HPV (human papillomavirus) typing primer probe composition and application thereof

Abstract

The invention provides an HPV (human papillomavirus) typing primer probe composition and application thereof, and relates to the technical field of biology. The HPV typing primer probe composition comprises specific primer pairs aiming at the following 23 types of HPV types: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82, and the nucleotide sequences of the specific primer pairs are shown as SEQ ID NO.1-15 and SEQ ID NO.18-40. According to the HPV typing primer probe composition, the defects that an existing HPV typing product is limited in HPV type distinguishing capacity and tedious in operation are overcome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an HPV typing primer probe composition and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Human papilloma virus (HPV) is a small molecule, non-enveloped circular double-stranded DNA virus with a genome length of about 8,000 base pairs (bp) and is widely present in nature. HPV that infects the reproductive tract and anus can be divided into two categories: low-risk and high-risk types, based on the pathogenicity or carcinogenicity of each genotype. Persistent high-risk HPV infection is the main cause of cervical intraepithelial neoplasia and cervical cancer. Studies have shown that high-risk HPV DNA is detected in 99.7% of cervical cancer patients. Low-risk HPV is generally associated with condyloma acuminatum or low-grade squamous intraepithelial lesions and rarely causes invasive cancer.

[0004] Based on the research results of the WHO International Agency for Research on Cancer (IARC) and other international organizations, the guidelines for human papillomavirus (HPV) nucleic acid testing, genotyping, and reagent technical review recommend that 13 genotypes, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, be listed as high-risk types, and 5 genotypes, including 26, 53, 66, 73, and 82, be listed as medium-risk types.

[0005] The main methods for detecting HPV include Pap smear, in situ hybridization, cytology, HPV nucleic acid testing, etc. HPV nucleic acid testing is the main detection method for cervical cancer screening. Compared with cytology screening methods, HPV testing can screen out high-risk groups to the greatest extent, thereby detecting precancerous lesions early and avoiding the occurrence of cervical cancer. At the same time, it can extend the screening interval and improve screening efficiency. Therefore, the demand for HPV typing products is increasing. However, the existing typing methods still have the following defects: low sensitivity; limited typing, most HPV typing reagents use multi-tube testing, which is costly; and typing uses multi-tube testing, and the experimental operation is complicated. Therefore, how to improve HPV typing products is a problem that needs to be solved at present.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an HPV typing primer-probe combination to alleviate the defects of existing HPV typing products, such as limited ability to distinguish HPV types and complicated operation.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, a HPV typing primer-probe composition is provided, comprising at least one pair of the following primer pairs: an HPV6 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.18; an HPV53 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.32; an HPV58 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.34; an HPV66 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.36; an HPV33 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.24; HPV56 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.33; HPV45 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.29; HPV11 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.19; HPV35 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.25; HPV18 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.21; HPV31 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. ID NO.2 and 11, and the nucleotide sequence of the probe is shown in SEQ ID NO.23; HPV52 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.31; HPV39 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.26; HPV42 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and 13, and the nucleotide sequence of the probe is shown in SEQ ID NO.27; HPV16 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.20; HPV26 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.22; HPV82 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.40; HPV51 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.30; HPV68 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 15, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.37; HPV81 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.4 and 7 respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.39; HPV73 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.4 and 13 respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.38; HPV59 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.5 and 14 respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.35; and, HPV43 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.6 and 11 respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.28. .

[0010] In a second aspect, there is provided a use of the HPV typing primer-probe combination described in the first aspect in the preparation of HPV typing detection products or HPV typing for non-diagnostic and treatment destinations.

[0011] In a third aspect, a kit for HPV typing detection is also provided, the kit comprising the HPV typing primer probe combination described in the first aspect.

[0012] In a fourth aspect, a non-diagnostic and treatment destination HPV typing method is also provided, wherein the HPV typing includes distinguishing the following types of HPV: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82, and the HPV typing method includes:

[0013] (A) Obtaining the Tm value temperature range of each primer pair in the HPV typing primer-probe composition described in the first aspect for amplifying the corresponding HPV type.

[0014] (B) The HPV typing primer-probe combination described in the first aspect or the kit described in the third aspect is used to amplify the sample to be tested, and primer pairs with overlapping Tm value ranges of corresponding types are assigned to different fluorescent channels.

