide sequence shown in SEQ ID NO. 24; probe I: the nucleotide sequence shown in SEQ ID NO. 29; probe G: the nucleotide sequence shown in SEQ ID NO. 34; probe K: the nucleotide sequence shown in SEQ ID NO. 39; probe L: the nucleotide sequence shown in SEQ ID NO. 44; probe M: the nucleotide sequence shown in SEQ ID NO. 49; probe N: the nucleotide sequence shown in SEQ ID NO. 54; probe O: the nucleotide sequence shown in SEQ ID NO. 59; and probe P: the nucleotide sequence shown in SEQ ID NO. 62.

[0010] Preferably, the 23 human papillomaviruses are HPV52, HPV39, HPV18, HPV16, HPV31, HPV26, HPV35, HPV45, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, HPV81, HPV6, HPV11, HPV51, HPV82, HPV73, HPV42, HPV56, and HPV66.

[0011] Preferably, the upstream primers for HPV52, HPV39, HPV18, HPV16, HPV31, HPV26, HPV35, and HPV45 in the E6 / E7 region are the nucleotide sequences shown in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 16, SEQ ID NO. 19, and SEQ ID NO. 22, respectively, and the corresponding downstream primers are the nucleotide sequences shown in SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 20, and SEQ ID NO. 23, respectively. The linear fluorescent probes used for symmetrical PCR amplification and genotyping are the sequences shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, and SEQ ID NO. 23, respectively. The nucleotide sequences shown in SEQ ID NO. 18, SEQ ID NO. 21, and SEQ ID NO. 24 are as follows:

[0012] The upstream primers for HPV68 and HPV59 in the L1 region are shown in SEQ ID NO. 25 and SEQ ID NO. 27, respectively, and the corresponding downstream primers are shown in SEQ ID NO. 26 and SEQ ID NO. 28, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 29.

[0013] The upstream primers for HPV33 and HPV58 in the L1 region are shown in SEQ ID NO. 30 and SEQ ID NO. 32, respectively, and the corresponding downstream primers are shown in SEQ ID NO. 31 and SEQ ID NO. 33, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 34.

[0014] The upstream primers for HPV53 and HPV43 in the L1 region are shown in SEQ ID NO. 35 and SEQ ID NO. 37, respectively, and the corresponding downstream primers are shown in SEQ ID NO. 36 and SEQ ID NO. 38, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 39.

[0015] The upstream primers for HPV6 and HP11 in the L1 region are shown in SEQ ID NO. 40 and SEQ ID NO. 42, respectively, and the downstream primers are shown in SEQ ID NO. 41 and SEQ ID NO. 43, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 44.

[0016] The upstream primers for HPV51 and HPV82 in the L1 region are shown in SEQ ID NO. 45 and SEQ ID NO. 47, respectively, and the corresponding downstream primers are shown in SEQ ID NO. 46 and SEQ ID NO. 48, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 49.

[0017] The upstream primers for HPV73 and HPV42 in the L1 region are shown in SEQ ID NO. 50 and SEQ ID NO. 52, respectively, and the corresponding downstream primers are shown in SEQ ID NO. 51 and SEQ ID NO. 53, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 54.

[0018] The upstream primers for HPV56 and HPV66 in the L1 region are shown in SEQ ID NO. 55 and SEQ ID NO. 57, respectively, and the corresponding downstream primers are shown in SEQ ID NO. 56 and SEQ ID NO. 58, respectively. They share a single melting curve probe for asymmetric PCR amplification and genotyping detection. The probe is shown in SEQ ID NO. 59.

[0019] The upstream and downstream primers corresponding to the L1 region of HPV81 are the nucleotide sequences shown in SEQ ID NO. 60 and SEQ ID NO. 61, respectively. Asymmetric PCR amplification and genotyping were performed using a melting curve probe, the probe being the nucleotide sequence shown in SEQ ID NO. 62.

[0020] Preferably, the probes in the probe group are labeled with different fluorescent luminescent groups.

[0021] A kit for genotyping detection of 23 types of human papillomaviruses, comprising the primer and probe composition described in this invention.

[0022] A method for genotyping detection of 23 human papillomaviruses, for non-disease diagnostic purposes, comprising the following steps:

[0023] S1. Collect cervical exfoliated cell samples for DNA extraction.

[0024] S2. Prepare a fluorescent PCR reagent, which includes the primer and probe composition described above, as well as DNA polymerase and PCR buffer.

[0025] S3. Add the extracted sample nucleic acid to the fluorescent PCR reagent. S4. Perform fluorescent PCR detection and determine whether the sample contains any of the 23 human papillomavirus types based on the detection results.

[0026] This invention targets the amplification regions of different HPV types located in different functional regions (E region and L region) of the human papillomavirus genome. In the E region, symmetric fluorescent PCR detection and typing are performed using single-type-specific primers and hydrolysis probes, while in the L region, asymmetric PCR-melting curve detection and typing are performed using single-type-specific primers and molecular beacons.

