Compositions and kits for single tube genotyping of 28 hpv subtypes

Abstract

The application relates to a composition and a kit for single-tube typing detection of 28 HPV subtypes. The composition comprises a primer composition whose detection result is judged by a melting curve and a primer probe composition whose detection result is judged by an amplification curve; the primer composition comprises 18 pairs of upstream and downstream primers; the base sequences of the 18 pairs of upstream and downstream primers are shown in SEQ ID NO: 1-36; the primer probe composition comprises one universal upstream primer, one universal downstream primer and 10 probes; the base sequence of the one universal upstream primer is shown in SEQ ID NO: 37, the base sequences of the one universal downstream primer are shown in SEQ ID NO: 38, and the base sequences of the 10 probes are shown in SEQ ID NO: 41-50. By using the specific composition, 28 different HPV virus subtypes can be detected by one tube, the combined analysis of real-time fluorescent PCR amplification detection technology and melting curve technology is realized, the detection flux is high, the specificity is strong, the time consumption is short and the sensitivity is high.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection technology, and in particular to a composition and kit for single-tube typing detection of 28 HPV subtypes. Background Technology

[0002] Human papillomavirus (HPV) belongs to the Papillomaviridae family and is a small, non-enveloped, circular, double-stranded DNA virus. HPV that infects the genital tract and anus can be divided into two main categories based on the pathogenicity or carcinogenic risk of each genotype: low-risk and high-risk. Persistent high-risk HPV infection is a major cause of cervical intraepithelial neoplasia and cervical cancer. Global studies show that high-risk HPV DNA is detected in 99.7% of cervical cancer patients, with HPV types 16, 18, 45, and 31 accounting for 80% of infections. Low-risk HPV is generally associated with condyloma acuminata or low-grade squamous intraepithelial lesions. Therefore, rapid and accurate detection of high-risk HPV infection is crucial for early treatment and reducing the incidence and mortality of cervical cancer.

[0003] With a deeper understanding of how HPV infection leads to cervical cancer and the development of molecular diagnostic technology, HPV gene testing has been more widely used as a screening method for cervical cancer. Compared with traditional cytological testing methods, molecular testing methods do not require waiting for changes in cells to identify HPV infection. They can screen for the virus in the incubation period when the virus infects cells but no symptoms appear, thus achieving the goal of early prevention and early treatment.

[0004] Current HPV genotyping methods include: In situ hybridization (ISH): This method uses nucleic acid probes (DNA or RNA) to hybridize with samples in situ. The advantages of ISH are its ability to locate suspicious cells and its semi-quantitative function; however, it suffers from low sensitivity, high sample quality requirements, high labor intensity, and high cost. Furthermore, each HPV type requires a specific probe. PCR reverse dot blot hybridization combines in vitro PCR amplification with DNA reverse dot blot hybridization. Specific primers designed based on HPV genotypes amplify target fragments containing HPV genotypes. The amplified products are then hybridized with genotyping probes immobilized on membrane strips. The presence or absence of hybridization signals indicates 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, affecting result interpretation. Flow cytometry hybridization: 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. The disadvantages are its complex and cumbersome operation, high cost, and the need for specialized technical personnel. PCR-Taqman MGB probe typing is a commonly used detection method due to its simplicity, accuracy, and closed-tube operation. However, due to limitations in the instrument's signal acquisition channels, only a limited number of HPV types can be detected per reaction, resulting in low throughput. Furthermore, multi-tube testing significantly increases both economic and time costs. Melting curve analysis involves adding probes labeled with fluorescent and quenching groups to the PCR system. During PCR, single-stranded oligonucleotide sequences complementary to the probe sequence are amplified. After amplification, a melting curve analysis is performed to obtain melting curves and determine the melting point (Tm value) for each type. However, a drawback is that the melting peaks of different targets are closely spaced, easily leading to interference between adjacent melting peaks and the appearance of fusion peaks.

[0005] Therefore, providing a method that can reduce the consumption of manpower, material resources, and financial resources, accurately and quickly detect HPV, and greatly reduce the complexity of PCR reaction, is of practical significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application provides a composition for single-tube genotyping detection of 28 HPV subtypes (including 18 intermediate- and high-risk types and 10 low-risk types). The composition comprises a primer composition for interpreting detection results using melting curves and a primer-probe composition for interpreting detection results using amplification curves. The primer composition is used to amplify the test sample, and the amplification products are used for melting curve analysis. The primer-probe composition is used to amplify the test sample, and the amplification process is used for amplification curve analysis. Using this composition, 28 different HPV subtypes can be detected in a single tube, with accurate genotyping of the 18 intermediate- and high-risk types and partial genotyping of the 10 low-risk types. This achieves combined analysis of real-time fluorescence PCR amplification detection technology and melting curve technology, offering advantages such as high throughput, strong specificity, short processing time, high sensitivity, and comprehensive detection site coverage. It can provide clinicians with auxiliary diagnostic references, enabling earlier treatment and has wide clinical applications.

[0007] Therefore, the first aspect of this application provides a composition for single-tube genotyping detection of 28 HPV subtypes, the composition comprising a primer composition for interpreting detection results using melting curves and a primer-probe composition for interpreting detection results using amplification curves; The primer composition comprises 18 pairs of upstream and downstream primers for genotyping 18 intermediate- to high-risk HPV subtypes: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82. The base sequences of the 18 pairs of upstream and downstream primers are shown in SEQ ID NO: 1-36, respectively. The primer-probe composition comprises one universal upstream primer, one universal downstream primer, and ten probes for genotyping detection of ten low-risk HPV subtypes: HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, and HPV83. The base sequence of the universal upstream primer is shown in SEQ ID NO:37, the base sequence of the universal downstream primer is shown in SEQ ID NO:38, and the base sequences of the ten probes are shown in SEQ ID NO:41-50, respectively.

[0008] The composition described in this application includes a set of primer compositions and a set of primer-probe compositions. The primer compositions are used to amplify target genes of 18 intermediate- and high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) in the test sample. The product is analyzed using melting curve analysis to determine whether the test sample is infected with the above 18 medium- and high-risk HPV virus subtypes. The primer and probe composition is used to amplify the target genes of HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, and HPV83 in the test sample. The amplification process is analyzed using real-time fluorescent PCR amplification curve analysis to determine whether the test sample may be infected with the above 10 low-risk HPV types.

