CN116606964A

CN116606964A - Composition and kit for single-tube typing detection of 28 HPV subtypes

Info

Publication number: CN116606964A
Application number: CN202310698056.8A
Authority: CN
Inventors: 赵瑞瑞; 童京京; 刘世娇; 刘勋; 李博茹; 张辉
Original assignee: Henan Huayuan Biotechnology Co ltd
Current assignee: Henan Huayuan Biotechnology Co ltd
Priority date: 2023-06-13
Filing date: 2023-06-13
Publication date: 2023-08-18

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 for judging a detection result by adopting a melting curve and a primer probe composition for judging a detection result by adopting 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 respectively shown as SEQ ID NO. 1-36; the primer probe group comprises 1 general upstream primer, 1 general downstream primer and 10 probes; the base sequence of the 1 general upstream primer is shown as SEQ ID NO. 37, the base sequence of the 1 general downstream primer is shown as SEQ ID NO. 38, and the base sequence of the 10 probes is shown as SEQ ID NO. 41-50. By utilizing the specific composition, 28 different HPV virus subtypes can be detected in one tube, and the combined analysis of a real-time fluorescence PCR amplification detection technology and a melting curve technology is realized, so that the detection flux is high, the specificity is strong, the time consumption is short and the sensitivity is high.

Description

Composition and kit for single-tube typing detection of 28 HPV subtypes

Technical Field

The application relates to the technical field of nucleic acid detection, in particular to a composition and a kit for single-tube typing detection of 28 HPV subtypes.

Background

Cervical cancer is the most common malignant tumor of female genital tract, the incidence rate is the second in the malignant tumor of female, the new cases of China are 13.15 ten thousand per year, the death number of cervical cancer is about 5.3 ten thousand per year, the death number of cervical cancer accounts for 18.4% of the death number of all the malignant tumor of female, the tendency of younger age is obvious, and the cases are increased in proportion of 2% per year. Early detection and treatment of cervical cancer is critical for effective treatment of cervical cancer.

Human papilloma virus (Human Papillomavirus, HPV) belongs to the papilloma virus family and is a small-molecule, non-enveloped circular double-stranded DNA virus. For HPV infecting genital tract and anus, the pathogenic or carcinogenic risk is classified into two major categories, low-risk and high-risk, depending on the genotype. Genital HPV infection is prevalent in women with a history of sexual life, and it is counted that 70% to 80% of women will have at least one HPV infection during their lifetime, but most infections are self-limiting, and over 90% of infected women will develop an effective immune response that will clear the infection between 6 and 24 months without any long-term healthy intervention. Persistent high-risk HPV infection is a major cause of cervical intraepithelial neoplasia and cervical cancer. The results of the global study showed that the presence of high-risk HPV DNA was detected in 99.7% of cervical cancer patients, with HPV type 16, 18, 45 and 31 infections accounting for 80%. Low-risk HPV is generally associated with condyloma acuminatum or low-grade squamous intraepithelial lesions. Therefore, the rapid and accurate detection of HPV high-risk infection has important significance for early treatment, reduction of cervical cancer morbidity and mortality, and the like.

Along with the deepening of knowledge of cervical cancer caused by HPV infection and the development of molecular diagnosis technology, HPV gene detection is widely applied as a screening means of cervical cancer. Compared with the traditional cytological detection mode, the molecular detection method does not need to wait until the change of cells to identify HPV infection, and can screen the virus at the earliest stage of virus infection in the incubation period of virus infection cells but no symptoms, thereby achieving the aim of early prevention and early treatment.

Current HPV genotyping methods, such as in situ hybridization techniques: the nucleic acid probe (DNA or RNA) and the sample are subjected to in-situ hybridization reaction, and the in-situ hybridization has the advantages of positioning suspicious cells and simultaneously having semi-quantitative function; the disadvantages are low sensitivity, high sample quality requirements, high labor capacity, high cost, and the need for corresponding probes for each type of HPV. PCR reverse dot hybridization: PCR in vitro amplification and DNA reverse dot hybridization were combined. The specific primer is designed by utilizing the gene characteristics of HPV, a target fragment containing HPV genotypes can be amplified, the amplified product is hybridized with a parting probe fixed on a membrane strip, and whether the HPV genotypes exist or not is judged according to the existence of hybridization signals, so that the method can be used for auxiliary diagnosis of clinical HPV infection. The greatest disadvantage of this method is that contamination is likely to occur during hybridization, thereby affecting the judgment of the result. Flow fluorescent hybridization method: the technology hybridizes the PCR amplified product with a fluorescent marker probe crosslinked on the microsphere, and finally detects a fluorescent signal on a multifunctional flow dot matrix instrument. The method has the disadvantages of complex and complicated operation, high price and need to be operated by a professional technician. The PCR-Taqman MGB probe typing method is a detection method commonly used at present due to the characteristics of simple operation, accurate typing, whole-course tube closing operation and the like. However, due to the limitation of the instrument acquisition signal channel, only limited HPV types can be detected in each reaction, the flux is low, and the economic cost and the time cost of detection are greatly increased when multi-tube detection is adopted. Melting curve method: the probe marked with fluorescent group and quenching group is added into PCR system, the single-chain oligonucleotide sequence complementary with probe sequence is amplified in PCR process, after amplification is completed, melting curve analysis process is added to obtain melting curve, and melting points (Tm values) of various types are obtained, but the defects are that melting point ranges of melting peaks between various targets are relatively close, adjacent melting peaks are easy to interfere with each other, fusion peaks appear, and the like.

Therefore, the method for accurately and rapidly detecting the HPV can reduce the consumption of manpower, material resources and financial resources, greatly reduces the complexity of PCR reaction and has practical significance.

Disclosure of Invention

In order to solve the defects in the prior art, the application provides a composition for single-tube typing detection of 28 HPV subtypes (including 18 medium-high-risk types and 10 low-risk types), wherein the composition comprises a primer composition for judging a set of detection results by using a melting curve and a primer probe composition for judging a set of detection results by using an amplification curve, the primer composition is used for amplifying a sample to be detected, an amplification product is used for melting curve analysis, the primer probe composition is used for amplifying the sample to be detected, and an amplification process is used for amplification curve analysis. By utilizing the composition, 28 different HPV virus subtypes can be detected in one tube, 18 medium-high risk types are accurately typed, 10 low risk types are partially typed, and the combined analysis of a real-time fluorescence PCR amplification detection technology and a melting curve technology is realized.