[0027] Conventional HPV genotyping techniques often employ either simple real-time fluorescence PCR amplification or simple melting curve detection. Simple real-time fluorescence PCR amplification can only achieve multiplex detection by increasing the number of wells, while simple melting curve detection is prone to interference between targets when there are too many targets. This invention combines melting curve detection with real-time fluorescence PCR amplification, detecting some target sequences simultaneously in addition to amplifying the DNA template during the amplification stage. This increases the throughput of single-well detection of targets in fluorescence PCR analysis. Simultaneously, it optimizes the melting temperature distribution of each target, better avoiding interference between targets. This provides a superior detection method for multiplex fluorescence PCR genotyping.

[0028] As a preferred option, the specific parameters for fluorescence PCR detection in S4 are set as follows: pre-denaturation at 95℃ for 15 minutes; denaturation at 95℃ for 30 seconds (fluorescence is collected during denaturation), denaturation at 55℃ for 20 seconds, denaturation at 58℃ for 20 seconds, repeated for 50 cycles; after PCR, melting curve analysis is performed at 95℃ for 10 minutes, 37℃ for 5 minutes, and 37℃-75℃ (fluorescence is collected), with a heating rate of 0.04℃ / second.

[0029] Preferably, the final concentration of the symmetrical fluorescent PCR amplification primers is 0.1µM-0.5µM, and the final concentration of the linear fluorescent probe is 0.01µM-0.5µM.

[0030] Preferably, the final concentration of the upstream primer (in the same direction as the probe) for asymmetric PCR amplification is 1 / 50 to 1 / 10 of that of the downstream primer (in the opposite direction to the probe), with the final concentration of the upstream primer being 0.01µM-0.05µM, the final concentration of the downstream primer being 0.1µM-1µM, and the final concentration of the melting curve probe being 0.05µM-0.5µM.

[0031] As a preferred embodiment, in S4, the fluorescence PCR detection involves simultaneous multicolor fluorescence collection and detection during both the amplification and melting curve stages; the fluorescence collection during the amplification stage is performed during the high-temperature denaturation step within the cyclic amplification.

[0032] Compared with existing HPV fluorescent PCR genotyping detection technology, the advantages of this invention are as follows:

[0033] By designing hydrolysis probes and molecular beacon probes in two different regions of the HPV gene sequence, E6 and L1, respectively, and setting the fluorescence collection point during the amplification stage to the high-temperature denaturation stage, the detection of the amplification stage and the melting curve stage are independent and do not interfere with each other. This enables the combined use of real-time fluorescence PCR amplification detection technology and melting curve technology. This technology improves the single-well detection throughput of fluorescence PCR analysis technology, reduces detection costs, and provides a better detection method for HPV multiplex fluorescence PCR genotyping. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the process of the present invention;

[0035] Figure 2 shows the genotyping results of 23 HPV types in Example 1. Figures 2-1 to 2-23 The images show positive results for HPV types 16, 18, 39, 52, 33, 58, 59, 68, 43, 53, 81, 31, 35, 26, 45, 6, 11, 73, 42, 51, 82, 56, and 66, in that order.

[0036] Figure 3 shows the detection results for HPV types other than those detected in Example 1. Figures 3-1 to 3-10 The images show the test results for HPV types 40, 44, 54, 61, 67, 69, 70, 71, 72, and 83, in that order.

[0037] Figure 4 shows the typing results of representative clinical samples in Example 2. Figures 4-1 to 4-23 The images show the test results for HPV types 16, 18, 39, 52, 33, 58, 59, 68, 43, 53, 81, 31, 35, 26, 45, 6, 11, 73, 42, 51, 82, 56, and 66, in that order. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0039] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0040] Example 1

[0041] The specific steps for designing a primer-probe mixture for the genotyping detection of 23 human papillomaviruses are as follows:

[0042] 1. Selection of amplification regions

[0043] Search the NCBI database for the full-length genome sequences of HPV52, HPV39, HPV18, HPV16, HPV31, HPV26, HPV35, HPV45, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, HPV81, HPV6, HPV11, HPV51, HPV82, HPV73, HPV42, HPV56, and HPV66. Perform alignment analysis.

[0044] We designed specific primers and probes for HPV52, HPV39, HPV18, HPV16, HPV31, HPV26, HPV35, and HPV45 in the E6 / E7 region, and performed symmetrical PCR amplification and genotyping using linear fluorescent probes.

[0045] HPV68 and HPV59 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0046] HPV33 and HPV58 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0047] HPV53 and HPV43 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0048] HPV81 type was determined by sequence comparison. The L1 region was selected as the target amplification fragment. Asymmetric PCR amplification and genotyping were performed using a melting curve probe.

[0049] HPV6 and HP11 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0050] HPV51 and HPV82 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0051] HPV73 and HPV42 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0052] HPV56 and HPV66 types were determined by sequence comparison. The L1 region was selected as the target amplification fragment, and a single melting curve probe was used for asymmetric PCR amplification and genotyping detection.