[0009] The composition described in this application combines real-time fluorescence PCR detection and melting curve analysis technologies. Without requiring upgrades to PCR instrument hardware, it doubles the number of detection targets in a single fluorescence channel. A standard quantitative PCR instrument is sufficient for single-tube, simultaneous detection of 28 HPV subtypes, accurately distinguishing between 18 medium- and high-risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) and 10 low-risk HPV subtypes. Partial typing of subtypes (including HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV83) takes 2 hours. It is easy to operate, does not require opening tubes, and reduces contamination.

[0010] Multiplex PCR, also known as multiplex primer PCR or complex PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments. Its basic principle is the same as conventional PCR, the difference being that multiplex PCR uses two or more pairs of primers, each pair binding to a corresponding site on the template, ultimately amplifying two or more target DNA fragments. Simultaneous detection of multiple pathogens in the same reaction tube significantly saves time, reagents, and costs. However, multiplex PCR is not simply a mixture of multiple pairs of specific primers. The difficulty of multiplex PCR lies in the incompatibility of amplification conditions between multiple targets; each target requires the cooperation of other adjacent primers. The amplification efficiency of different target sequences in the same reaction well is inconsistent, especially with high GC template amplification, where competitive inhibition occurs, resulting in inconsistent copy numbers of single-stranded products from different target sequences. This leads to significantly different detection sensitivities for different target sequences in the same reaction well, limiting the multiplex detection of HPV subtypes. Therefore, simplifying the complexity of the HPV multiplex reaction system while ensuring the performance of HPV multiplex detection is key to solving the application of HPV multiplex detection.

[0011] Therefore, this application employs a special universal upstream and downstream primer design for the 10 HPV virus subtypes (HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, and HPV83) analyzed by fluorescence probe amplification curves. That is, the single-tube fluorescence probe method for detecting the 10 HPV virus subtypes only requires one universal upstream primer and one universal downstream primer to complete the multiplex PCR detection of the 10 HPV virus subtypes. This greatly simplifies the complexity of the HPV multiplex reaction system while ensuring the amplification efficiency of HPV multiplex PCR detection, and at the same time improves the sensitivity and specificity of HPV single-tube multiplex detection.

[0012] In this application, the term "primer" refers to an oligonucleotide that is capable of "initiating" DNA synthesis by a template-dependent DNA polymerase, i.e., the 3'-terminus of the oligonucleotide provides a free 3'-OH group, which can be linked to more "nucleotides" by the template-dependent DNA polymerase to establish a 3' to 5' phosphodiester bond, thereby using deoxynucleoside triphosphate and thereby releasing pyrophosphate.

[0013] In this application, the term "upstream primer" refers to an oligonucleotide that extends continuously along the negative strand; the term "downstream primer" refers to an oligonucleotide that extends continuously along the positive strand. It should be understood that when the designations of the positive and negative strands are interchanged, the corresponding names of the upstream and downstream primers can also be interchanged. That is, the upstream and downstream primers in this application are relative terms.

[0014] In some embodiments, the primer-probe composition further includes a pair of upstream and downstream primers and a probe for detecting the internal standard gene β-globin, wherein the base sequence of the upstream primer is shown in SEQ ID NO:39, the base sequence of the downstream primer is shown in SEQ ID NO:40, and the base sequence of the probe is shown in SEQ ID NO:51.

[0015] In this application, by setting the internal standard gene β-globin, the sampling situation and the entire detection process can be monitored to ensure the accuracy of the detection process.

[0016] In some embodiments, all upstream primers in the 18 pairs of upstream and downstream primers used for genotyping 18 intermediate- and high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) are labeled with a fluorescent quencher at their 5' ends, and a fluorescent reporter group is labeled at the middle position of each upstream primer.

[0017] This application labels each of the 18 upstream primers in the aforementioned 18 pairs of upstream and downstream primers with one fluorescent reporter group and one fluorescent quencher group. After PCR amplification, amplification products with different lengths and base compositions of fluorescent quencher and reporter groups are formed. After amplification, melting curve analysis is performed on the product. As the temperature increases, the fluorescence signal intensity of the product will decrease as it melts. Therefore, a melting peak related to the Tm value of the amplification product can be formed for melting curve analysis, thereby determining whether the sample to be tested is infected with the aforementioned 18 medium- and high-risk HPV virus subtypes.

[0018] In some embodiments, the distance between the fluorescent reporter group and the fluorescent quencher group is 15~18 bp.

[0019] This application optimizes the spacing between the fluorescent reporter group and the fluorescent quencher group, controlling the distance between them to 15-18 bp. This ensures that the fluorescent quencher group does not affect the luminescence effect of the fluorescent reporter group when primer amplification forms double-stranded DNA, resulting in a higher target peak signal in the detection results. At the same time, it ensures that when the amplified product is analyzed by melting curve, as the temperature rises, the DNA melts to form single-stranded DNA. Due to the hairpin structure design, the physical positions of the fluorescent quencher group and the fluorescent reporter group are relatively close, and the fluorescence is fully quenched, greatly reducing the background signal and thus facilitating the interpretation of the detection results, improving the sensitivity and accuracy of the detection results.

[0020] In some implementations, the 5' end of all upstream primers in the 18 pairs of upstream and downstream primers used for genotyping 18 intermediate- and high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) is designed to contain a 4-5 bp sequence complementary to the 3' end of the fluorescent reporter group labeled in the middle position, thus forming a hairpin structure.

[0021] This application designs a 4-5 bp sequence at the 5' end of the upstream primers (18 upstream primers) for 18 intermediate- to high-risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) used for melting curve analysis. This sequence is complementary to the 3' end of the fluorescent reporter group labeled in the middle of the upstream primer. This allows the 18 upstream primers to form a hairpin structure. This structure, on the one hand, makes the Tm value of the primers higher than the annealing temperature of the PCR reaction system by 2~ At 3℃, during annealing, the primer preferentially maintains its hairpin structure and does not form double strands with other multiplex primers, thus preventing primer dimer formation. This ensures that the amplification of different target gene detection primers in the HPV single-tube multiplex reaction system does not interfere with each other, expanding the number of detectable targets and improving HPV multiplex detection capability. On the other hand, this structure design can solve the problem of small signal differences between the amplification products labeled by the fluorescent reporter group and the fluorescent quencher group in the hybridization and free states. The hairpin structure design makes the physical positions of the fluorescent quencher group and the fluorescent reporter group relatively close, and the fluorescence is fully quenched, which greatly reduces the background signal of multiplex detection. This design has obvious advantages for the amplification and melting curve analysis of multiplex primers.