To this end, the first aspect of the present application provides a composition for single-tube typing detection of 28 HPV subtypes, the composition comprising a primer composition whose detection results are interpreted using a melting curve and a primer probe composition whose detection results are interpreted using an amplification curve;

the primer composition comprises 18 pairs of upstream and downstream primers for typing detection of high-risk HPV subtypes in 18 types of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV 82; the base sequences of the 18 pairs of upstream and downstream primers are respectively shown as SEQ ID NO. 1-36;

the primer probe composition comprises 1 universal upstream primer, 1 universal downstream primer and 10 probes for typing 10 low-risk HPV subtypes of HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV 83; the base sequence of the 1 general upstream primer is shown as SEQ ID NO. 37, the base sequence of the 1 general downstream primer is shown as SEQ ID NO. 38, and the base sequence of the 10 probes is shown as SEQ ID NO. 41-50.

The composition comprises a group of primer compositions and a group of primer probe compositions, wherein the primer compositions are used for amplifying target genes of 18 types of high-risk HPV in HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV82 in a sample to be detected, and the amplified products are analyzed by adopting a melting curve so as to judge whether the 18 types of high-risk HPV virus are infected in the sample to be detected; the primer probe composition is used for amplifying target genes of HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV83 in a sample to be detected, and real-time fluorescence PCR amplification curve analysis is adopted in the amplification process, so that whether the 10 low-risk HPVs are possibly infected in the sample to be detected is judged.

According to the composition, two technologies of real-time fluorescence PCR detection and melting curve analysis are combined, under the condition that hardware upgrading of a PCR instrument is not needed, the detection target number of a single fluorescent channel can be doubled, 28 types of HPV subtypes can be detected at one time by only using a conventional fluorescence quantitative PCR instrument, 18 types of medium-high-risk types (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV 82) can be accurately distinguished, and 10 types of low-risk types (including HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV 83) can be partially typed, the whole process takes 2 hours, the operation is easy, the tube opening is not needed, and the pollution is reduced.

The multiplex PCR (multiplex PCR), also called multiplex primer PCR or composite PCR, is a PCR reaction in which more than two pairs of primers are added in the same PCR reaction system, and simultaneously multiple nucleic acid fragments are amplified, and its basic principle is identical to that of conventional PCR, and is characterized by that more than two pairs of primers are added in the multiplex PCR reaction system, and each pair of primers is respectively combined with correspondent position of template, so that more than two target DNA fragments can be finally amplified. Multiple pathogens are detected in the same reaction tube at the same time, so that the time and the reagent are greatly saved, and the expense is saved. Multiplex PCR, however, is not a simple mixing of pairs of specific primers into one system. Multiplex PCR is difficult because of the incompatibility of amplification conditions between multiple targets, each requiring additional primer matching alongside. The amplification efficiency of different target sequences in the same reaction well is inconsistent, particularly the problem of high GC template amplification, mutual competitive inhibition exists, so that the copy numbers of single-stranded products of different target sequences are inconsistent, the detection sensitivity of different target sequences in the same reaction well is obviously different, and the multiplex detection of HPV virus subtypes is limited. Therefore, how to simplify the complexity of the HPV multiplex reaction system and ensure the performance of HPV multiplex detection is the key for solving the application of HPV multiplex detection.

Therefore, the application adopts the design of special general upstream and downstream primers for the 10 HPV virus subtypes (HPV 6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV 83) analyzed by the fluorescent probe amplification curve, namely the multiplex PCR detection of the 10 HPV virus subtypes can be completed by only 1 general upstream primer and 1 general downstream primer for the single tube fluorescent probe detection of the 10 HPV virus subtypes, thereby greatly simplifying the complexity of an HPV multiplex reaction system, ensuring the amplification efficiency of HPV multiplex PCR detection and improving the sensitivity and specificity of HPV single tube multiplex detection.

In the present application, the term "primer" means such an oligonucleotide: it is capable of "priming" DNA synthesis by a template dependent DNA polymerase, i.e. e.g. the 3 '-end of an oligonucleotide provides a free 3' -OH group to which more "nucleotides" can be attached by the template dependent DNA polymerase, creating a 3 'to 5' phosphodiester linkage, thereby using deoxynucleoside triphosphates, and thereby releasing pyrophosphate.

In the present application, the term "upstream primer" is an oligonucleotide that extends uninterruptedly along the negative strand; the term "downstream primer" is an oligonucleotide that extends uninterrupted along the forward strand. It will be appreciated that when the designations of forward and reverse strands are interchanged, the designations of the corresponding upstream and downstream primers may also be interchanged. That is, the upstream primer and the downstream primer in the present application are relatively speaking.

In some embodiments, the primer probe composition further comprises 1 pair of upstream primer and downstream primer for detecting the internal standard gene beta-globin, wherein the base sequence of the upstream primer is shown as SEQ ID NO. 39, the base sequence of the downstream primer is shown as SEQ ID NO. 40, and the base sequence of the probe is shown as SEQ ID NO. 51. .

According to the application, the sampling condition and the detection process of the whole flow can be monitored by setting the internal standard gene beta-globin, so that the accuracy of the detection process is ensured.

In some embodiments, the 5' ends of all upstream primers in 18 pairs of upstream and downstream primers used for typing detection of high-risk HPV genotypes among 18 types of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV82 are each labeled with 1 fluorescence quenching group, and the intermediate positions of the upstream primers are each labeled with 1 fluorescence reporting group.

According to the application, 1 fluorescence report group and 1 fluorescence quenching group are marked on 18 upstream primers of the 18 pairs of upstream and downstream primers, amplification products with different lengths and base compositions and marked with the fluorescence quenching groups and the fluorescence report groups can be formed after PCR amplification, after amplification is completed, melting curve analysis is carried out on the products, and along with the rise of temperature, the fluorescence signal intensity of the products is weakened along with melting, so that melting peaks related to Tm values of the amplification products can be formed for melting curve analysis, and further whether the 18 high-risk HPV virus subtypes are infected in a sample to be detected is judged.

In some embodiments, the distance between the fluorescent reporter group and the fluorescence quenching group is 15-18 bp.

According to the application, the distance between the fluorescent reporter group and the fluorescent quenching group is controlled to be 15-18 bp by optimizing the distance between the fluorescent reporter group and the fluorescent quenching group, so that the fluorescent quenching group can not influence the luminous effect of the fluorescent reporter group when the primer is amplified to form double-chain DNA, the target peak signal of a detection result is higher, and simultaneously, when the amplified product is subjected to melting curve analysis, the DNA is melted to form single-chain DNA along with the rise of temperature, the physical positions of the fluorescent quenching group and the fluorescent reporter group are relatively close due to the design of a hairpin structure, and fluorescence is sufficiently quenched, so that the detection background signal is greatly weakened, further, the interpretation of the detection result is facilitated, and the sensitivity and the accuracy of the detection result are improved.