[0015] (C) Obtaining the melting curve of the sample to be tested in each fluorescent channel and comparing it with the Tm value range of step (A); if the primer pair of a fluorescent channel has a melting peak within the Tm value range of the corresponding type, it is determined that the HPV type corresponding to the primer pair exists in the sample to be tested.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a kind of HPV typing primer probe composition, and the composition comprises specific primer pairs for the following 23 kinds of HPV types: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82. Through these primer pairs, the above 23 kinds of HPV types can be distinguished. The HPV typing primer probe composition of the present invention can carry out multiple amplification reaction in fewer reaction systems. In a preferred embodiment, only two reaction systems are needed to simply, quickly and accurately detect these 23 kinds of HPV DNA, thereby achieving the purpose of typing. This design simplifies the operation process, making the detection easier to implement and realize. Furthermore, when detecting multiple HPV types simultaneously, the HPV typing primer probe combination provided by the present invention can still maintain high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The Tm value and melting peak curve of HPV type 6 in the CY5 channel in Example 3;

[0020] Figure 2 The Tm value and melting peak curve of HPV type 11 in the ROX channel in Example 3;

[0021] Figure 3 The Tm value and melting peak curve of HPV 16 type in CY5 channel in Example 3;

[0022] Figure 4 The Tm value and melting peak curve of HPV type 18 in the FAM channel in Example 3;

[0023] Figure 5 The Tm value and melting peak curve of HPV type 26 in the HEX channel in Example 3;

[0024] Figure 6 The Tm value and melting peak curve of HPV type 31 in the HEX channel in Example 3;

[0025] Figure 7 The Tm value and melting peak curve of HPV type 33 in the FAM channel in Example 3;

[0026] Figure 8 The Tm value and melting peak curve of HPV 35 type in the HEX channel in Example 3;

[0027] Fig. 9 The Tm value and melting peak curve of HPV type 39 in the ROX channel in Example 3;

[0028] Fig.10 The Tm value and melting peak curve of HPV 42 type in CY5 channel in Example 3;

[0029] Fig.11 The Tm value and melting peak curve of HPV 43 type in CY5 channel in Example 3;

[0030] Fig.12 The Tm value and melting peak curve of HPV type 45 in the ROX channel in Example 3;

[0031] Fig.13 The Tm value and melting peak curve of HPV 51 type in the FAM channel in Example 3;

[0032] Fig.14 The Tm value and melting peak curve of HPV 52 type in ROX channel in Example 3;

[0033] Fig.15 The Tm value and melting peak curve of HPV 53 type in the HEX channel in Example 3;

[0034] Fig.16 The Tm value and melting peak curve of HPV 56 type in CY5 channel in Example 3;

[0035] Fig.17 The Tm value and melting peak curve of HPV 58 in the ROX channel in Example 3;

[0036] Fig.18 The Tm value and melting peak curve of HPV 59 type in ROX channel in Example 3;

[0037] Fig.19 The Tm value and melting peak curve of HPV type 66 in the FAM channel in Example 3;

[0038] Fig. 20 The Tm value and melting peak curve of HPV type 68 in the HEX channel in Example 3;

[0039] Fig.21 The Tm value and melting peak curve of HPV 73 type in the HEX channel in Example 3;

[0040] Fig. 22 The Tm value and melting peak curve of HPV 81 in the ROX channel in Example 3;

[0041] Fig.23 The Tm value and melting peak curve of HPV type 82 in the ROX channel in Example 3. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] It should be noted that:

[0044] In this document, unless otherwise specified, all implementation methods and preferred implementation methods mentioned in this document can be combined with each other to form a new technical solution.

[0045] In this document, unless otherwise specified, all technical features and preferred features mentioned in this document can be combined with each other to form a new technical solution.

[0046] Herein, unless otherwise specified, percentage (%) or part refers to percentage by weight or part by weight relative to the composition.

[0047] Herein, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0048] In this document, unless otherwise specified, the numerical range "a-b" is an abbreviation for any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" are listed in this document, and "6-22" is just an abbreviation for these numerical combinations.

[0049] The “range” disclosed in the present invention is in the form of a lower limit and an upper limit, which can be one or more lower limits, and one or more upper limits, respectively.

[0050] Herein, unless otherwise specified, each reaction or operation step may be performed sequentially or non-sequentially. Preferably, the reaction method herein is performed sequentially.