[0022] In some embodiments, upstream primers (first group) for HPV16, HPV18, HPV31, and HPV33 are labeled with the FAM fluorescent reporter group; upstream primers (second group) for HPV39, HPV35, HPV45, HPV52, and HPV51 are labeled with the VIC fluorescent reporter group; upstream primers (third group) for HPV66, HPV26, HPV53, HPV82, and HPV73 are labeled with the ROX fluorescent reporter group; and upstream primers (fourth group) for HPV58, HPV56, HPV59, and HPV68 are labeled with the CY5 fluorescent reporter group.

[0023] This application divides the above 18 upstream primers into 4 groups. The upstream primers in each group are labeled with the same fluorescent reporter group, and the fluorescent reporter groups labeled in different groups are different. This ensures that the fluorescent reporter groups labeled in the primer group do not interfere with each other, and thus different fluorescence channels can be used for melting curve detection.

[0024] In this application, the fluorescence quenching groups labeled on the aforementioned 18 upstream primers can be, for example, BHQ1 or BHQ2.

[0025] In some embodiments, each of the 10 probes is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

[0026] This application uses fluorescent reporter groups labeled at the 5' end and fluorescent quencher groups labeled at the 3' end of the aforementioned 10 probes. During PCR amplification using these fluorescent probes, the probes are cleaved by Taq enzyme during extension, releasing the fluorescent groups into the solution, allowing for real-time detection and the formation of amplification curves. Based on the shape of the fluorescence passing through the amplification curves, it is possible to determine whether the sample may be infected with any of the aforementioned 10 low-risk HPV subtypes.

[0027] In some implementations, the length of each of the 10 probes is independently 13-15 bp.

[0028] This application optimizes the length of the fluorescent probe for amplification curve detection, controlling the distance between the fluorescent reporter group and the fluorescent quencher group to 13-15 bp. This ensures that the fluorescence of the probe before hydrolysis is fully quenched, resulting in a lower fluorescence background and reduced signal interference. This design ensures that even when the probe is not hydrolyzed and hybridizes to the target sequence, the fluorescent quencher group can quench the fluorescence emitted by the fluorescent reporter group. During the melting curve analysis stage, as the temperature increases, the fluorescent probe hybridized with the template will also unwind. However, under the action of its fluorescent quencher group, the fluorescent probe's fluorescence signal is weak whether it is bound to the target sequence or free in solution. The signal intensity of the cleaved fluorescent reporter group free in solution does not change significantly with temperature, thus avoiding a temperature-dependent melting curve. Therefore, the interference of the fluorescent probe signal in the amplification curve on the melting curve analysis results can be resolved, ultimately enabling the identification of one target through the amplification curve and another target through the post-amplification melting curve.

[0029] In some embodiments, when performing amplification curve analysis using the primer-probe composition, the fluorescence acquisition during the amplification phase is set to acquire fluorescence signals during the high-temperature denaturation phase at 92-95°C.

[0030] This application sets the fluorescence acquisition of the amplification stage in the PCR reaction program using the fluorescent probe method for amplification curve analysis to a high-temperature denaturation stage of 92~95℃ (e.g., 95℃). This ensures that even if the primers labeled with fluorescent groups used for melting curve analysis produce products with fluorescent signals during the PCR amplification stage, the fluorescently labeled amplification products are in a dissociated state during real-time fluorescence acquisition due to the high denaturation temperature of 92~95℃ (e.g., 95℃). This is the same as the original single-stranded primer state and will not show an amplification curve that changes with cycle amplification. This makes the detection of the amplification stage and the melting curve stage independent and non-interfering, thus realizing the combined use of real-time fluorescence PCR amplification detection technology and melting curve technology.

[0031] In some implementations, probes targeting HPV6, HPV11, HPV40, and HPV42 (Group 1) are labeled with the FAM fluorescent reporter group; probes targeting HPV43, HPV44, and HPV54 (Group 2) are labeled with the VIC fluorescent reporter group; probes targeting HPV61, HPV81, and HPV83 (Group 3) are labeled with the ROX fluorescent reporter group; and probes targeting the internal standard gene β-globin (Group 4) are labeled with the CY5 fluorescent reporter group.

[0032] This application divides the above-mentioned 10 probes for detecting 10 low-risk HPV virus subtypes and probes for detecting internal standard genes into 4 groups. The fluorescent reporter groups labeled on the probes in each group are the same, and the fluorescent reporter groups labeled on different groups are different, so that the fluorescent reporter groups labeled on the primer probe groups do not interfere with each other, and thus different fluorescence channels can be used for amplification curve detection.

[0033] In this application, the fluorescence quenching group labeled on the above 10 probes can be, for example, MGB.

[0034] The second aspect of this application provides a kit for single-tube genotyping detection of 28 HPV subtypes. The kit includes a PCR reaction solution containing the composition described in the first aspect of this application. The final concentration of each primer in the primer composition in the reaction solution is 0.1-0.3 μM, the final concentration of each primer in the primer-probe composition in the reaction solution is 0.05-0.08 μM, and the final concentration of each probe is 0.02-0.05 μM.

[0035] The kit described in this application contains the composition described in this application. Therefore, using the kit, a single tube of multiplex HPV virus subtypes can be detected using only a conventional real-time PCR instrument. It can accurately distinguish between 18 medium- and high-risk HPV subtypes and 10 low-risk HPV subtypes, which can solve the throughput problem and facilitate rapid promotion and use, and has good application prospects.