In some embodiments, the 5 'ends of all upstream primers of 18 pairs of upstream and downstream primers for typing detection of high-risk HPV genotypes among 18 of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV82 are designed to contain a sequence of 4-5 bp in length complementary pairing to the 3' end of the centrally-labeled fluorescent reporter group to form a hairpin structure.

According to the application, by designing a sequence which is 4-5 bp long and is complementarily paired with the 3 'end of a fluorescent reporter group marked at the middle position of the upstream primer at the 5' end of the upstream primer (18 upstream primers) 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 HPV 82) for melting curve analysis, the above 18 upstream primers can form a hairpin structure, on one hand, the Tm value of the primers is higher than the annealing temperature of a PCR reaction system by 2-3 ℃, and the primers form self-formed hairpin structure preferentially in the annealing process without forming double chains with other multiplex primers, so that primer dimers are not formed, the amplification of different target gene detection primers in the single tube multiplex reaction system can not generate interference, the detectable number is expanded to more, and the multiplex detection capability is improved; on the other hand, the design of the structure can solve the problem that the signal difference of the amplified products marked by the fluorescence reporting group and the fluorescence quenching group is smaller in a hybridization state and a free state, the physical positions of the fluorescence quenching group and the fluorescence reporting group are relatively close, fluorescence is sufficiently quenched, the background signal of multiplex detection is greatly weakened, and the design has obvious advantages for amplification of multiplex primers and melting curve analysis.

In some embodiments, FAM fluorescent reporter groups are labeled on the upstream primers (first set) for HPV16, HPV18, HPV31 and HPV33, VIC fluorescent reporter groups are labeled on the upstream primers (second set) for HPV39, HPV35, HPV45, HPV52 and HPV51, ROX fluorescent reporter groups are labeled on the upstream primers (third set) for HPV66, HPV26, HPV53, HPV82 and HPV73, and CY5 fluorescent reporter groups are labeled on the upstream primers (fourth set) for HPV58, HPV56, HPV59 and HPV 68.

The application divides the 18 upstream primers into 4 groups, the fluorescence reporter groups marked on the upstream primers in each group are the same, and the fluorescence reporter groups marked among different groups are different, so that the fluorescence reporter groups marked in the primer groups are not interfered with each other, and further, different fluorescence channels can be used for detecting melting curves.

In the present application, the fluorescence quenching groups labeled on the 18 upstream primers may be, for example, BHQ1 or BHQ2.

In some embodiments, the 5 'ends of the 10 probes are each labeled with 1 fluorescent reporter group, and the 3' ends are each labeled with 1 fluorescent quencher group.

According to the application, the 5 'ends of the 10 probes are marked with fluorescent reporter groups, the 3' ends are marked with fluorescent quenching groups, and when the fluorescent probes are used for PCR amplification, the fluorescent probes are cut by Taq enzyme and release the fluorescent groups into solution in the extending process, so that real-time detection can be performed, and an amplification curve is formed. Thus, according to the shape of the fluorescence passing internal amplification curve, whether the sample to be tested is likely to be infected with the 10 low-risk HPV virus subtypes can be judged.

In some embodiments, the 10 probes are each independently 13 to 15bp in length.

According to the probe for detecting the amplification curve, the length of the fluorescent probe is optimized, and the distance between the fluorescent reporter group and the fluorescent quenching group is controlled to be 13-15 bp, so that the probe fluorescence when not hydrolyzed can be sufficiently quenched, a lower fluorescence background is presented, and signal interference is reduced. The design can ensure that when the probe is not hydrolyzed, even if hybridized on a target sequence, the fluorescent quenching group can quench fluorescence emitted by the fluorescent reporter group, and the fluorescent probe hybridized with the template can be melted along with the rise of temperature in the analysis stage of a melting curve, but under the action of the fluorescent quenching group, the fluorescent probe is combined on the target sequence or is free in solution, the signal of the fluorescent group is weaker, and the signal intensity of the fluorescent reporter group which is cut and free in solution is not changed obviously along with the change of temperature, so that the melting curve graph along with the change of temperature is not displayed. Therefore, the interference of fluorescent probe signals in the amplification curve on analysis results in the melting curve stage can be solved, and finally, one target can be distinguished through the amplification curve graph, and the other target can be distinguished through the amplified melting curve graph.

In some embodiments, the fluorescence acquisition during the amplification phase is set to acquire a fluorescence signal during the high temperature denaturation phase at 92-95 ℃ when the primer probe composition is used for amplification curve analysis.

The application sets the fluorescence collection of the amplification stage to collect fluorescence signals in the high-temperature denaturation stage of 92-95 ℃ (such as 95 ℃), ensures that the fluorescent group-marked primer for melting curve stage analysis generates a product with fluorescence signals in the PCR amplification stage, but when in real-time fluorescence collection, the amplified product with fluorescent marks is in a melting state as the high-temperature denaturation temperature of 92-95 ℃ (such as 95 ℃) is higher, and does not present an amplification curve changing along with the cyclic amplification as the original single-stranded primer, so that the detection of the amplification stage and the melting curve stage are mutually independent and do not interfere, thereby realizing the combined use of the real-time fluorescence PCR amplification detection technology and the melting curve technology.

In some embodiments, probes (first set) for HPV6, HPV11, HPV40 and HPV42 are each labeled with a FAM fluorescent reporter, probes (second set) for HPV43, HPV44 and HPV54 are each labeled with a VIC fluorescent reporter, probes (third set) for HPV61, HPV81 and HPV83 are each labeled with a ROX fluorescent reporter, and probes (fourth set) for the internal standard gene β -globin are each labeled with a CY5 fluorescent reporter.

The application divides the 10 probes for detecting the 10 low-risk HPV virus subtypes and the probes for detecting the internal standard genes into 4 groups, wherein the fluorescent reporter groups marked on the probes in each group are the same, and the fluorescent reporter groups marked among the different groups are different, so that the fluorescent reporter groups marked in the primer probe groups are not interfered with each other, and further, different fluorescent channels can be utilized for detecting an amplification curve.

In the present application, the fluorescence quenching group labeled on the 10 probes may be, for example, MGB.

In a second aspect, the application provides a kit for single-tube typing detection of 28 HPV subtypes, which comprises a PCR reaction solution, wherein the PCR reaction solution comprises the composition according to the first aspect of the application, the final concentration of each primer in a primer composition in the reaction solution is 0.1-0.3 mu M, the final concentration of each primer in a primer probe composition in the reaction solution is 0.05-0.08 mu M, and the final concentration of each probe is 0.02-0.05 mu M.