[0051] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0052] As used herein, "optionally", "optional", "optional" or "optional" is intended to mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0053] In this disclosure, "includes," "comprising," "having," "may," "containing," and variations thereof are generally intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "consisting of" generally indicates that no other components (or, similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, nouns with indefinite number also include plural referents.

[0054] Herein, "each... is independently selected from" is interchangeable with "... are independently selected from" and "... are independently selected from" and should be understood in a broad sense, referring to the range or options that each member of a group of variables or components can be independently selected, that is, the selection of each variable or component is independent and is not affected by the selection of other variables or components.

[0055] In the first aspect, a HPV typing primer-probe composition is provided, wherein the HPV typing primer-probe composition designs primers and probes for the L1 segment of HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82, wherein the primers are common primers, and some types share one primer. In addition, each primer has a certain degree of superposition effect for each type. The probe is a specific probe. The HPV typing primer-probe composition includes at least one of the following primer pairs:

[0056] HPV6 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 18;

[0057] HPV53 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.32;

[0058] HPV58 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 34;

[0059] HPV66 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.36;

[0060] HPV33 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.24;

[0061] HPV56 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.33;

[0062] HPV45 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.29;

[0063] HPV11 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 19;

[0064] HPV35 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 25;

[0065] HPV18 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 21;

[0066] HPV31 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 23;

[0067] HPV52 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 31;

[0068] HPV39 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 26;

[0069] HPV42 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 27;

[0070] HPV16 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.20;

[0071] HPV26 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.22;

[0072] HPV82 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.40;

[0073] HPV51 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.30;

[0074] HPV68 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 15, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.37;

[0075] HPV81 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 39;

[0076] HPV73 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 38;

[0077] HPV59 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.5 and 14, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.35;

[0078] and, HPV43 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.6 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.28.

[0079] In an optional embodiment, each probe in the primer pair is independently modified with a luminescent group and a quenching group.

[0080] In an optional embodiment, the luminescent group includes but is not limited to one or more of quantum dots, FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX and Texas Red.

[0081] In an optional embodiment, the luminescent group is modified at the 5' end of the probe.

[0082] In an optional embodiment, the quenching group includes one or more of BHQ1, BHQ2, BHQ3 and MGB.

[0083] In an optional embodiment, at least one probe in at least one primer pair contains at least one modified deoxynucleotide. The modification includes all changes in nucleic acid by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another chemical moiety. The modification includes, but is not limited to, changes to at least one or more of the sugar ring, phosphate group, and nitrogenous base of the deoxynucleotide.

[0084] In an optional embodiment, the modification includes replacing at least one deoxynucleotide in the probe with a locked nucleic acid (LNA). A locked nucleic acid is a deoxynucleotide with a connecting bridge added between the 2' and 4' positions of the ribose ring. The locked nucleic acid limits the flexibility of the sugar ring, reduces the flexibility of the ribose structure, and increases the stability of the local structure of the phosphate backbone, which can improve the affinity of the probe for the target fragment.

[0085] In an optional embodiment, the 10th G of the probe of the HPV26 typing primer pair (SEQ ID NO. 22) is a locked nucleic acid.

[0086] In an optional embodiment, the probe of the HPV31 typing primer pair (SEQ ID NO. 23) is a locked nucleic acid at position 9 G and is a locked nucleic acid at position 12 A.

[0087] In an optional embodiment, the probe of the HPV35 typing primer pair (SEQ ID NO. 25) is a locked nucleic acid at position 5 T and a locked nucleic acid at position 12 G.

[0088] In an optional embodiment, the 12th C of the probe of the HPV39 typing primer pair (SEQ ID NO. 26) is a locked nucleic acid.

[0089] In an optional embodiment, the 11th G of the probe of the HPV53 typing primer pair (SEQ ID NO. 32) is a locked nucleic acid.

[0090] In an optional embodiment, the 10th A of the probe of the HPV56 typing primer pair (SEQ ID NO. 33) is a locked nucleic acid.

[0091] In an optional embodiment, the ninth T of the probe of the HPV59 typing primer pair (SEQ ID NO. 35) is a locked nucleic acid.

[0092] In an optional embodiment, the 12th C of the probe of the HPV66 typing primer pair (SEQ ID NO. 36) is a locked nucleic acid.