[0036] The technical principle of the genotyping detection method using the kit described in this application is as follows: In a monochromatic fluorescence channel, one target is detected and interpreted using an amplification curve, while the other target is analyzed and interpreted using a melting curve. This method is based on two technical principles: the fluorescence signal generated by enzyme hydrolysis probes and the fluorescence changes of amplification products carrying fluorescent labels in double-stranded and unstranded states, to distinguish and detect two targets in the same fluorescence channel. The enzyme hydrolysis probe method is similar to the TaqMan probe method, using unlabeled conventional primers. The probe ends are labeled with a fluorescent group and a quencher group, respectively, with the distance between the two groups within a suitable range. Only during the extension phase, when the fluorescent group at the 5' end of the probe is hydrolyzed by Taq enzyme, does the free fluorescent group emit strong fluorescence. For the other target analyzed using melting curves, the 5' end of the corresponding upstream or downstream specific primer is labeled with a fluorescent quencher group, and the middle position of the primer is labeled with a fluorescent reporter group. The distance between the two groups is adjusted to ensure that the fluorescent quencher group does not affect the luminescence effect of the fluorescent reporter group when the primer amplifies to form double strands. Thus, when the labeled primers amplify to form an amplification product with a fluorescent quencher and a fluorescent reporter group, the fluorescent reporter and the fluorescent quencher are relatively far apart in physical position because the double-stranded DNA is in an extended state. Therefore, the fluorescent signal emitted by the fluorescent reporter can be detected. If this amplification product is heated for detection, the DNA unwinds to form single-stranded DNA as the temperature rises. Due to the molecular flexibility of oligonucleotides, the fluorescent quencher and the fluorescent reporter are relatively close in physical position, and the fluorescence is quenched, resulting in a weakened detection signal. By analyzing the melting curve of this process, the presence of the target can be determined based on the Tm value.

[0037] In some embodiments, the PCR reaction solution further includes PCR buffer, PCR enhancer, dNTPs, and enzyme mixture; the PCR enhancer contains 15-20 mM tetramethylammonium chloride, 1-2 wt% dimethyl sulfoxide, and 0.5-1.0 mg / mL bovine serum albumin; the enzyme mixture contains DNA polymerase and UNG enzyme.

[0038] The PCR buffer described in this application is a conventional buffer, and those skilled in the art can make conventional selections as needed.

[0039] The PCR reaction solution described in this application includes a PCR enhancer with a specific composition. Adding this PCR enhancer to the PCR reaction solution can further improve the efficiency of HPV multiplex PCR and enhance the specificity of the PCR reaction. In some specific embodiments, the PCR enhancer contains 20 mM tetramethylammonium chloride, 2 wt% dimethyl sulfoxide, and 0.8 mg / mL bovine serum albumin.

[0040] In some specific embodiments, the DNA polymerase is Taq DNA polymerase.

[0041] In this application, the Taq DNA polymerase is a thermostable DNA polymerase; the UNG enzyme is uracil-N-glycosylation enzyme, also known as uracil DNA glycosylation enzyme (UDG enzyme), whose function is to selectively hydrolyze and break the uracil glycosidic bonds in double-stranded or single-stranded DNA containing dU, forming DNA strands with missing bases. These strands are further hydrolyzed and broken under alkaline media and high temperatures, thus being eliminated. The optimal activity temperature of the UNG enzyme is 37°C, and it is inactivated at 95°C. By employing Taq enzyme and UNG enzyme, this application can ensure the accuracy of PCR results and prevent non-specific PCR amplification and contamination.

[0042] In this application, the kit may further include positive and negative controls. The positive control may be a DNA fragment of 28 HPV viral subtypes and an internal standard gene, and the negative control may be purified water.

[0043] The kit described in this application can be used to perform single-tube typing detection of 28 HPV virus subtypes in unknown samples such as exfoliated cells on the surface of warts, cervical epithelial cells of women, and genital tract secretions.

[0044] The third aspect of this application provides an application of the kit described in the second aspect of this application for single-tube detection of 28 different HPV viral subtypes, accurate typing of 18 intermediate- and high-risk types, and partial typing of 10 low-risk types. The beneficial technical effects of this application are as follows: This application provides a composition for single-tube genotyping detection of 28 HPV subtypes (including 18 medium- and high-risk types and 10 low-risk types). The composition includes a set of primer compositions and a set of primer-probe compositions. The primer compositions are used to amplify the test sample, and the amplification products are used for melting curve analysis. The primer-probe compositions are used to amplify the test sample, and the amplification process is used for amplification curve analysis. This method for HPV subtype detection using a kit containing the aforementioned composition combines real-time fluorescence PCR detection and melting curve analysis. Without requiring upgrades to PCR instrument hardware, it doubles the number of detection targets in a single fluorescence channel, enabling single-tube detection of 28 HPV subtypes. It accurately distinguishes 18 medium- and high-risk HPV subtypes and partially genotypes 10 low-risk subtypes. The entire process takes 2 hours, is easy to operate, requires no tube opening, reduces contamination, and boasts advantages such as high specificity, short processing time, high sensitivity, and comprehensive detection site coverage. It can provide clinicians with auxiliary diagnostic references, enabling earlier treatment and has wide clinical applications. Attached Figure Description

[0045] Figure 1 This is a schematic diagram illustrating the technical principle of the reagent kit in this application, which uses melting curve analysis to perform single-tube typing detection of 18 medium- and high-risk HPV subtypes.

[0046] Figure 2 This is a schematic diagram illustrating the technical principle of the reagent kit in this application, which uses amplification curve analysis to perform single-tube typing detection of 10 low-risk HPV subtypes.

[0047] Figure 3 The image shows the target melting peak detected in Example 4 using the PCR reaction system with two upstream primers, 18F1 and 18F2, for HPV18.

[0048] Figure 4 The image shows the target melting peak after detection using the PCR reaction system with three upstream primers 18F3, 18F4 and 18F5 for HPV18 in Example 5.

[0049] Figure 5 The figure shows the amplification curves of the multiplex PCR reaction system used in Example 6 to detect 10 low-risk HPV subtypes using primer-probe composition 1 (containing 1 universal upstream primer, 1 universal downstream primer, 10 probes and internal standard gene primers and probes).

[0050] Figure 6 The figure shows the amplification curves of the multiplex PCR reaction system for detecting 10 low-risk HPV subtypes in Example 6, which uses primer-probe composition 2 (containing 10 pairs of separate upstream and downstream primers and 10 probes).

[0051] Figure 7 The image shows the target melting peak of HPV18 subtype after detection using the PCR reaction system containing the HPV6 probe 6P1 in Example 7.

[0052] Figure 8 The image shows the target melting peak of HPV18 subtype after detection using the PCR reaction system containing the HPV6 probe 6P2 in Example 7.

[0053] Figure 9 The image shows the target melting peak of HPV18 subtype after detection using the PCR reaction system containing the HPV6 probe 6P3 in Example 7. Detailed Implementation

[0054] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0055] Example 1: Design of a composition for single-tube genotyping of 28 HPV subtypes The known gene sequences of 18 intermediate- and high-risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82), 10 low-risk HPV subtypes (HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, and HPV83), and an internal control gene were compared and analyzed to obtain their respective differentially expressed gene sequences. Upstream and downstream primers and probes were designed to target these differentially expressed gene sequences. The designed compositions included a primer composition for melting curve analysis and a primer-probe composition for amplification curve analysis.