[0053] The designed primer and probe sequences are shown in Table 1:

[0054] Table 1

[0055]

[0056]

[0057] 2. Validation of the designed primers and probes

[0058] 2.1 Construction of positive plasmids

[0059] Search the NCBI database for full-length DNA sequences of the E6 / E7 regions for HPV types 31, 26, 35, 45, 52, 39, 18, and 16, and full-length DNA sequences of the L1 regions for HPV types 68, 59, 33, 58, 43, 53, 81, 6, 11, 51, 82, 73, 42, 56, and 66. Submit these sequences to a plasmid synthesis company (Nanjing Qingke Biotechnology Co., Ltd.) for insertion into the pUC57 plasmid vector for plasmid synthesis.

[0060] 2.2 Determination of the concentration of positive plasmids

[0061] Concentration calibration was performed using a digital PCR instrument (A300 / Drop Mark M1 / ChipReader R1) from Xinyi Manufacturing Technology (Beijing) Co., Ltd.

[0062] 2.3 Validation of the effectiveness of the designed primers and probes

[0063] 1) Testing system

[0064] PCR buffer (containing 3 mM magnesium chloride, 2 U Taq DNA polymerase, and 200 µM dNTPs), primers and probes (containing 0.2 µM of symmetrical upstream primer, 0.2 µM of symmetrical downstream primer, and 0.01 µM of linear fluorescent probe; 0.02 µM of asymmetrical upstream primer, 0.5 µM of symmetrical downstream primer, and 0.08 µM of melting curve probe).

[0065] 2) Prepare the PCR reaction system

[0066]

[0067] In the system, A represents 11 types of primers and probes, and B represents another 12 types of primers and probes. A+B detects a total of 23 types (the same applies below).

[0068] 3) Add sample

[0069] Aliquot the prepared PCR reaction system into PCR tubes, adding 10 µL of the pre-constructed and calibrated concentration (1×10⁻⁶) to each reaction system. 5Positive plasmids for HPV52, HPV39, HPV18, HPV16, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, HPV81, HPV52, HPV39, HPV18, HPV16, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, and HPV81 were measured in copies / mL.

[0070] 4) Set up the fluorescent PCR detection program

[0071] Pre-denaturation at 95℃ for 15 minutes; denaturation at 95℃ for 30 seconds (fluorescence collected during denaturation), denaturation at 55℃ for 20 seconds, denaturation at 58℃ for 20 seconds, repeated for 50 cycles; after PCR, melting curve analysis was performed at 95℃ for 10 minutes, 37℃ for 5 minutes, and 37℃-75℃ (fluorescence collected), with a heating rate of 0.04℃ / second.

[0072] 5) Result Interpretation

[0073] According to Table 2, the HPV types present in the sample can be determined based on the specific detection method and detection channel for each type of HPV. If there is no detection signal, it means that the sample does not contain any of the 23 HPV types required for the detection purpose.

[0074] Table 2. Determination Table for 23 HPV Types

[0075]

[0076] 6) Test results

[0077]

[0078] The results for each type are shown in Figure 2.

[0079] 2.4 Specificity confirmation of the designed primers and probes

[0080] 1) Testing system

[0081] PCR buffer (containing 3 mM magnesium chloride, 2 U Taq DNA polymerase, and 200 µM dNTPs), primers and probes (containing 0.2 µM of symmetrical upstream primer, 0.2 µM of symmetrical downstream primer, and 0.01 µM of linear fluorescent probe; 0.02 µM of asymmetrical upstream primer, 0.5 µM of symmetrical downstream primer, and 0.08 µM of melting curve probe).

[0082] 2) Prepare the PCR reaction system

[0083]

[0084] 2) Add sample

[0085] The specificity of the primers and probes was confirmed by selecting types other than those described in this invention from the human papillomavirus L1 genotyping reference and whole genotyping reference (excluding HPV52, HPV39, HPV18, HPV16, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, HPV81, HPV52, HPV39, HPV18, HPV16, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, and HPV81).

[0086] National reference samples for HPV types 40, 44, 54, 61, 67, 69, 70, 71, 72, and 83 were selected at a concentration of 1×10⁻⁶. 7 copies / mL.

[0087] Aliquot the prepared PCR reaction mixture into PCR tubes, adding 10 µL of a 1×10⁻⁶ solution to each reaction mixture. 7 National reference samples of HPV types 40, 44, 54, 61, 67, 69, 70, 71, 72, and 83, in copies / mL.

[0088] 3) Set up the fluorescent PCR detection program

[0089] Pre-denaturation at 95℃ for 15 minutes; denaturation at 95℃ for 30 seconds (fluorescence collected during denaturation), denaturation at 55℃ for 20 seconds, denaturation at 58℃ for 20 seconds, repeated for 50 cycles; after PCR, melting curve analysis was performed at 95℃ for 10 minutes, 37℃ for 5 minutes, and 37℃-75℃ (fluorescence collected), with a heating rate of 0.04℃ / second.