[0093] In an optional embodiment, the HPV typing primer probe composition further comprises a primer pair for detecting an internal standard.

[0094] In an optional embodiment, the nucleotide sequences of the upstream primer and the downstream primer of the primer pair for detecting the internal standard are shown as SEQ ID NO.16 and 17 respectively, and the nucleotide sequence of the probe is shown as SEQ ID NO.41.

[0095] In an optional embodiment, the 10th C of the probe of the primer pair for detecting the internal standard (SEQ ID NO. 41) is a locked nucleic acid.

[0096] In an optional embodiment, the upstream primer in the HPV typing primer probe composition is a limiting primer, and the downstream primer is a non-limiting primer. A limiting primer refers to a primer used in a smaller amount in a PCR reaction system, and a non-limiting primer refers to a primer used in a larger amount in a PCR reaction system.

[0097] In an optional embodiment, the working concentration of the upstream primer in each primer pair is independently 0.01-0.05 μM, for example, but not limited to 0.01, 0.02, 0.03, 0.04 or 0.05 μM.

[0098] In an optional embodiment, the working concentration of the downstream primer in each primer pair is independently 0.05-0.5 μM, for example, but not limited to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 μM.

[0099] In an optional embodiment, the working concentration of the probe in each primer pair is independently 0.05-0.3 μM, for example, but not limited to 0.05, 0.1, 0.15, 0.2, 0.25 or 0.3 μM, preferably 0.18 μM.

[0100] In an optional embodiment, the working concentration of the upstream primer in the HPV18, HPV66, HPV53, HPV35, HPV26, HPV52, HPV11, HPV81, HPV59, HPV43 and HPV16 typing primer pairs is 0.02 μM, and the working concentration of the downstream primer is 0.25 μM.

[0101] In an optional embodiment, the working concentration of the upstream primer in the HPV33, HPV51, HPV68, HPV73, HPV31, HPV45, HPV58, HPV82, HPV39, HPV6, HPV56 and HPV42 typing primer pairs is 0.015 μM, and the working concentration of the downstream primer is 0.35 μM.

[0102] In an optional embodiment, the HPV typing primer probe composition includes a first primer probe composition and / or a second primer probe composition. The first primer probe composition can be subjected to an amplification reaction in the same reaction system, and the second primer probe composition can be subjected to an amplification reaction in the same reaction system.

[0103] The first primer-probe composition includes HPV18, HPV66, HPV53, HPV35, HPV26, HPV52, HPV11, HPV81, HPV59, HPV43 and HPV16 typing primer pairs. In an optional embodiment, the first primer-probe composition also includes a primer pair for detecting an internal standard.

[0104] In an optional embodiment, the probes in the HPV18 and HPV66 typing primer pairs are all labeled with a first luminescent group, the probes in the HPV53, HPV35 and HPV26 typing primer pairs are all labeled with a second luminescent group, the probes in the HPV52, HPV11, HPV81 and HPV59 typing primer pairs are all labeled with a third luminescent group, and the probes in the HPV43 and HPV16 typing primer pairs are all labeled with a fourth luminescent group; and the first luminescent group, the second luminescent group, the third luminescent group and the fourth luminescent group are different from each other.

[0105] In an optional embodiment, the first luminescent group is FAM, the second luminescent group is HEX, the third luminescent group is ROX and the fourth luminescent group is CY5. In an optional embodiment, the first luminescent group is FAM and the second luminescent group is HEX, and the quenching group modified by the probe is BHQ1. In an optional embodiment, the third luminescent group is ROX and the fourth luminescent group is CY5, and the quenching group modified by the probe is BHQ2.

[0106] The second primer-probe composition includes HPV33, HPV51, HPV68, HPV73, HPV31, HPV45, HPV58, HPV82, HPV39, HPV6, HPV56 and HPV42 typing primer pairs. In an optional embodiment, the second primer-probe composition also includes a primer pair for detecting an internal standard.

[0107] In an optional embodiment, the probes in the HPV33 and HPV51 typing primer pairs are all labeled with the fifth luminescent group, the probes in the HPV68, HPV73 and HPV31 typing primer pairs are all labeled with the sixth luminescent group, the probes in the HPV45, HPV58, HPV82 and HPV39 typing primer pairs are all labeled with the seventh luminescent group, and the probes in the HPV6, HPV56 and HPV42 typing primer pairs are all labeled with the eighth luminescent group; and the fifth luminescent group, the sixth luminescent group, the seventh luminescent group and the eighth luminescent group are different from each other.