[0056] Meanwhile, a fluorescent quencher group was labeled at the 5' end of each of the 18 upstream primers targeting 18 high-risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) analyzed by melting curve analysis, and a fluorescent reporter group was labeled at the middle position of each primer. The distance between the fluorescent reporter group and the fluorescent quencher group on each upstream primer was 15-18 bp. In addition, a 4-5 bp sequence was added to the 5' end of the above 18 upstream primer base sequences, which was complementary to the 3' end of the fluorescent reporter group labeled at the middle position of the upstream primer.

[0057] In addition, for the 10 low-risk HPV subtypes (HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, HPV83) analyzed by fluorescent probe amplification curves, a special universal upstream and downstream primer design was adopted. That is, the single-tube fluorescent probe method for detecting the 10 HPV virus subtypes only requires one universal upstream primer and one universal downstream primer. At the same time, the length of the 10 probes for the 10 low-risk HPV subtypes was controlled to 13~15bp, and a fluorescent reporter group was labeled at the 5' end of each of the 10 probes, and a fluorescent quencher group was labeled at the 3' end of each of the 10 probes, so that the distance between the fluorescent reporter group and the fluorescent quencher group on the probe was controlled to 13~15bp.

[0058] The base sequences of each primer in the final primer composition designed for single-tube genotyping of 28 HPV subtypes are shown in Table 1, and the base sequences of each primer and probe in the primer-probe composition are shown in Table 2.

[0059] Table 1: Base sequences of primers in primer compositions used for melting curve analysis

[0060] Table 2: Base sequences of primers and probes in primer-probe compositions used for amplification curve analysis

[0061] Example 2: A kit for single-tube typing detection of 28 HPV viral subtypes The kit consists of the following components: HPV PCR reaction solution: The single-person dosage of the PCR reaction solution consists of 0.5 μL of 10 mM dNTPs and 5 mM Mg2+. 2+ The mixture consisted of 2 μL of 10*Ace Taq buffer (Mg+plus), 2.5 μL of PCR enhancer, 5.0 μL of the PCR enhancer, 7.5 μL of the composition designed and synthesized in Example 1, and purified water to a final volume of 19.7 μL. The PCR enhancer formulation consisted of 20 mM tetramethylammonium chloride (TMAC), 2 wt% dimethyl sulfoxide (DMSO), and 0.8 mg / mL bovine serum albumin (BSA). HPV enzyme mixture: A single dose consists of 0.25 μL of Ace Taq DNA polymerase at a concentration of 5 U / μL for multiplex amplification and 0.05 μL of E. coli UDG enzyme at a concentration of 1 U / μL; Positive control samples: DNA fragments of 28 HPV viral subtypes and internal standard genes; Negative control: purified water.

[0062] Example 3: The kit from Example 2 was used to perform typing detection of 28 HPV viral subtypes in the sample. 1. Reagent preparation (1) Remove the PCR reaction solution from the kit, thaw it at room temperature, shake to mix, and centrifuge briefly to ensure that all liquid on the tube wall is centrifuged to the bottom of the tube. Calculate the number of reactions N required for this experiment (N = number of samples + 1 positive control + 1 negative control), calculate the required volume of PCR reaction solution according to the PCR reaction system preparation table shown in Table 3, prepare the reaction system, and then centrifuge briefly to ensure that all liquid on the tube wall is centrifuged to the bottom of the tube.

[0063] Table 3: Preparation of Single-Person PCR Reaction System

[0064] (2) Dispense the prepared PCR reaction system into N reaction wells at a dispensing volume of 20 μL / well, and transfer the dispensed PCR tubes to the sample preparation area.

[0065] 2. Sample processing and nucleic acid extraction Samples suitable for detection include exfoliated cells from the surface of warts, cervical epithelial cells from women, and unknown samples such as genital tract secretions. Nucleic acid extraction is performed using commercially available HPV nucleic acid extraction or purification reagents, following the instructions. Genomic DNA extracted according to the kit steps can be added immediately or stored at -20±5℃ for later use. Genomic DNA can be stored at -20±5℃ for up to 8 months; avoid repeated freeze-thaw cycles.

[0066] Note: The negative control in this kit should be extracted for nucleic acid simultaneously with the sample; the positive control should not be used for nucleic acid extraction.

[0067] 3. Adding samples (1) Take 5 μL of the extracted genomic DNA into the PCR reaction tube, cap the tube, centrifuge at 1000 rpm or shake gently to remove air bubbles at the bottom of the tube; the template used for the positive control reaction is the positive control in the kit, and the template used for the blank control reaction is the negative control in the kit.

[0068] (2) Cap the PCR reaction tube and record the sample loading details. Transfer the PCR reaction tube to the nucleic acid amplification area for instrumental testing. If the PCR reaction tube cannot be immediately used after adding the template due to unforeseen circumstances, it is recommended to temporarily store the PCR reaction tube with the template added at 2-8 ℃ and test it as soon as possible within 24 hours.

[0069] 4. PCR reaction program settings (nucleic acid amplification area) Place the PCR tubes into the instrument's sample slots, and set the sample names, negative controls, and positive controls in the corresponding order. Set the reaction volume to 25 μL. The amplification curve and melting curve analysis steps are performed as a single program, continuously completed on a fully automated medical PCR analysis system (SLAN-96P, Shanghai Hongshi Medical Technology Co., Ltd.). The parameter settings for the reaction program (PCR reaction program and melting curve analysis program) are shown in Table 4. In this application, the fluorescence acquisition during the amplification stage of the PCR reaction program using the fluorescent probe method for amplification curve analysis is set to acquire fluorescence signals during the 95℃ high-temperature denaturation stage. This ensures that the detection of the amplification stage and the melting curve stage are independent and do not interfere with each other, thereby realizing the combined use of real-time fluorescent PCR amplification detection technology and melting curve technology.

[0070] Table 4: Amplification Cycling Conditions and Fluorescence Acquisition Settings

[0071] 5. Result Interpretation The results of the tests for 18 medium- and high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) were interpreted according to the melting curve analysis method interpretation table for 18 medium- and high-risk HPV genotypes shown in Table 5.