[0090] 4) Result Interpretation

[0091] According to Table 2, the HPV types present in the sample can be determined based on the specific detection method and detection channel for each type of HPV. If there is no detection signal, it means that the sample does not contain any of the 23 HPV types required for the detection purpose.

[0092] 5) Test results

[0093]

[0094]

[0095]

[0096] The detection results for HPV types outside the detection range are shown in Figure 3.

[0097] Example 2

[0098] The application of 23 human papillomavirus (HPV) typing detection methods, and the specific steps are as follows:

[0099] 1. Testing System

[0100] PCR buffer (containing 3 mM magnesium chloride, 2 U Taq DNA polymerase, and 200 µM dNTPs), primers and probes (containing 0.2 µM of symmetrical upstream primer, 0.2 µM of symmetrical downstream primer, and 0.01 µM of linear fluorescent probe; 0.02 µM of asymmetrical upstream primer, 0.5 µM of symmetrical downstream primer, and 0.08 µM of melting curve probe).

[0101] 2. Testing Procedure

[0102] Pre-denaturation at 95℃ for 15 minutes; denaturation at 95℃ for 30 seconds (fluorescence collected during denaturation), denaturation at 55℃ for 20 seconds, denaturation at 58℃ for 20 seconds, repeated for 50 cycles; after PCR, melting curve analysis was performed at 95℃ for 10 minutes, 37℃ for 5 minutes, and 37℃-75℃ (fluorescence collected), with a heating rate of 0.04℃ / second.

[0103] 3. Clinical sample validation

[0104] 3.1 Preparation of PCR reaction system

[0105]

[0106] 3.2 Nucleic acid extraction

[0107] Take 200µL of cervical secretion sample (for HPV genotyping using the Human Papillomavirus Genotyping (Type 23) Detection Kit (PCR-Reverse Dot Hybridization) produced by Yaneng Biotechnology Co., Ltd.), and extract nucleic acid according to the viral nucleic acid extraction kit (product registration number: Zhehangxiebei 20190045) produced by Hangzhou Danwei Biotechnology Co., Ltd., and collect the extracted products.

[0108] 3.3 Sample Addition

[0109] The prepared PCR reaction system was dispensed into PCR tubes, and 10 µL of the extracted product of the above sample was added to each reaction system for detection. The detection procedure was as described above.

[0110] 3.4 Setting up the fluorescent PCR detection program

[0111] Pre-denaturation at 95℃ for 15 minutes; denaturation at 95℃ for 30 seconds (fluorescence collected during denaturation), denaturation at 55℃ for 20 seconds, denaturation at 58℃ for 20 seconds, repeated for 50 cycles; after PCR, melting curve analysis was performed at 95℃ for 10 minutes, 37℃ for 5 minutes, and 37℃-75℃ (fluorescence collected), with a heating rate of 0.04℃ / second.

[0112] 3.5 Result Interpretation

[0113] Interpret according to Table 2.

[0114] 3.6 Summary of Results

[0115]

[0116] The above experimental results demonstrate that, through the detection of various types of HPV samples in clinical settings, the human papillomavirus detection and typing method of the present invention can accurately detect 23 types of human papillomavirus, and the melting curve peaks of each type are far apart and do not interfere with each other.

[0117] The test results of representative clinical samples are shown in Figure 4.

[0118] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to in the method section.