[0108] In an optional embodiment, the fifth luminescent group is FAM, the sixth luminescent group is HEX, the seventh luminescent group is ROX, and the eighth luminescent group is CY5. In an optional embodiment, the fifth luminescent group is FAM and the sixth luminescent group is HEX, and the quenching group modified by the probe is BHQ1. In an optional embodiment, the seventh luminescent group is ROX and the eighth luminescent group is CY5, and the quenching group modified by the probe is BHQ2.

[0109] In a second aspect, there is provided a use of the HPV typing primer-probe combination described in the first aspect in the preparation of HPV typing detection products or HPV typing for non-diagnostic and treatment destinations.

[0110] In a third aspect, a kit for HPV typing detection is also provided, the kit comprising the HPV typing primer probe combination described in the first aspect.

[0111] In an optional embodiment, the kit includes the first primer-probe combination and the second primer-probe combination, which are separately packaged.

[0112] It is understandable that, in the test kit, the HPV typing primer-probe composition is removed, and the material or reagent for nucleic acid amplification acceptable in the art is optionally included, including but not limited to one or more of buffer, DMSO, preservative, salt, dATP, dCTP, dGTP, dTTP, dUTP, DNA polymerase, negative control and positive control. The above-mentioned material for nucleic acid amplification is optionally packaged separately or premixed in the same reagent, for example, premixed as a reaction solution reagent. Each material or each reagent in the test kit is optionally present in the form of a working reagent or a reserve reagent. When it is a reserve reagent, it is diluted to a working concentration for use.

[0113] In an optional embodiment, the kit includes a reaction solution A, and the reaction solution A includes: Tris-HCl, DMSO, magnesium ions and dNTPs and the HPV typing primer probe composition.

[0114] In an optional embodiment, the reaction solution A includes reaction solution A1 and reaction solution A2, the HPV typing primer probe composition in the reaction solution A1 is the first primer probe composition; the HPV typing primer probe composition in the reaction solution A2 is the second primer probe composition.

[0115] In an optional embodiment, the kit includes a reaction solution B, and the reaction solution B includes a DNA polymerase and an enzyme for preventing false positives.

[0116] In an optional embodiment, the reaction solution B includes Tris-HCl, KCl, an enzyme for preventing false positives and a DNA polymerase; the DNA polymerase includes Taq DNA polymerase, and the enzyme for preventing false positives includes UNG enzyme (Uracil-N-glycosylase).

[0117] In an optional embodiment, the reaction solution A or B may further include a preservative to extend the shelf life, such as sodium azide.

[0118] In an optional embodiment, the negative control contains the target gene of the primer pair for detecting the internal standard. Further optionally, the negative control is a plasmid containing the target gene of the primer pair for detecting the internal standard.

[0119] In an optional embodiment, the positive control contains at least one target gene of the HPV typing primer-probe combination and a target gene of a primer pair for detecting an internal standard; further optionally, the negative control is a plasmid containing at least one target gene of the HPV typing primer-probe combination and a plasmid containing a target gene of a primer pair for detecting an internal standard. When the positive control contains target genes of multiple primer pairs, each plasmid contains one or more target genes.

[0120] In a fourth aspect, a non-diagnostic and treatment destination HPV typing method is also provided, wherein the HPV typing includes distinguishing the following types of HPV: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82, and the HPV typing method includes:

[0121] (A) Obtaining the Tm value temperature range of each primer pair in the HPV typing primer-probe composition described in the first aspect for amplifying the corresponding HPV type.

[0122] (B) The HPV typing primer-probe combination described in the first aspect or the kit described in the third aspect is used to amplify the sample to be tested, and the primer pairs with overlapping Tm value ranges of corresponding types are assigned to different fluorescence channels to avoid the melting peaks in the same fluorescence channel falling into the Tm value ranges of multiple HPV types.

[0123] (C) Obtaining the melting curve of the sample to be tested in each fluorescent channel and comparing it with the Tm value range of step (A); if a melting peak appears in a fluorescent channel within the Tm value range of the corresponding type, it is determined that the HPV type corresponding to the primer pair exists in the sample to be tested.