[0072] Interpretation of whether a sample carries one of the 18 intermediate- to high-risk HPV genotypes: If a melting peak is present in the FAM, VIC, ROX, or Cy5 channels within the Tm reference range for a specific genotype, the sample is considered positive for that genotype. If melting peaks are present in two or more Tm reference ranges simultaneously, the sample carries two or more corresponding genotypes. If the CT value of the CY5 channel in the amplification curve is ≤36, and there are no melting peaks within the Tm reference range in the FAM, VIC, ROX, and Cy5 channels of the melting curve, the sample is considered negative for one of the 18 intermediate- to high-risk HPV genotypes. If the sample has no melting peaks in the four fluorescence channels, but the negative control in this test has a specific melting peak, the sample is considered invalid, and resampling or re-extraction is recommended for retesting. If the sample has no melting peaks in the four fluorescence channels, and the positive control in this test has no melting peaks, the kit is considered invalid.

[0073] Interpretation of positive controls: If there is a corresponding melting peak in the FAM, VIC, ROX, and Cy5 channels, the positive control is considered qualified. If there is no melting peak in any fluorescence channel, the kit is considered invalid.

[0074] Interpretation of negative controls: The CT value of the CY5 channel in the amplification curve is ≤41, and there are no melting peaks in any fluorescence channel of the melting curve. Negative controls can be used to control whether aerosol contamination occurred during sample loading. If a melting peak appears in any fluorescence channel of the negative control, it indicates possible nucleic acid contamination in the operating environment, and false positive results may occur in samples tested simultaneously.

[0075] The HPV type in a sample can be determined by using specific detection methods and channels for each HPV subtype. The kit in this application employs melting curve analysis for single-tube genotyping of 18 high-risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82). The technical principle diagram is shown below. Figure 1 As shown.

[0076] Table 5: Interpretation of Melting Curve Analysis for 18 Medium- and High-Risk HPV Genotypes

[0077] The Tm values ​​above are common values ​​obtained from a fully automated medical PCR analysis system (SLAN-96P; Shanghai Hongshi Medical Technology Co., Ltd.) and are used as reference values. The Tm value obtained automatically by the instrument should be considered the definitive value. When the instrument provides more than one Tm value, please refer to the peak shapes of the positive and negative controls to select the valid Tm value. If the instrument cannot automatically provide a Tm value, it can be obtained by adjusting the baseline or through manual interpretation.

[0078] After the experiment is completed, the instrument will provide the corresponding Ct value for each sample. Generally, the results can be judged based on the Ct value and ΔCt value.

[0079] Internal standard result determination: If the CY5 channel of the amplification curve shows a clear logarithmic amplification curve and the CT value is ≤41, and this condition is met, then proceed to determine the results of other channels; if this condition is not met, this experiment is invalid, and it is recommended to retest after confirming the nucleic acid or sample quality.

[0080] The test results for the 10 low-risk HPV subtypes (HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, and HPV83) were interpreted according to the interpretation table of amplification curve analysis for the 10 low-risk HPV subtypes shown in Table 6. Based on the specific detection method and detection channel for each HPV subtype, the type of low-risk HPV infecting the sample can be determined. The technical principle diagram of the kit in this application for single-tube typing detection of 10 low-risk HPV viral subtypes using amplification curve analysis is shown below. Figure 2 As shown.

[0081] Table 6: Interpretation Table of Amplification Curve Analysis for 10 Low-Risk HPV Subtypes

[0082] Example 4: The effect of a specially designed hairpin structure on the upstream primer of the primer composition used for melting curve analysis on the detection results. Test sample: HPV18 positive sample from a hospital.

[0083] The design sequence of the HPV18 downstream primer 18R is the same as the HPV18 downstream primer 18R sequence in Example 1 (sequence number: SEQ ID NO.4).

[0084] HPV18 upstream primer 1, namely 18F1: The base sequence of 18F1 is the same as that of 18F in Example 1 (sequence number: SEQ ID NO. 3). A 4bp sequence is added to the 5' end of its upstream primer, which is complementary to the 3' end of the fluorescent reporter group labeled in the middle of the upstream primer, so as to form a hairpin structure.

[0085] The base sequence of the HPV18 upstream primer 2, i.e., 18F2:18F2, is shown in Table 7 (Sequence number: SEQ ID NO.52). The 5' end of the upstream primer does not have a 4-5 bp sequence that is complementary to the 3' end of the fluorescent reporter group labeled in the middle of the upstream primer. Therefore, the primer structure cannot form a hairpin structure.

[0086] Table 7: Design of hairpin structure in HPV18 upstream primer

[0087] PCR reaction systems containing two upstream primers (18F1 and 18F2) for HPV18 were prepared according to Table 8-9. Amplification was performed under the amplification cycle conditions described in Table 4 of Example 3. The HPV18 positive samples from Example 3 were then tested. The influence of the artificially designed hairpin structure on the upstream primer of the primer composition used for melting curve analysis on the detection results was determined based on the melting peak diagram. The results are as follows: Figure 3 As shown.

[0088] Table 8: PCR reaction system for 18F1

[0089] Table 9: PCR reaction system for 18F2

[0090] Figure 3 The results of the target melting peak diagrams of HPV18 subtypes are presented, obtained by detecting HPV18 subtypes using reaction systems containing two upstream primers, 18F1 and 18F2, respectively. Figure 3It can be seen that, compared to the absence of a specially designed hairpin structure, adding a 4-5 bp sequence complementary to the 3' end of the fluorescent reporter group labeled in the middle of the upstream primer at the 5' end—that is, a specially designed hairpin structure—significantly increases the signal value of the target melting peak and greatly reduces the interference of background signals. This indicates that the hairpin structure formed by adding a 4-5 bp sequence complementary to the 3' end of the fluorescent reporter group labeled in the middle of the upstream primer ensures that during melting curve analysis of the amplified product, as the temperature increases, the DNA unwinds to form single-stranded DNA. The physical positions of the fluorescent quencher and reporter groups are relatively close due to the hairpin structure design, resulting in sufficient fluorescence quenching. This significantly reduces the background signal, increases the signal value of the target melting peak in HPV detection, and improves the sensitivity and accuracy of the detection results.

[0091] Example 5: The effect of the distance between the fluorescence quencher group and the fluorescence reporter group in the upstream primer of the primer composition used for melting curve analysis on the detection results. Test sample: HPV18 positive sample from a hospital.