The kit provided by the application comprises the composition, so that the kit can be used for detecting single-tube multiple HPV virus subtypes by using a conventional fluorescent quantitative PCR instrument, 18 medium-high-risk types and 10 low-risk types of HPV subtypes can be accurately distinguished, the flux problem can be solved, the rapid popularization and the use are convenient, and the kit has a good application prospect.

The technical principle of the method for parting detection by using the kit of the application is as follows: in the monochromatic fluorescent channel, one target is judged by adopting amplification curve detection, and the other target is judged by adopting melting curve analysis. The method is based on two technical principles of generating fluorescent signals by an enzyme hydrolysis probe and fluorescence change of amplification products carrying fluorescent markers in double-chain and melting states, and is used for distinguishing and detecting two targets in the same fluorescent channel. The method for hydrolyzing the probe by the enzyme is the same as the TaqMan probe method, the primer is a conventional primer without a mark, the two ends of the probe are respectively marked with a fluorescent group and a quenching group, the distance between the two groups is in a proper range, and the free fluorescent group can emit stronger fluorescence only when the fluorescent group at the 5' end of the probe is hydrolyzed by the Taq enzyme in the extension stage. And the other target analyzed by adopting a melting curve is characterized in that the 5' end of the corresponding upstream or downstream specific primer is marked with a fluorescence quenching group, the middle position of the primer is marked with a fluorescence reporting group, and the distance between the two groups is adjusted to a proper position so as to ensure that the fluorescence quenching group does not influence the luminous effect of the fluorescence reporting group when the primer is amplified to form a double chain. Thus, when the labeled primer is amplified to form an amplification product with a fluorescence quenching group and a fluorescence reporter group, the fluorescence signal emitted by the fluorescence reporter group can be detected because the double-stranded DNA is in an extended state, the fluorescence reporter group is in a relatively remote physical position from the fluorescence quenching group; if the amplification product is subjected to temperature rise detection, DNA is melted to form single-stranded DNA along with the temperature rise, due to the molecular flexibility of the oligonucleotide, the physical positions of the fluorescence quenching group and the fluorescence reporting group are relatively close, fluorescence is quenched, so that a detection signal is weakened, and if the melting curve analysis is performed in the process, whether a target exists or not can be determined according to a Tm value.

In some embodiments, the PCR reaction solution further comprises a PCR buffer, a PCR enhancer, dNTPs, and an enzyme cocktail; the PCR synergist 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.

The PCR buffer described in the present application is a conventional buffer, and those skilled in the art can perform conventional selection as needed.

The PCR reaction liquid comprises the PCR synergist with specific composition, and the PCR reaction liquid is added with the PCR synergist, so that the efficiency of HPV multiplex PCR reaction and the specificity of the PCR reaction can be further improved. In some embodiments, the PCR synergist comprises 20mM tetramethylammonium chloride, 2wt% dimethyl sulfoxide, and 0.8mg/mL bovine serum albumin.

In some embodiments, the DNA polymerase is Taq DNA polymerase.

In the application, the Taq DNA polymerase is a DNA polymerase with thermal stability; the UNG enzyme is uracil-N-glycosylase, also known as uracil DNA glycosylase (UDG enzyme), and functions to selectively hydrolyze uracil glycosidic bonds in double-stranded or single-stranded DNA containing dU, and the DNA strand formed with the missing base is further hydrolyzed and cleaved under alkaline medium and high temperature, thereby being eliminated. The optimal activation temperature of UNG enzyme is 37℃and 95℃for inactivation. The application can ensure the accuracy of PCR results and prevent non-specific PCR amplification and pollution by adopting Taq enzyme and UNG enzyme.

The kit can also comprise a positive quality control product and a negative quality control product. The positive quality control product can be DNA fragments of 28 HPV virus subtypes and internal standard genes, and the negative quality control product can be purified water.

The kit can be used for single-tube typing detection of 28 HPV virus subtypes in unknown samples such as wart surface exfoliated cells, female cervical epithelial cells, genital secretion and the like.

The third aspect of the application provides a kit for detecting 28 different HPV virus subtypes in a single tube, accurately typing 18 medium-high risk types and partially typing 10 low risk types, which has the following beneficial technical effects: the application provides a composition for single-tube typing detection of 28 HPV subtypes (including 18 medium-high-risk types and 10 low-risk types), wherein the composition comprises a group of primer compositions and a group of primer probe compositions, the primer compositions are used for amplifying a sample to be detected, an amplified product is used for melting curve analysis, the primer probe compositions are used for amplifying the sample to be detected, and an amplification process is used for amplification curve analysis. The method for typing and detecting HPV subtypes by using the kit containing the composition combines two technologies of real-time fluorescence PCR detection and melting curve analysis, can double the number of detection targets of a single fluorescence channel under the condition of no need of hardware upgrading of a PCR instrument, realizes single-tube disposable detection of 28 HPV subtypes, accurately distinguishes 18 medium-high-risk HPV subtypes and carries out partial typing on 10 low-risk types, takes 2 hours in the whole process, is easy to operate, does not need to open a tube, reduces pollution, has the advantages of high specificity, short time consumption, high sensitivity, comprehensive coverage of detection sites and the like, can provide auxiliary diagnosis reference for clinicians, carries out treatment in advance, and has wide clinical application.

Drawings

FIG. 1 is a schematic diagram of a single tube typing detection technique for 18 medium-high risk HPV subtypes using melting curve analysis in the kit of the present application.

FIG. 2 is a schematic diagram of the single tube typing detection of 10 low-risk HPV subtypes using amplification curve analysis in the kit of the present application.

FIG. 3 is a graph showing melting peaks of targets detected by a PCR reaction system using two upstream primers 18F1 and 18F2 of HPV18 in example 4.

FIG. 4 is a graph showing the melting peaks of the targets detected by the PCR reaction system using the three upstream primers 18F3, 18F4 and 18F5 of HPV18 in example 5.

FIG. 5 is a graph showing the amplification curve of a multiplex PCR reaction system for detecting 10 low-risk HPV subtypes using the reaction system comprising primer probe composition 1 (comprising 1 universal upstream primer, 1 universal downstream primer, 10 probes, and an internal standard gene primer probe) in example 6.

FIG. 6 is a graph showing the amplification curve of a multiplex PCR reaction system for detecting 10 low-risk HPV subtypes using the reaction system containing primer probe composition 2 (10 pairs of separate upstream and downstream primers and 10 probes) in example 6.