[0124] In an optional embodiment, the step (B) further comprises amplifying a negative control and a positive control, and step (B) is performed when the melting curve data of the negative control and the positive control are correct. The correct melting curve data of the negative control and the positive control include that the melting peaks of the negative control and the positive control fall within the Tm value range of the respective target fragments.

[0125] In an optional embodiment, each reaction system contains at least one primer pair for detecting the internal standard, and step (B) is performed when the melting curve data of the internal standard is correct. The correct melting curve data of the internal standard includes that the melting peaks of the internal standard fall within the Tm value range of the internal standard target fragment.

[0126] In an optional embodiment, the step (B) comprises two reaction systems, and the two reaction systems respectively use the first primer-probe combination and the second primer-probe combination described in the first aspect for amplification.

[0127] In an optional embodiment, the Tm value range corresponding to the HPV18 typing primer pair is 54.5-57.5°C; and / or, the Tm value range corresponding to the HPV66 typing primer pair is 60.5-63.5°C; and / or, the Tm value range corresponding to the HPV53 typing primer pair is 56.5-59.5°C; and / or, the Tm value range corresponding to the HPV35 typing primer pair is 62.5-65.5°C; and / or, the Tm value range corresponding to the HPV26 typing primer pair is 67.5-70.5°C; and / or, the Tm value range corresponding to the HPV52 typing primer pair is 56.5-59.5°C; and / or, the Tm value range corresponding to the HPV35 typing primer pair is 62.5-65.5°C; and / or, the Tm value range corresponding to the HPV26 typing primer pair is 67.5-70.5°C; and / or, the Tm value range corresponding to the HPV52 typing primer pair is 56.5-59.5°C. The range of Tm values ​​for the HPV11 typing primer pair is 52.5-55.5°C; and / or, the range of Tm values ​​for the HPV81 typing primer pair is 60.5-63.5°C; and / or, the range of Tm values ​​for the HPV59 typing primer pair is 66.5-69.5°C; and / or, the range of Tm values ​​for the HPV43 typing primer pair is 43.5-48.5°C; and / or, the range of Tm values ​​for the HPV16 typing primer pair is 64.0-70.0°C; and / or, the range of Tm values ​​for the HPV33 typing primer pair is 61.5-62.5°C; and / or, the range of Tm values ​​for the HPV59 typing primer pair is 66.5-69.5°C; and / or, the range of Tm values ​​for the HPV43 typing primer pair is 43.5-48.5°C; and / or, the range of Tm values ​​for the HPV16 typing primer pair is 64.0-70.0°C; and / or, the range of Tm values ​​for the HPV33 typing primer pair is 63.5-64.5°C; and / or, the range of Tm values ​​for the HPV33 typing primer pair is 66.5-69.5°C; and / or, the range of Tm values ​​for the HPV43 typing primer pair is 43.5-48.5°C; and / or, the range of Tm values ​​for the HPV16 typing primer pair is 64.0-70.0°C; and / or, The corresponding Tm value range is 52.5-55.5°C; and / or, the corresponding Tm value range of the HPV51 typing primer pair is 58.5-61.5°C; and / or, the corresponding Tm value range of the HPV68 typing primer pair is 41.5-44.5°C; and / or, the corresponding Tm value range of the HPV73 typing primer pair is 53.5-56.5°C; and / or, the corresponding Tm value range of the HPV31 typing primer pair is 62.5-67.5°C; and / or, the corresponding Tm value range of the HPV45 typing primer pair is 43.5-46.5°C; and / or, The Tm value range corresponding to the V58 typing primer pair is 50.5-53.5°C; and / or, the Tm value range corresponding to the HPV82 typing primer pair is 57.5-60.5°C; and / or, the Tm value range corresponding to the HPV39 typing primer pair is 64.5-69.5°C; and / or, the Tm value range corresponding to the HPV6 typing primer pair is 51.5-56.5°C; and / or, the Tm value range corresponding to the HPV56 typing primer pair is 58.5-61.5°C; and / or, the Tm value range corresponding to the HPV42 typing primer pair is 67.5-72.5°C.

[0128] In an optional embodiment, the nucleotide sequences of the upstream primer and the downstream primer of the primer pair for detecting the internal standard are shown as SEQ ID NO.16 and 17 respectively, the nucleotide sequence of the probe is shown as SEQ ID NO.41, and the Tm value range corresponding to the internal standard typing primer pair is 71.5-74.5°C.