[0119] The foregoing has provided a detailed description of a primer and probe composition, kit, and detection method for genotyping detection of 23 types of human papillomaviruses (HPV). Specific examples have been used to illustrate the principles and implementation methods of the invention. These examples are merely illustrative and are intended to aid in understanding the method and core concepts of the invention. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. sequence list <110> Hangzhou Danwei Biotechnology Co., Ltd. <120> Primer and probe compositions, kits, and detection methods for genotyping detection of 23 types of human papillomaviruses. <130> DWSW2022-03 <160> 62 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Synthetic sequence <400> 1 agagacaatr atccatatg 19 <210> 2 <211> 20 <212> DNA <213> Synthetic sequence <400> 2 acagtgaata tygataatgc 20 <210> 3 <211> twenty two <212> DNA <213> Synthetic sequence <400> 3 ggcgtgtgta ttatgtgcct ac 22 <210> 4 <211> 15 <212> DNA <213> Synthetic sequence <400> 4 acaccacctt gcagg 15 <210> 5 <211> 16 <212> DNA <213> Synthetic sequence <400> 5 acctcrgttt gctgta 16 <210> 6 <211> 20 <212> DNA <213> Synthetic sequence <400> 6 atagcctgtg tctattgcag 20 <210> 7 <211> 18 <212> DNA <213> Synthetic sequence <400> 7 acgatttcac aacatagc 18 <210> 8 <211> 17 <212> DNA <213> Synthetic sequence <400> 8 tcgttggagt cgttcct 17 <210> 9 <211> 20 <212> DNA <213> Synthetic sequence <400> 9 cactatagag gccagtgcca 20 <210> 10 <211> 20 <212> DNA <213> Synthetic sequence <400> 10 ggaacaacat tagaacagca 20 <210> 11 <211> 17 <212> DNA <213> Synthetic sequence <400> 11 tgtccagatg tctttgc 17 <210> 12 <211> twenty three <212> DNA <213> Synthetic sequence <400> 12 aacaaaccgt tgtgtgtgat ttg 23 <210> 13 <211> 20 <212> DNA <213> Synthetic sequence <400> 13 caatagtata tagggacgac 20 <210> 14 <211> 18 <212> DNA <213> Synthetic sequence <400> 14 acggtctttg acacgtta 18 <210> 15 <211> twenty four <212> DNA <213> Synthetic sequence <400> 15 cggagtgtgt acaaaatgtt taag 24 <210> 16 <211> twenty four <212> DNA <213> Synthetic sequence <400> 16 gacctaagag tagtatatat agag 24 <210> 17 <211> twenty one <212> DNA <213> Synthetic sequence <400> 17 gccttggtct ccaacaattt g 21 <210> 18 <211> twenty two <212> DNA <213> Synthetic sequence <400> 18 tggtgcaaca ttagaagcct ta 22 <210> 19 <211> 17 <212> DNA <213> Synthetic sequence <400> 19 aactgcatga tttgtgc 17 <210> 20 <211> 17 <212> DNA <213> Synthetic sequence <400> 20 ctcactccgc tgtaatt 17 <210> twenty one <211> twenty two <212> DNA <213> Synthetic sequence <400> twenty one gcatccatga aatttgtttg aa 22 <210> twenty two <211> 18 <212> DNA <213> Synthetic sequence <400> twenty two agcagaaaaa crtagaca 18 <210> twenty three <211> 19 <212> DNA <213> Synthetic sequence <400> twenty three acaacatgta ttacactgc 19 <210> twenty four <211> twenty one <212> DNA <213> Synthetic sequence <400> twenty four cgaagatttc acagcatagc t 21 <210> 25 <211> 18 <212> DNA <213> Synthetic sequence <400> 25 gacagtaggg acaatgtt 18 <210> 26 <211> 18 <212> DNA <213> Synthetic sequence <400> 26 cattggtagg cttacaag 18 <210> 27 <211> 20 <212> DNA <213> Synthetic sequence <400> 27 catctgctgt tgataccaaa 20 <210> 28 <211> 19 <212> DNA <213> Synthetic sequence <400> 28 aattctagtg gaggacaat 19 <210> 29 <211> 33 <212> DNA <213> Synthetic sequence <400> 29 ccggctgtat tataggctgt gttcctgcgc cgg 33 <210> 30 <211> twenty one <212> DNA <213> Synthetic sequence <400> 30 tgcacaaggt cataacaatg g 21 <210> 31 <211> twenty two <212> DNA <213> Synthetic sequence <400> 31 catattagtg stacgagtgg ta 22 <210> 32 <211> twenty one <212> DNA <213> Synthetic sequence <400> 32 tgcacaaggt cataataatg g 21 <210> 33 <211> twenty two <212> DNA <213> Synthetic sequence <400> 33 catattggta ctgcgagtgg ta 22 <210> 34 <211> 34 <212> DNA <213> Synthetic sequence <400> 34 ccggccaatc aggtatttgt tactgtggtg ccgg 34 <210> 35 <211> 18 <212> DNA <213> Synthetic sequence <400> 35 ggttacttct gattctca 18 <210> 36 <211> 18 <212> DNA <213> Synthetic sequence <400> 36 ggtatctacc actgtaac 18 <210> 37 <211> 20 <212> DNA <213> Synthetic sequence <400> 37 taagccatat tggctgcaac 20 <210> 38 <211> 19 <212> DNA <213> Synthetic sequence <400> 38 gtagacatag actgtgtgg 19 <210> 39 <211> 34 <212> DNA <213> Synthetic sequence <400> 39 ccggcaggga cataataatg gcatttgttg ccgg 34 <210> 40 <211> 19 <212> DNA <213> Synthetic sequence <400> 40 ggtagtggaa atcgaacgt 19 <210> 41 <211> 17 <212> DNA <213> Synthetic sequence <400> 41 atgacgcatg tactctt 17 <210> 42 <211> twenty one <212> DNA <213> Synthetic sequence <400> 42 gtagtatttw tgtacataca c 21 <210> 43 <211> twenty two <212> DNA <213> Synthetic sequence <400> 43 gtatctacca cagtaacaaa ca 22 <210> 44 <211> 32 <212> DNA <213> Synthetic sequence <400> 44 ccggctacat gcgtcatgtg gaagagtgcc gg 32 <210> 45 <211> 20 <212> DNA <213> Synthetic sequence <400> 45 tcgaattgtg aatacagaag 20 <210> 46 <211> twenty one <212> DNA <213> Synthetic sequence <400> 46 gttgaggttt taggtattgg a 21 <210> 47 <211> 19 <212> DNA <213> Synthetic sequence <400> 47 gcaccggcat atattatta 19 <210> 48 <211> 17 <212> DNA <213> Synthetic sequence <400> 48 aaaggcagat accttag 17 <210> 49 <211> 36 <212> DNA <213> Synthetic sequence <400> 49 ccggcactaa taacattagg acatccctat tgccgg 36 <210> 50 <211> 20 <212> DNA <213> Synthetic sequence <400> 50 ggatacatat acagcttcta 20 <210> 51 <211> twenty four <212> DNA <213> Synthetic sequence <400> 51 catattgtgt atatatgaca taac 24 <210> 52 <211> 17 <212> DNA <213> Synthetic sequence <400> 52 agatactaga agcacta 17 <210> 53 <211> 20 <212> DNA <213> Synthetic sequence <400> 53 acaaactgta aatcaaactc 20 <210> 54 <211> 33 <212> DNA <213> Synthetic sequence <400> 54 ccggcagaca tgctgaagaa tatgatgtgc cgg 33 <210> 55 <211> 20 <212> DNA <213> Synthetic sequence <400> 55 cgctatggac tttaaggtgt 20 <210> 56 <211> 18 <212> DNA <213> Synthetic sequence <400> 56 ctctgcaggt attgtttc 18 <210> 57 <211> 20 <212> DNA <213> Synthetic sequence <400> 57 gacggtgaca tggtggacac 20 <210> 58 <211> 19 <212> DNA <213> Synthetic sequence <400> 58 ttgccaccct tccaataca 19 <210> 59 <211> 35 <212> DNA <213> Synthetic sequence <400> 59 ccggccttta gacattgtac aatccacctg gccgg 35 <210> 60 <211> 20 <212> DNA <213> Synthetic sequence <400> 60 aataatggca tttgttggtt 20 <210> 61 <211> twenty three <212> DNA <213> Synthetic sequence <400> 61 gsagaaattc cttaaagtta gag 23 <210> 62 <211> 30 <212> DNA <213> Synthetic sequence <400> 62 ccggcactac cagaagcacc aatttgccgg 30