FIG. 7 is a graph showing the peak of the target melting of HPV18 subtype detected by the PCR reaction system of example 7 using HPV 6-containing probe 6P 1.

FIG. 8 is a graph showing the peak of the target melting of HPV18 subtype detected by the PCR reaction system of example 7 using HPV 6-containing probe 6P 2.

FIG. 9 is a graph showing the peak melting targets of HPV18 subtypes detected by the PCR reaction system using HPV 6-containing probe 6P3 in example 7.

Detailed Description

In order that the application may be more readily understood, the application will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present application may be prepared by commercial or conventional methods unless specifically indicated.

Example 1: design of compositions for single-tube typing detection of 28 HPV subtypes known gene sequences of 18 medium-high risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV 82), 10 low risk HPV subtypes (HPV 6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, HPV 83) and internal standard genes were compared and analyzed to obtain respective differential gene sequences, and the upstream and downstream primers and probes required for detection were designed for the differential gene sequences. The designed composition includes a set of primer compositions for melting curve analysis and a set of primer probe compositions for amplification curve analysis.

Meanwhile, the 5' ends of 18 upstream primers aiming at 18 high-risk HPV subtypes (including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV 82) analyzed by adopting a melting curve are respectively marked with 1 fluorescence quenching group, the middle positions of the primers are respectively marked with 1 fluorescence reporting group, and the distance between the fluorescence reporting group and the fluorescence quenching group on each upstream primer is 15-18 bp; and a sequence which is 4-5 bp in length and is complementary and paired with the 3 '-end of the fluorescence reporter group marked at the middle position of the upstream primer is added at the 5' -end of the 18 upstream primer base sequences.

In addition, for 10 low-risk HPV subtypes (HPV 6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV 83) analyzed by a fluorescent probe amplification curve, a special general upstream primer and a special general downstream primer are designed, namely only 1 general upstream primer and 1 general downstream primer are needed for detection of 10 HPV virus subtypes by a single tube fluorescent probe method; meanwhile, the length of 10 probes aiming at 10 low-risk HPV subtypes is controlled to be 13-15 bp, 1 fluorescence report group is marked at the 5 'end of the 10 probes, and 1 fluorescence quenching group is marked at the 3' end of the 10 probes, so that the distance between the fluorescence report groups and the fluorescence quenching groups on the probes is controlled to be 13-15 bp.

The base sequences of the primers in the primer compositions in the final designed compositions for single-tube typing detection of 28 HPV subtypes are shown in Table 1, and the base sequences of the primers and probes in the primer probe compositions are shown in Table 2.

Table 1: base sequence of each primer in primer composition for melting curve analysis

Table 2: base sequences of each primer and probe in primer probe composition for amplification curve analysis

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Example 2: kit for single-tube typing detection of 28 HPV virus subtypes

The kit comprises the following components:

HPV PCR reaction solution: the single-person dosage of the PCR reaction liquid comprises 10mM dNTPs 0.5 mu L and 5mM Mg ²⁺ 2. Mu.L, 10*Ace Taq buffer (Mg+plus) 2.5. Mu.L, 5.0. Mu.L of PCR synergist, 7.5. Mu.L of the composition designed and synthesized in example 1, and 19.7. Mu.L of purified water were made up. The formula of the PCR synergist is as follows: 20mM tetramethylammonium chloride (TMAC), 2wt% dimethyl sulfoxide (DMSO), and 0.8mg/mL Bovine Serum Albumin (BSA); HPV enzyme cocktail: the single-person dosage consists of 0.25 mu L of Ace Taq DNA polymerase with the concentration of 5U/mu L and 0.05 mu L of E.coli UDG enzyme with the concentration of 1U/mu L;

controlling the nature of yang: DNA fragments of 28 HPV virus subtypes and internal standard genes;

Negative quality control: purifying the water.

Example 3: typing detection of 28 HPV subtypes in sample Using the kit of example 2 1, reagent preparation

(1) And taking out the PCR reaction liquid from the kit, melting at room temperature, shaking, mixing uniformly, and performing instantaneous centrifugation to ensure that the liquid on the tube wall is completely centrifuged to the bottom of the tube. The number of reactions N (n=number of samples+1 positive control+1 negative control) required for the current experiment was counted, the volume of the required PCR reaction solution was calculated according to the PCR reaction system formulation table shown in table 3, and the reaction system was prepared, and then, instantaneous centrifugation was performed so that the liquid on the tube wall was all centrifuged to the bottom.

Table 3: single-person PCR reaction system preparation table

(2) The prepared PCR reaction system is respectively split-packed into N reaction holes according to the volume of 20 mu L/Kong Fenzhuang, and the split-packed PCR tubes are transferred to a sample preparation area.

2. Sample processing and nucleic acid extraction

The sample for detection can be unknown samples such as wart surface abscission cells, female cervical epithelial cells, genital secretion and the like. Nucleic acid extraction was performed using commercial HPV nucleic acid extraction or purification reagents, operating according to instructions. The genomic DNA extracted according to the kit steps can be immediately sampled or placed at-20+/-5 ℃ for standby. The genome DNA can be preserved for 8 months at-20+ -5deg.C, and repeated freezing and thawing is avoided.

And (3) injection: the negative quality control product in the kit should be used for synchronously extracting nucleic acid with the sample, and the positive quality control product does not participate in the extraction of nucleic acid.

3. Sample addition

(1) Adding 5 mu L of genomic DNA of the extracted sample into a PCR reaction tube, covering a tube cover, centrifuging at 1000rpm or lightly throwing, and removing bubbles at the bottom of the tube; the template used in the positive control reaction is a positive quality control product in the kit, and the template used in the blank control reaction is a negative quality control product in the kit.

(2) And (5) covering the PCR reaction tube, and recording the sample loading condition. And transferring the PCR reaction tube to a nucleic acid amplification region for on-machine detection. If the template is added into the PCR reaction tube and then the machine cannot be started immediately under the temporary condition, the PCR reaction tube with the template is put into a condition of 2-8 ℃ for temporary storage, and the machine is started as soon as possible within 24 hours for detection.

4. PCR reaction program set-up (nucleic acid amplification region)

The PCR tube was placed in the instrument sample tank, the sample name, negative quality control and positive quality control were set in the corresponding order, the reaction volume was set to 25. Mu.L, and the amplification curve and melting curve analysis steps were one procedure, which was continuously completed on a full-automatic medical PCR analysis system (SLAN-96P Shanghai Marble medical science and technology Co., ltd.). The reaction program (PCR reaction program and melting curve analysis program) parameters were set as shown in Table 4. The application sets the fluorescence acquisition of the amplification stage to acquire fluorescence signals in the high-temperature denaturation stage at 95 ℃ for the PCR reaction program adopting the fluorescent probe method amplification curve analysis, so that the detection of the amplification stage and the detection of the melting curve are mutually independent and noninterfere, thereby realizing the combined use of the real-time fluorescent PCR amplification detection technology and the melting curve technology.