[0129] In an optional embodiment, the PCR amplification program and the melting analysis program are as shown in Table 1.

[0130] Table 1

[0131]

[0132] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0133] Example 1 Primer probe design

[0134] This embodiment provides 23 primer pairs, each of which comprises a pair of upstream primers and downstream primers, and also comprises a probe, as follows:

[0135] (1) HPV6 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 18.

[0136] (2) HPV53 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 32.

[0137] (3) HPV58 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 34.

[0138] (4) HPV66 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 36.

[0139] (5) HPV33 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 24.

[0140] (6) HPV56 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 33.

[0141] (7) HPV45 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 29.

[0142] (8) HPV11 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 19.

[0143] (9) HPV35 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 25.

[0144] (10) HPV18 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 21.

[0145] (11) HPV31 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 23.

[0146] (12) HPV52 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 31.

[0147] (13) HPV39 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 26.

[0148] (14) HPV42 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 27.

[0149] (15) HPV16 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 3 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 20.

[0150] (16) HPV26 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 22.

[0151] (17) HPV82 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 40.

[0152] (18) HPV51 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 3 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 30.

[0153] (19) HPV68 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 3 and 15, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 37.

[0154] (20) HPV81 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 39.

[0155] (21) HPV73 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 38.

[0156] (22) HPV59 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 5 and 14, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 35.

[0157] (23) HPV43 typing primer pair: The nucleotide sequences of the upstream primer and the downstream primer are shown as SEQ ID NO. 6 and 11, respectively, and the nucleotide sequence of the probe is shown as SEQ ID NO. 28.

[0158] (24) Primer pair for detecting internal standard: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 16 and 17, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 41.

[0159] The nucleotide sequences of the upstream primer and the downstream primer are shown in Table 2, and the probe sequence is shown in Table 3.

[0160] Table 2 Nucleotide sequences of upstream primers and downstream primers

[0161]

[0162]

[0163] Table 3 Probe sequences

[0164]

[0165] In Table 3, (LNA) indicates that the nucleotide represented by a capital letter adjacent to its left side is a locked nucleic acid.

[0166] Example 2 PCR amplification reaction

[0167] 1. Reagents:

[0168] 1. PCR reaction solution A1:

[0169] Tris-HCl (pH 8.3) 60mM, DMSO 2% (v / v), MgCl2 4mM, dATP, dGTP, dCTP, dUTP 0.4mM each, each primer and probe (the final concentration of each primer and probe is: 0.02μM for the limiting primer, 0.25μM for the non-limiting primer, and 0.18μM for each specific probe), and the balance is ultrapure water. The reaction system uses the UNG enzyme anti-pollution system, so dTTP is replaced by dUTP.

[0170] The primers and probes in the PCR reaction solution A1 are the primers and probes in the HPV18, HPV66, HPV53, HPV35, HPV26, HPV52, HPV11, HPV81, HPV59, HPV43 and HPV16 typing primer pairs provided in Example 1 and the primer pair used to detect the internal standard.

[0171] PCR reaction solution A2: Tris-HCl (pH 8.3) 60 mM, DMSO 2% (v / v), MgCl2 4 mM, dATP, dGTP, dCTP, dUTP 0.4 mM each, each primer and probe (the final concentration of each primer and probe is: limiting primer is 0.015 μM, non-restrictive primer is 0.35 μM, specific probe is 0.18 μM each), and the balance is ultrapure water;

[0172] The primers and probes in the PCR reaction solution A2 are the primers and probes in the HPV33, HPV51, HPV68, HPV73, HPV31, HPV45, HPV58, HPV82, HPV39, HPV6, HPV56 and HPV42 typing primer pairs provided in Example 1 and the primer pair used to detect the internal standard.

[0173] 2. PCR reaction solution B: Tris-HCl (pH 8.3) 6mM, KCl 60mM, 0.2U of UNG enzyme (per 15μL PCR reaction solution B), 5U of Taq DNA polymerase (per 15μL PCR reaction solution B), and the balance is ultrapure water.

[0174] 3. Negative control: plasmid solution containing internal standard DNA fragment.

[0175] Positive control: plasmid solution containing internal standard DNA fragment and plasmid solution containing HPV35, HPV52, HPV16, HPV31, HPV58, HPV56 typing primers for target genes.