[0092] The downstream primer sequence for HPV18 is the same as the downstream primer sequence for HPV18 in Example 1 (sequence number: SEQ ID NO. 4).

[0093] The upstream primer 3 for HPV18, namely 18F3, has the same base sequence as the upstream primer sequence for HPV18 in Example 1 (sequence number: SEQ ID NO.3). The distance between the fluorescent reporter group labeled in the middle of the primer and the fluorescent quencher group labeled at the 5' end of the primer is 16 bp.

[0094] The base sequence of the upstream primer 4 of HPV18, namely 18F4:18F4, is shown in Table 10 (sequence number: SEQ ID NO.53). The distance between the fluorescent reporter group labeled in the middle of the primer and the fluorescent quencher group labeled at the 5' end of the primer is 12 bp.

[0095] The base sequence of the upstream primer 5 of HPV18, namely 18F5:18F5, is shown in Table 10 (Sequence number: SEQ ID NO.54). The distance between the fluorescent reporter group labeled in the middle of the primer and the fluorescent quencher group labeled at the 5' end of the primer is 20 bp.

[0096] Table 10: Design of the distance between the fluorescence quencher group and the fluorescence reporter group in the upstream primer of HPV18

[0097] Three upstream primers containing HPV18 (18F3, 18F4, and 18F5) were used, and PCR reaction systems were prepared according to Table 11-13. Amplification was performed according to the amplification cycle conditions in Table 4 of Example 3. Then, the HPV18 positive samples from Example 5 were detected. The influence of the distance between the fluorescence quencher group and the fluorescence reporter group in the upstream primers on the detection results was determined based on the peak diagram. The results are as follows: Figure 4 As shown.

[0098] Table 11: PCR reaction system for 18F3

[0099] Table 12: PCR reaction system for 18F4

[0100] Table 13: PCR reaction system for 18F5

[0101] Figure 4 The results show the target melting peak diagrams of HPV18 subtypes after detection using reaction systems containing three upstream primers (18F3, 18F4, and 18F5) respectively. Figure 4 It can be seen that the signal value of the target melting peak is highest when the distance between the fluorescent quencher and the fluorescent reporter group in 18F3 is 16 bp; while the signal values ​​of the target melting peak decrease significantly when the distance between the fluorescent quencher and the fluorescent reporter group in 18F4 is 13 bp and the distance between the fluorescent quencher and the fluorescent reporter group in 18F5 is 20 bp. This indicates that when the distance between the fluorescent reporter group and the fluorescent quencher group labeled on the upstream primer is controlled at 15-18 bp, it can ensure that the fluorescent quencher group does not affect the luminescence effect of the fluorescent reporter group during melting curve analysis, resulting in a high signal of the target melting peak in the detection results. On the other hand, it can also ensure that the fluorescently labeled single-stranded DNA formed by DNA melting is fully quenched as the temperature increases during melting curve analysis, reducing the interference of background signal, which is beneficial to the interpretation of detection results and improves the sensitivity and accuracy of the detection results.

[0102] Example 6: The effect of using universal upstream and downstream primers in primer-probe compositions for amplification curve analysis on detection results. Test sample: The calibrated concentration was 1×10⁻⁶. 5 Positive plasmids containing 10 low-risk HPV subtypes (HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, HPV83) at copies / mL.

[0103] Primer-probe composition 1: Same as in Example 1; Primer-probe composition 2: For each of the 10 low-risk HPV subtypes to be detected, a pair of separate upstream and downstream primers corresponding to the HPV type are designed in primer-probe composition 2; the probe sequence of primer-probe composition 2 is the same as in Example 1.

[0104] PCR reaction systems containing the two primer / probe compositions mentioned above, namely primer / probe composition 1 and primer / probe composition 2, were prepared according to Table 14-15. The positive plasmids containing 10 low-risk HPV subtypes from Example 6 were then tested. The amplification curves of the detection results were used to determine the impact of using a pair of universal upstream and downstream primers in the primer / probe composition on the detection results. The results are as follows: Figure 5-6 As shown.

[0105] Table 14: PCR reaction system of primer and probe composition 1

[0106] Table 15: PCR reaction system of primer and probe composition 2

[0107] Figure 5 The amplification curves of a multiplex PCR reaction system for detecting 10 low-risk HPV subtypes using a reaction system containing primer-probe composition 1 (containing one universal upstream primer and one universal downstream primer) are shown. Figure 6 The amplification curves of a multiplex PCR reaction system using primer-probe composition 2 (containing 10 pairs of individual upstream and downstream primers) to detect 10 low-risk HPV subtypes are shown. Figure 5 and 6 It is evident that amplification using the universal primer design scheme described in this application resulted in higher signal values ​​in the amplification curve, reduced background signal interference, and improved the accuracy of the detection results. Therefore, the upstream and downstream primer design described in this application can significantly simplify the complexity of the HPV multiplex PCR reaction system and improve the sensitivity and specificity of HPV single-tube multiplex detection.

[0108] Example 7: The interference effect of the spacing between the fluorescent reporter group and the fluorescent quencher group on the probe in the primer-probe composition used for amplification curve analysis on the melting curve detection results. Test samples: Positive samples from a hospital with mixed infection of high-risk HPV18 and low-risk HPV6.

[0109] The upstream and downstream primer sequences for HPV6 are shown in Table 16 (sequence numbers: SEQ ID NO.57 and 58).

[0110] The base sequence of the HPV6 probe 6P1 is the same as the HPV6 probe sequence in Example 1 (sequence number: SEQ ID NO. 41), and the distance between the fluorescent group and the quencher group of this probe is 14 bp.

[0111] The base sequence of HPV6 probe 6P2 is shown in Table 16 (sequence number: SEQ ID NO.55). The distance between the fluorescent group and the quencher group of this probe is 17 bp.

[0112] The base sequence of HPV6 probe 6P3 is shown in Table 16 (Sequence number: SEQ ID NO.56). The distance between the fluorescent group and the quencher group of this probe is 20 bp.