Table 4: amplification cycle conditions and fluorescent acquisition settings

5. Interpretation of results

The detection results of the high-risk HPV genotypes among 18 types of 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 tables of the 18 high-risk HPV genotypes shown in Table 5.

Judging whether 18 medium-high-risk HPV genotypes are carried in the sample: judging that the sample is positive for the genotype when a melting peak exists in a specific genotype Tm reference value range in a FAM, VIC, ROX or Cy5 channel; when melting peaks exist in the range of two or more Tm reference values at the same time, judging that the sample carries two or more corresponding genotypes at the same time; if the CT value of a CY5 channel of the amplification curve is less than or equal to 36 and no melting peak exists in the reference value range of Tm values of FAM, VIC, ROX and Cy5 channels of the melting curve, judging that the sample is 18 medium-high risk HPV genotype negative samples; if the sample does not have any melting peak in the four fluorescent channels, but when the negative control of the test has a specific melting peak, judging that the sample is invalid, and suggesting resampling or re-extraction and then detection; if the sample does not have any melting curve peaks in the four fluorescent channels, and when the positive control of the secondary test does not have any melting peaks, the kit is judged to be invalid.

Interpretation of positive control: the corresponding melting peaks in both FAM, VIC, ROX and Cy5 channels can be judged as positive control, and if any fluorescent channel has no melting peak, the kit is judged to be invalid.

Interpretation of negative control: the CT value of a CY5 channel of the amplification curve is less than or equal to 41, and each fluorescent channel of the melting curve has no melting peak. The negative control can control whether the sample is polluted by aerosol in the air or not in the sample adding operation process, and if the negative control has melting peaks in any fluorescent channel, the negative control prompts that nucleic acid pollution possibly exists in the operation environment, and the synchronously detected sample possibly has false positive results.

According to the specific detection method and detection channel of each HPV subtype, the infected HPV type in the sample can be judged, and the technical principle diagram of single-tube typing detection 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 HPV 82) is adopted by the kit disclosed by the application and is shown in figure 1.

Table 5: melting curve analysis method interpretation table for 18 medium-high-risk HPV genotypes

The above Tm values are common values obtained on a full automatic medical PCR analysis system (SLAN-96P; shanghai Marble medical science and technology Co., ltd.) as reference values. The Tm values are based on the automatic interpretation by the instrument, and when the instrument gives more than one Tm value, please select the effective Tm value with reference to the peaks of the positive control and the negative control. When the instrument cannot automatically give the Tm value, the Tm value can be obtained by adjusting a baseline or directly and manually judging.

After the experiment operation is finished, the instrument gives corresponding Ct values of all samples, and the result judgment can be generally carried out according to the Ct values and delta Ct values.

And (3) internal standard result judgment: the CY5 channel of the amplification curve has an obvious logarithmic amplification curve, the CT value is less than or equal to 41, and the judgment of the results of other channels is continued if the condition is met; if this condition is not satisfied, the experiment is not effective, and it is recommended to confirm the quality of the nucleic acid or sample and then re-detect.

According to the amplification curve analysis method of the 10 low-risk HPV subtypes shown in Table 6, the detection results of the 10 low-risk HPV subtypes (HPV 6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81 and HPV 83) are interpreted, and according to the specific detection method and detection channel of each HPV subtype, the low-risk HPV types infected in the sample can be determined.

Table 6: interpretation table of amplification curve analysis method of 10 low-risk HPV subtypes

Example 4: influence of artificially designed specific hairpin structures on the upstream primer of a primer composition for melting curve analysis on the detection result

Detecting a sample: HPV type 18 positive samples from a hospital.

The design sequence of the HPV18 downstream primer 18R was identical to that of the HPV18 downstream primer 18R of example 1 (SEQ ID NO: 4).

HPV18 upstream primer 1, 18F1: the base sequence of 18F1 was the same as that of 18F (SEQ ID NO: 3) in example 1, and the 5 '-end of the upstream primer was added with a sequence having a length of 4bp and complementary to the 3' -end of the fluorescent reporter group labeled in the middle of the upstream primer, so that a hairpin structure was formed.

HPV18 upstream primer 2, 18F2: the base sequence of 18F2 is shown in Table 7 (SEQ ID NO: 52), the 5 '-end of the upstream primer is not designed with a sequence of 4-5 bp added and complementary paired with the 3' -end of the fluorescent reporter group labeled in the middle of the upstream primer, and the primer structure is not formed into a hairpin structure.

Table 7: design of hairpin structure in HPV18 upstream primer

PCR reaction systems were prepared using two upstream primers, 18F1 and 18F2, each containing HPV18 described above according to tables 8 to 9, and amplification was performed according to the amplification cycle conditions of Table 4 in example 3, followed by detection of HPV type 18 positive samples in example 3. And judging the influence of a special hairpin structure designed manually on an upstream primer of the primer composition for melting curve analysis on the detection result according to a melting peak diagram of the detection result, wherein the result is shown in figure 3.

Table 8:18F1 PCR reaction system

Table 9: PCR reaction system of 18F2

FIG. 3 shows the results of a target melting peak of HPV18 subtype detected using a reaction system containing the two upstream primers 18F1 and 18F2 of HPV18, respectively. As can be seen from FIG. 3, compared with a special hairpin structure without artificial design, a sequence which is 4-5 bp in length and is complementary to the 3 'end of the fluorescence reporting group marked in the middle position of the upstream primer is designed and added at the 5' end of the upstream primer, namely, the special hairpin structure is designed manually, so that the signal value of a target melting peak is obviously improved, and the interference of a background signal value is greatly reduced. The hairpin structure formed by adding the sequence which is 4-5 bp in length and complements and pairs with the 3 'end of the fluorescent reporter group marked at the middle position of the upstream primer at the 5' end of the upstream primer sequence can ensure that when the melting curve analysis is carried out on an amplified product, DNA is melted to form single-stranded DNA along with the rise of temperature, the physical positions of the fluorescent quenching group and the fluorescent reporter group are relatively close due to the design of the hairpin structure, fluorescence is sufficiently quenched, so that the detection background signal is greatly weakened, the target melting peak signal value of HPV detection is improved, and the sensitivity and the accuracy of the detection result are improved.