Claims

1. A primer probe composition for detecting 23 human papillomavirus genotypes by combining real-time fluorescent PCR amplification detection with melting curve, the composition comprising a primer set and a probe set, characterized in that: the primer set comprises: primer 1: a nucleotide sequence as shown in SEQ ID NO. 1, primer 2: a nucleotide sequence as shown in SEQ ID NO. 2, primer 3: a nucleotide sequence as shown in SEQ ID NO. 4, primer 4: a nucleotide sequence as shown in SEQ ID NO. 5, primer 5: a nucleotide sequence as shown in SEQ ID NO. 7, primer 6: a nucleotide sequence as shown in SEQ ID NO. 8, primer 7: a nucleotide sequence as shown in SEQ ID NO. 10, primer 8: a nucleotide sequence as shown in SEQ ID NO. 11, primer 9: a nucleotide sequence as shown in SEQ ID NO. 13, primer 10: a nucleotide sequence as shown in SEQ ID NO. 14, primer 11: a nucleotide sequence as shown in SEQ ID NO. 16, primer 12: a nucleotide sequence as shown in SEQ ID NO. 17, primer 13: a nucleotide sequence as shown in SEQ ID NO. 19, primer 14: a nucleotide sequence as shown in SEQ ID NO. 20, primer 15: a nucleotide sequence as shown in SEQ ID NO. 22, primer 16: a nucleotide sequence as shown in SEQ ID NO. 23, primer 17: a nucleotide sequence as shown in SEQ ID NO. 25, primer 18: a nucleotide sequence as shown in SEQ ID NO. 26, primer 19: a nucleotide sequence as shown in SEQ ID NO. 27, primer 20: a nucleotide sequence as shown in SEQ ID NO. 28, primer 21: a nucleotide sequence as shown in SEQ ID NO. 30, primer 22: a nucleotide sequence as shown in SEQ ID NO. 31, primer 23: a nucleotide sequence as shown in SEQ ID NO. 32, primer 24: a nucleotide sequence as shown in SEQ ID NO. 33, primer 25: a nucleotide sequence as shown in SEQ ID NO. 35, primer 26: a nucleotide sequence as shown in SEQ ID NO. 36, primer 27: a nucleotide sequence as shown in SEQ ID NO. 37, primer 28: a nucleotide sequence as shown in SEQ ID NO. 38, primer 29: a nucleotide sequence as shown in SEQ ID NO. 40, primer 30: a nucleotide sequence as shown in SEQ ID NO. 41, primer 31: a nucleotide sequence as shown in SEQ ID NO. 42, primer 32: a nucleotide sequence as shown in SEQ ID NO. 43, primer 33: a nucleotide sequence as shown in SEQ ID NO. 45, and the probe set comprises: probe 1: a nucleotide sequence as shown in SEQ ID NO. 3, probe 2: a nucleotide sequence as shown in SEQ ID NO. 6, probe 3: a nucleotide sequence as shown in SEQ ID NO. 9, probe 4: a nucleotide sequence as shown in SEQ ID NO. 12, probe 5: a nucleotide sequence as shown in SEQ ID NO. 15, probe 6: a nucleotide sequence as shown in SEQ ID NO. 18, probe 7: a nucleotide sequence as shown in SEQ ID NO. 21, probe 8: a nucleotide sequence as shown in SEQ ID NO. 24, probe 9: a nucleotide sequence as shown in SEQ ID NO. 34, probe 10: a nucleotide sequence as shown in SEQ ID NO.

44. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Primer 34: a nucleotide sequence as shown in SEQ ID NO. 46, Primer 35: a nucleotide sequence as shown in SEQ ID NO. 47, Primer 36: a nucleotide sequence as shown in SEQ ID NO. 48, Primer 37: a nucleotide sequence as shown in SEQ ID NO. 50, Primer 38: a nucleotide sequence as shown in SEQ ID NO. 51, Primer 39: a nucleotide sequence as shown in SEQ ID NO. 52, Primer 40: a nucleotide sequence as shown in SEQ ID NO. 53, Primer 41: a nucleotide sequence as shown in SEQ ID NO. 55, Primer 42: a nucleotide sequence as shown in SEQ ID NO. 56, Primer 43: a nucleotide sequence as shown in SEQ ID NO. 57, Primer 44: a nucleotide sequence as shown in SEQ ID NO. 58, Primer 45: a nucleotide sequence as shown in SEQ ID NO. 60, Primer 46: a nucleotide sequence as shown in SEQ ID NO. 61, The probe set comprises: Probe A: a nucleotide sequence as shown in SEQ ID NO. 3, Probe B: a nucleotide sequence as shown in SEQ ID NO. 6, Probe C: a nucleotide sequence as shown in SEQ ID NO. 9, Probe D: a nucleotide sequence as shown in SEQ ID NO. 12, Probe E: a nucleotide sequence as shown in SEQ ID NO. 15, Probe F: a nucleotide sequence as shown in SEQ ID NO. 18, Probe G: a nucleotide sequence as shown in SEQ ID NO. 21, Probe H: a nucleotide sequence as shown in SEQ ID NO. 24, Probe I: a nucleotide sequence as shown in SEQ ID NO. 29, Probe G: a nucleotide sequence as shown in SEQ ID NO. 34, Probe K: a nucleotide sequence as shown in SEQ ID NO. 39, Probe L: a nucleotide sequence as shown in SEQ ID NO. 44, Probe M: a nucleotide sequence as shown in SEQ ID NO. 49, Probe N: a nucleotide sequence as shown in SEQ ID NO. 54, Probe O: a nucleotide sequence as shown in SEQ ID NO. 59, Probe P: a nucleotide sequence as shown in SEQ ID NO. 62; The corresponding upstream primers of HPV52, HPV39, HPV18, HPV16, HPV31, HPV26, HPV35 and HPV45 in the E6 / E7 region are respectively the nucleotide sequences shown in SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 16, SEQ ID NO. 19 and SEQ ID NO. 22, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14, SEQ ID NO. 17, SEQ ID NO. 20 and SEQ ID NO. 23, the linear fluorescent probes for symmetrical PCR amplification and typing detection are respectively the nucleotide sequences shown in SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 21 and SEQ ID NO. 24, The corresponding upstream primers of HPV68 and HPV59 in the L1 region are respectively the nucleotide sequences shown in SEQ ID NO. 25 and SEQ ID NO. 27, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 26 and SEQ ID NO. 28, and a melting curve probe is shared for asymmetrical PCR amplification and typing detection, and the probe is the nucleotide sequence shown in SEQ ID NO. 29, The corresponding upstream primers of HPV33 and HPV58 in the L1 region are respectively the nucleotide sequences shown in SEQ ID NO. 30 and SEQ ID NO. 32, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 31 and SEQ ID NO. 33, and a melting curve probe is shared for asymmetrical PCR amplification and typing detection, and the probe is the nucleotide sequence shown in SEQ ID NO. 34, The corresponding upstream primers of HPV53 and HPV43 in the L1 region are respectively the nucleotide sequences shown in SEQ ID NO. 35 and SEQ ID NO. 37, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 36 and SEQ ID NO. 38, and a melting curve probe is shared for asymmetrical PCR amplification and typing detection, and the probe is the nucleotide sequence shown in SEQ ID NO. 39, The corresponding upstream primers of HPV68 and HPV59 in the L1 region are respectively the nucleotide sequences shown in SEQ ID NO. 25 and SEQ ID NO. 27, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 26 and SEQ ID NO. 28, and a melting curve probe is shared for asymmetrical PCR amplification and typing detection, and the probe is the nucleotide sequence shown in SEQ ID NO. 29, The corresponding upstream primers of HPV33 and HPV58 in the L1 region are respectively the nucleotide sequences shown in SEQ ID NO. 30 and SEQ ID NO. 32, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 31 and SEQ ID NO. 33, and a melting curve probe is shared for asymmetrical PCR amplification and typing detection, and the probe is the nucleotide sequence shown in SEQ ID NO. 34, The corresponding upstream primers of HPV53 and HPV43 in the L1 region are respectively the nucleotide sequences shown in SEQ ID NO. 35 and SEQ ID NO. 37, the corresponding downstream primers are respectively the nucleotide sequences shown in SEQ ID NO. 36 and SEQ ID NO. 38, and a melting curve probe is shared for asymmetrical PCR amplification and typing detection, and the probe is the nucleotide sequence shown in SEQ ID NO. 39, The upstream primers corresponding to the L1 region of HPV6 and HPV11 are respectively the nucleotide sequences as shown in SEQ ID NO. 40 and SEQ ID NO. 42, the corresponding downstream primers are respectively the nucleotide sequences as shown in SEQ ID NO. 41 and SEQ ID NO. 43, and a melting curve probe as shown in SEQ ID NO. 44 is used for asymmetric PCR amplification and typing detection, The upstream primers corresponding to the L1 region of HPV51 and HPV82 are respectively the nucleotide sequences as shown in SEQ ID NO. 45 and SEQ ID NO. 47, the corresponding downstream primers are respectively the nucleotide sequences as shown in SEQ ID NO. 46 and SEQ ID NO. 48, and a melting curve probe as shown in SEQ ID NO. 49 is used for asymmetric PCR amplification and typing detection, The upstream primers corresponding to the L1 region of HPV73 and HPV42 are respectively the nucleotide sequences as shown in SEQ ID NO. 50 and SEQ ID NO. 52, the corresponding downstream primers are respectively the nucleotide sequences as shown in SEQ ID NO. 51 and SEQ ID NO. 53, and a melting curve probe as shown in SEQ ID NO. 54 is used for asymmetric PCR amplification and typing detection, The upstream primers corresponding to the L1 region of HPV56 and HPV66 are respectively the nucleotide sequences as shown in SEQ ID NO. 55 and SEQ ID NO. 57, the corresponding downstream primers are respectively the nucleotide sequences as shown in SEQ ID NO. 56 and SEQ ID NO. 58, and a melting curve probe as shown in SEQ ID NO. 59 is used for asymmetric PCR amplification and typing detection, The upstream and downstream primers corresponding to the L1 region of HPV81 are respectively the nucleotide sequences as shown in SEQ ID NO. 60 and SEQ ID NO. 61, and a melting curve probe as shown in SEQ ID NO. 62 is used for asymmetric PCR amplification and typing detection.