[0113] The upstream and downstream primer sequences for HPV18 are the same as those for HPV18 in Example 1 (sequence numbers: SEQ ID NO.3 and 4). Table 16: Design of the spacing between the fluorescence quencher group and the fluorescence reporter group in the HPV6 fluorescent probe

[0114] Using three probes containing HPV6 (6P1, 6P2, and 6P3), a multiplex PCR reaction system was prepared according to Table 17-19. Amplification was performed according to the amplification cycle conditions in Table 4 of Example 3. Then, the positive samples from Example 7, which contained a mixed infection of high-risk HPV18 and low-risk HPV6, were tested. The peak diagram of the detection results is shown below. Figure 7-9 As shown in the figure. Based on the peak diagram of the detection results, the interference effect of the spacing between the fluorescent reporter group and the fluorescent quencher group on the probe in the primer-probe composition used for amplification curve analysis on the melting curve detection results is determined.

[0115] Table 17: PCR reaction system for 6P1

[0116] Table 18: PCR reaction system for 6P2

[0117] Table 19: PCR reaction system for 6P3

[0118] Figure 7-9 The results show the target melting peak diagrams of HPV18 subtypes after detection using reaction systems containing three probes (6P1, 6P2, and 6P3) specifically for HPV6. Figure 7-9It can be seen that when the distance between the fluorescent quencher group and the fluorescent reporter group in 6P1 is 14 bp, the signal value of the target melting peak of HPV18 subtype is the highest, and no non-specific melting peak caused by the 6P1 fluorescent probe appears. However, when the distance between the fluorescent quencher group and the fluorescent reporter group in 6P2 is 17 bp and the distance between the fluorescent quencher group and the fluorescent reporter group in 6P3 is 20 bp, the signal value of the target melting peak decreases significantly, and non-specific melting peaks caused by the 6P2 and 6P3 fluorescent probes appear. This indicates that by optimizing the probe length and controlling the distance between the fluorescent reporter group and the fluorescent quencher group to 13-15 bp, the fluorescence of the unhydrolyzed fluorescent probe can be fully quenched, resulting in a lower fluorescence background. This ensures that the fluorescent probe, whether bound to the target sequence or free in solution, will not exhibit a melting curve that changes with temperature, reducing non-specific signal interference and improving the sensitivity and accuracy of the detection results. Therefore, the cross-interference of fluorescent probe signals in the amplification curve with the analysis results of the melting curve stage can be solved, and ultimately, it is possible to identify one target by the amplification curve and another target by the melting curve after amplification.

[0119] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A composition for single-tube genotyping of 28 HPV subtypes, characterized in that, The composition includes a primer composition for interpreting detection results using a melting curve and a primer-probe composition for interpreting detection results using an amplification curve; The primer composition comprises 18 pairs of upstream and downstream primers for genotyping 18 intermediate- to high-risk HPV subtypes: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82. The base sequences of the 18 pairs of upstream and downstream primers are shown in SEQ ID NO: 1-36, respectively. The primer-probe composition comprises one universal upstream primer, one universal downstream primer, and ten probes for genotyping detection of ten low-risk HPV subtypes: HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, and HPV83. The base sequence of the universal upstream primer is shown in SEQ ID NO:37, the base sequence of the universal downstream primer is shown in SEQ ID NO:38, and the base sequences of the ten probes are shown in SEQ ID NO:41-50. The 18 pairs of upstream and downstream primers used for genotyping 18 medium- and high-risk HPV subtypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) all have a fluorescent quencher group labeled at the 5' end and a fluorescent reporter group labeled at the middle position. The distance between the fluorescent reporter group and the fluorescent quencher group is 15~18 bp.

2. The composition according to claim 1, characterized in that, The primer-probe composition further includes a pair of upstream and downstream primers and a probe for detecting the internal standard gene β-globin, wherein the base sequence of the upstream primer is shown in SEQ ID NO:39, the base sequence of the downstream primer is shown in SEQ ID NO:40, and the base sequence of the probe is shown in SEQ ID NO:

51.

3. The composition according to claim 1, characterized in that, The 18 pairs of upstream and downstream primers used for genotyping 18 intermediate- to high-risk HPV subtypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, and HPV82) all contain a 4-5 bp sequence at their 5' end that is complementary to the 3' end of the fluorescent reporter group labeled in the middle position, forming a hairpin structure.

4. The composition according to claim 1, characterized in that, The upstream primers for HPV16, HPV18, HPV31, and HPV33 were all labeled with the FAM fluorescent reporter group; the upstream primers for HPV39, HPV35, HPV45, HPV52, and HPV51 were all labeled with the VIC fluorescent reporter group; the upstream primers for HPV66, HPV26, HPV53, HPV82, and HPV73 were all labeled with the ROX fluorescent reporter group; and the upstream primers for HPV58, HPV56, HPV59, and HPV68 were all labeled with the CY5 fluorescent reporter group.

5. The composition according to claim 1 or 2, characterized in that, Each of the 10 probes has a fluorescent reporter group labeled at its 5' end and a fluorescent quencher group labeled at its 3' end.

6. The composition according to claim 1 or 2, characterized in that, When performing amplification curve analysis using the primer-probe composition, the fluorescence acquisition during the amplification stage is set to acquire fluorescence signals during the high-temperature denaturation stage at 92~95℃.

7. The composition according to claim 1 or 2, characterized in that, Probes targeting HPV6, HPV11, HPV40, and HPV42 are all labeled with the FAM fluorescent reporter group; probes targeting HPV43, HPV44, and HPV54 are all labeled with the VIC fluorescent reporter group; probes targeting HPV61, HPV81, and HPV83 are all labeled with the ROX fluorescent reporter group; and probes targeting the internal standard gene β-globin are labeled with the CY5 fluorescent reporter group.

8. A kit for single-tube genotyping detection of 28 HPV subtypes, characterized in that, The kit includes a PCR reaction solution, which includes the composition as described in any one of claims 1-7; the final concentration of each primer in the primer composition in the reaction solution is 0.1-0.3 μM, the final concentration of each primer in the primer-probe composition in the reaction solution is 0.05-0.08 μM, and the final concentration of each probe is 0.02-0.05 μM.

9. The reagent kit according to claim 8, characterized in that, The PCR reaction solution also includes PCR buffer, PCR enhancer, dNTPs and enzyme mixture; the PCR enhancer contains 15-20 mM tetramethylammonium chloride, 1-2 wt% dimethyl sulfoxide and 0.5-1.0 mg / mL bovine serum albumin; the enzyme mixture contains DNA polymerase and UNG enzyme.

10. The application of the kit as described in claim 8 or 9 in the preparation of a reagent for single-tube detection of 28 different HPV viral subtypes, said application being for accurate typing of 18 intermediate- and high-risk types and partial typing of 10 low-risk types.

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