Example 5: influence of distance separation of fluorescence quenching group and fluorescence reporting group in upstream primer of primer composition for melting curve analysis on detection result

Detecting a sample: HPV type 18 positive samples from a hospital.

The downstream primer design sequence of HPV18 was identical to that of HPV18 in example 1 (SEQ ID NO: 4).

HPV18 upstream primer 3, 18F3: the base sequence of 18F3 is identical to the upstream primer sequence of HPV18 in example 1 (SEQ ID NO. 3), and the distance between the fluorescent reporter group labeled in the middle of the primer and the fluorescent quenching group labeled at the 5' -end of the primer is 16bp.

HPV18 upstream primer 4, 18F4: the base sequence of 18F4 is shown in Table 10 (SEQ ID NO: 53), and the distance between the fluorescent reporter group labeled at the intermediate position of the primer and the fluorescent quenching group labeled at the 5' -end of the primer is 12bp.

HPV18 upstream primer 5, 18F5: the base sequence of 18F5 is shown in Table 10 (SEQ ID NO: 54), and the distance between the fluorescent reporter group labeled at the intermediate position of the primer and the fluorescent quenching group labeled at the 5' -end of the primer is 20bp.

Table 10: distance design of fluorescence quenching group and fluorescence reporting group in HPV18 upstream primer

PCR reaction systems were prepared according to tables 11 to 13 using three upstream primers 18F3, 18F4 and 18F5 containing HPV18, respectively, and amplification was performed according to the amplification cycle conditions of Table 4 in example 3, followed by detection of HPV type 18 positive samples in example 5, respectively. And judging the influence of the distance between the fluorescence quenching group and the fluorescence reporting group in the upstream primer on the detection result according to the peak diagram of the detection result, wherein the result is shown in figure 4.

Table 11: PCR reaction system of 18F3

Table 12: PCR reaction system of 18F4

Table 13: PCR reaction system of 18F5

FIG. 4 shows the results of target melting peak patterns of HPV18 subtypes detected using a reaction system containing the three upstream primers of HPV18 described above, namely 18F3, 18F4 and 18F5, respectively. As can be seen from FIG. 4, the signal value of the target melting peak is highest when the distance between the fluorescence quenching group and the fluorescence reporting group in 18F3 is 16 bp; and when the distance between the fluorescence quenching group and the fluorescence reporting group in 18F4 is 13bp and the distance between the fluorescence quenching group and the fluorescence reporting group in 18F5 is 20bp, the signal value of the target melting peak is obviously reduced. The method has the advantages that when the distance between the fluorescent reporter group and the fluorescent quenching group marked on the upstream primer is controlled to be 15-18 bp, on one hand, the fluorescent quenching group can be ensured not to influence the luminous effect of the fluorescent reporter group in melting curve analysis, so that the target melting peak signal of a detection result is higher, on the other hand, the fluorescent marked single-stranded DNA formed by DNA melting along with the increase of temperature in melting curve analysis can be ensured to be sufficiently quenched, the interference of background signals is reduced, the interpretation of the detection result is facilitated, and the sensitivity and the accuracy of the detection result are improved.

Example 6: primer probe compositions for amplification curve analysis detect samples using the effect of the universal upstream and downstream primer on detection results: build scaled concentration of 1×10 ⁵ The copies/mL positive plasmid comprising 10 low risk HPV subtypes (HPV 6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV81, HPV 83).

Primer probe composition 1: as in example 1;

primer probe composition 2: designing independent upstream and downstream primers corresponding to HPV types in the primer probe composition 2 aiming at 10 low-risk HPV subtypes to be detected; the probe sequence of primer probe composition 2 was the same as in example 1.

PCR reaction systems were prepared according to tables 14 to 15 using primer probe compositions 1 and 2 containing the above two primer probe compositions, respectively, and then positive plasmids containing 10 low-risk HPV subtypes in example 6 were detected, respectively. And judging the influence of a pair of general upstream and downstream primer designs on the detection result according to the amplification curve graph of the detection result, wherein the result is shown in figures 5-6.

Table 14: PCR reaction system of primer probe composition 1

Table 15: PCR reaction system of primer probe composition 2

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FIG. 5 shows the amplification curve results of a multiplex PCR reaction for detecting 10 low risk HPV subtypes using a reaction system containing primer probe composition 1 (1 universal upstream primer and 1 universal downstream primer); FIG. 6 shows the amplification curve results of a multiplex PCR reaction for detecting 10 low risk HPV subtypes using a reaction system containing primer probe composition 2 (10 pairs of separate upstream and downstream primers). As can be seen from FIGS. 5 and 6, after the amplification is performed by adopting the design scheme of the universal primer, the signal value of the amplification curve is higher, the interference of background signals is reduced, and the accuracy of the detection result is improved. Therefore, by adopting the design of the upstream and downstream primers, the complexity of an HPV multiplex PCR reaction system can be greatly simplified, and the sensitivity and the specificity of HPV single tube multiplex detection can be improved.

Example 7: interference of the spacing between the fluorescent reporter group and the fluorescent quencher group on the probe in the primer probe composition for amplification curve analysis on the melting curve detection results affects the detection sample: a positive sample from a hospital infected with HPV18 high-risk type and HPV6 low-risk type.

The design sequences of the primers for HPV6 are shown in Table 16 (SEQ ID NOS.57 and 58).

The base sequence of probe 6P1 of HPV6 is identical to the probe sequence of HPV6 in example 1 (SEQ ID NO. 41), and the distance between the fluorescent group and the quenching group of this probe is 14bp.

The nucleotide sequence of probe 6P2 of HPV6 is shown in Table 16 (SEQ ID NO: 55), and the distance between the fluorescent group and the quenching group of the probe is 17bp.

The nucleotide sequence of probe 6P3 of HPV6 is shown in Table 16 (SEQ ID NO: 56), and the distance between the fluorescent group and the quenching group of the probe is 20bp.

The design sequences of the upstream and downstream primers of HPV18 are the same as those of HPV18 in example 1 (SEQ ID NOS: 3 and 4)

Table 16: design of distance between fluorescence quenching group and fluorescence reporting group in HPV6 fluorescent probe

A multiplex PCR reaction system was prepared by using the three probes 6P1, 6P2 and 6P3 containing HPV6 described above according to tables 17 to 19, amplification was performed according to the amplification cycle conditions of Table 4 in example 3, and then positive samples mixed with HPV18 high-risk type and HPV6 low-risk type in example 7 were detected, and the peak patterns of the detection results were shown in FIGS. 7 to 9. And judging the interference influence of the space between the fluorescent reporter group and the fluorescent quenching group on the probe in the primer probe composition for amplification curve analysis on the melting curve detection result according to the detection result peak diagram.