2. The primer probe composition of claim 1, wherein: The 23 types of human papillomavirus are HPV52, HPV39, HPV18, HPV16, HPV31, HPV26, HPV35, HPV45, HPV68, HPV59, HPV33, HPV58, HPV43, HPV53, HPV81, HPV6, HPV11, HPV51, HPV82, HPV73, HPV42, HPV56, and HPV66.

3. The primer probe composition of claim 1, wherein: The probes in the probe set are respectively labeled with different fluorescent light emitting groups.

4. A kit for 23 types of human papillomavirus genotyping detection, the kit comprising the primer probe combination of claim 1.

5. A method for the detection of 23 human papillomavirus genotypes for non-diagnostic purposes, characterized in that The method comprises the following steps: The method comprises the following steps: S1, taking cervical exfoliated cell samples for DNA extraction; S2, preparing a fluorescent PCR reagent, the fluorescent PCR reagent comprising the primer probe composition of claim 1, and a DNA polymerase and a PCR buffer; S3, adding the extracted sample nucleic acid to the fluorescent PCR reagent; S4, fluorescent PCR detection, wherein multicolor fluorescence collection detection is performed simultaneously in the amplification stage and the melting curve stage; the collected fluorescence in the amplification stage is performed in the high-temperature denaturation step within the cyclic amplification, and whether the sample contains 23 human papilloma virus types is determined according to the detection result.

6. The method of claim 5, wherein: The specific parameter settings of the fluorescent PCR detection in S4 are as follows: 95℃ pre-denaturation for 15 minutes; 95℃ denaturation for 30 seconds, fluorescence collection during denaturation, 55℃ denaturation for 20 seconds, 58℃ denaturation for 20 seconds, and repeating 50 cycles; after the PCR is completed, melting curve analysis is performed, 95℃ for 10 minutes, 37℃ for 5 minutes, 37℃-75℃ for melting curve analysis, fluorescence collection, and a heating rate of 0.04℃ / second.

7. The method of claim 5, wherein: The final concentration of the symmetric fluorescent PCR amplification primer is 0.1µM-0.5µM, and the final concentration of the linear fluorescent probe is 0.01µM-0.5µM.

8. The method of claim 5, wherein: The final concentration of the upstream primer of the asymmetric PCR amplification is 0.01µM-0.05µM, the final concentration of the downstream primer is 0.1µM-1µM, and the final concentration of the melting curve probe is 0.05µM-0.5µM.

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