Table 17: PCR reaction system of 6P1

Sequence number	Names of raw and auxiliary materials	1 person (mu L)
			1	10*Ace Taq buffer(Mg+plus)	2.5
2	dNTPs	0.5
			3	Ace Taq DNA Polymerase	0.25
4	6F	0.2
			5	6R	0.2
6	6P1	0.1
			7	18F	0.2
8	18R	0.2
			9	ddH ₂ O	15.85
10	Genomic DNA	5

Table 18: PCR reaction system of 6P2

Sequence number	Names of raw and auxiliary materials	1 person (mu L)
			1	10*Ace Taq buffer(Mg+plus)	2.5
2	dNTPs	0.5
			3	Ace Taq DNA Polymerase	0.25
4	6F	0.2
			5	6R	0.2
6	6P2	0.1
			7	18F	0.2
8	18R	0.2
			9	ddH ₂ O	15.85
10	Genomic DNA	5

Table 19: PCR reaction system of 6P3

Sequence number	Names of raw and auxiliary materials	1 person (mu L)
			1	10*Ace Taq buffer(Mg+plus)	2.5
2	dNTPs	0.5
			3	Ace Taq DNA Polymerase	0.25
4	6F	0.2
			5	6R	0.2
6	6P3	0.1
			7	18F	0.2
8	18R	0.2
			9	ddH ₂ O	15.85
10	Genomic DNA	5

FIGS. 7-9 show the results of target melting peak patterns of HPV18 subtypes detected using a reaction system containing the three probes of HPV6 described above, namely 6P1, 6P2 and 6P3, respectively. 7-9, when the distance between the fluorescence quenching group and the fluorescence reporting group in the 6P1 is 14bp, the signal value of the target melting peak of HPV18 subtype is highest and no non-specific melting peak caused by the 6P1 fluorescent probe appears; and when the distance between the fluorescence quenching group and the fluorescence reporting group in the 6P2 is 17bp and the distance between the fluorescence quenching group and the fluorescence reporting group in the 6P3 is 20bp, the signal value of the target melting peak is obviously reduced, and the non-specific melting peak caused by the 6P2 and 6P3 fluorescent probes appears. By optimizing the length of the probe, the distance between the fluorescent reporter group and the fluorescent quenching group is controlled to be 13-15 bp, so that the fluorescence of the unhydrolyzed fluorescent probe can be fully quenched, a lower fluorescence background is presented, the fluorescent probe is ensured not to present a melting curve graph changing with temperature no matter being combined on a target sequence or being dissociated in a solution, the non-specific signal interference is reduced, and the sensitivity and the accuracy of a detection result are improved. Therefore, the cross interference of fluorescent probe signals in the amplification curve to analysis results in the melting curve stage can be solved, and finally, one target can be distinguished through the amplification curve graph, and the other target can be distinguished through the amplified melting curve graph.

It should be noted that the above-described embodiments are only for explaining the present application and do not constitute any limitation of the present application. The application has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the application as defined in the appended claims, and the application may be modified without departing from the scope and spirit of the application. Although the application is described herein with reference to particular means, materials and embodiments, the application is not intended to be limited to the particulars disclosed herein, as the application extends to all other means and applications which perform the same function.

Claims

1. A composition for single-tube typing detection of 28 HPV subtypes, characterized in that the composition comprises a primer composition for judging by using a melting curve as a detection result and a primer probe composition for judging by using an amplification curve as a detection result;

2. The composition of claim 1, wherein the primer probe composition further comprises 1 pair of upstream primer and downstream primer for detecting the internal standard gene beta-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 or 2, wherein 5' ends of all upstream primers in 18 pairs of upstream and downstream primers for typing detection of high-risk HPV subtypes of 18 types of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV82 are each labeled with 1 fluorescence quenching group, and intermediate positions are each labeled with 1 fluorescence reporting group.

4. The composition of claim 3, wherein the distance between the fluorescent reporter group and the fluorescent quenching group is 15-18 bp.

5. A composition according to claim 3 wherein the 5 'ends of all upstream primers in 18 pairs of upstream and downstream primers for the typing detection of high risk HPV subtypes of 18 of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73 and HPV82 comprise a sequence of length 4 to 5bp complementary to the 3' end of the centrally located labelled fluorescent reporter group to form a hairpin structure.

6. A composition according to claim 3 wherein the upstream primers for HPV16, HPV18, HPV31 and HPV33 are each labelled with a FAM fluorescent reporter group, the upstream primers for HPV39, HPV35, HPV45, HPV52 and HPV51 are each labelled with a VIC fluorescent reporter group, and the upstream primers for HPV66, HPV26, HPV53, HPV82 and HPV73 are each labelled with a ROX fluorescent reporter group, and the upstream primers for HPV58, HPV56, HPV59 and HPV68 are each labelled with a CY5 fluorescent reporter group.

7. The composition of claim 1 or 2, wherein the 5 'ends of the 10 probes are each labeled with 1 fluorescent reporter group, and the 3' ends are each labeled with 1 fluorescent quencher group; preferably, the lengths of the 10 probes are 13-15 bp independently.

8. The composition of claim 1 or 2, wherein the fluorescence signal is collected during a high temperature denaturation phase at 92-95 ℃ during amplification curve analysis using the primer probe composition.

9. The composition of claim 1 or 2, wherein FAM fluorescent reporter groups are labeled on probes for HPV6, HPV11, HPV40 and HPV42, VIC fluorescent reporter groups are labeled on probes for HPV43, HPV44 and HPV54, ROX fluorescent reporter groups are labeled on probes for HPV61, HPV81 and HPV83, and CY5 fluorescent reporter groups are labeled on probes for the internal standard gene β -globin.

10. A kit for single tube genotyping of 28 HPV subtypes, comprising a PCR reaction solution comprising the composition of any one of claims 1-8; the final concentration of each primer in the primer composition in the reaction solution is 0.1-0.3 mu M, the final concentration of each primer in the primer probe composition in the reaction solution is 0.05-0.08 mu M, and the final concentration of each probe is 0.02-0.05 mu M.

11. The kit according to claim 9, wherein the PCR reaction solution further comprises a PCR buffer solution, a PCR synergist, dNTPs and an enzyme mixed solution; the PCR synergist 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.

12. Use of a kit according to claim 10 or 11 for single tube detection of 28 different HPV viral subtypes, and for accurate typing of 18 medium-high risk types and for partial typing of 10 low risk types.