[0176] 2. Amplification method:

[0177] 1. Nucleic acid extraction: Use the magnetic bead method to extract sample nucleic acid (nucleic acid extraction or purification reagent: Henan Zhengxiebei 20180037), and the negative control and positive control are also involved in the extraction. After nucleic acid extraction, it can be used for PCR detection immediately, or the extracted nucleic acid can be transferred to a centrifuge tube and stored at 20°C for subsequent detection.

[0178] 2. Reaction system preparation:

[0179] Each tube of reaction system is 50 μL. Add 15 μL of PCR reaction solution A1 or PCR reaction solution A2, 15 μL of PCR reaction solution 3, and 20 μL of nucleic acid extract to the amplification tube in order, cover the reaction tube tightly, mark it, centrifuge it instantly, and transfer it to the PCR amplification area for PCR detection.

[0180] 3. Machine testing:

[0181] The test was performed on a MacroSLAN 96P or SLAN 96S. The project type was selected as "Standard Melting Curve" and the FAM, HEX, ROX and Cy5 channels were selected at the same time. The PCR amplification procedure was as follows:

[0182] Table 4 Reaction procedure

[0183]

[0184] 4. Result analysis:

[0185] After the amplification reaction is completed, the instrument automatically saves the results, which can be automatically analyzed using the software, or the baseline and threshold line values ​​can be manually adjusted for analysis.

[0186] The negative control should meet the following requirements: Tube 1 and Tube 2 have peaks at 71.5-74.5°C in the FAM channel, and no melting peaks in the HEX, ROX, and CY5 channels.

[0187] The positive control should meet the following requirements: Tube 1 and Tube 2 have peaks at 71.5-74.5°C in the FAM channel, melting peaks in the HEX, ROX, and CY5 channels, and the typing results are HPV35, HPV52, HPV16, HPV31, HPV58, and HPV56, respectively.

[0188] The Tm value ranges of each type of tube 1 in each channel are shown in Table 5, and the Tm value ranges of each type of tube 2 in each channel are shown in Table 6.

[0189] Table 5 Tm value range of each type of tube 1 in each channel

[0190]

[0191] Table 6 Tm value range of each type of tube 2 in each channel

[0192]

[0193] The analysis method is as follows:

[0194] (1) After both negative and positive controls meet the requirements, the sample results are analyzed. If the negative and positive controls do not meet the requirements, the test is invalid and needs to be repeated.

[0195] (2) If the internal standard has a melting peak at the corresponding Tm value, and each type of the FAM, HEX, ROX, and CY5 channels has a melting curve at the corresponding Tm value, it can be judged as positive for the corresponding type;

[0196] (3) If the internal standard has a melting peak at the corresponding Tm value, and each type of the FAM, HEX, ROX, and CY5 channels has no melting curve at the corresponding Tm value, it can be judged as negative for the corresponding type;

[0197] (4) If the internal standard has no melting peak at the corresponding Tm value, the test is not of reference value and needs to be repeated.

[0198] Example 3: Detection experiment of various types of samples

[0199] A positive plasmid sample containing one of the types of HPV 6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82 was tested according to the method described in Example 2. The results are shown in Figure 1 to Figure 23 From the figure, it can be seen that the positive samples all have corresponding melting peaks within the corresponding Tm value range of each type.

[0200] Example 4: Sensitivity and specificity experiments

[0201] 1. Sensitivity experiment: The HPV types within the detection range of the second-generation human papillomavirus whole genome typing national reference material were diluted to 100 copies / mL using TE buffer (10 mM Tris-HCl, 1 mM EDTA PH = 8.0) for detection. The results are shown in Table 7 (only the target Tm value is shown), indicating that the HPV types within the detection range of the method provided in Example 2 are all detected and can be accurately typed.

[0202] Table 7 Second-generation human papillomavirus whole genome typing national reference test results

[0203] Type FAM(℃) HEX(℃) ROX(℃) CY5(℃) HPV6 Negative Negative Negative 55.61 HPV11 Negative Negative 54.16 Negative HPV16 Negative Negative Negative 67.32 HPV18 56.21 Negative Negative Negative HPV26 Negative 68.93 Negative Negative HPV31 Negative 66.21 Negative Negative HPV33 53.92 Negative Negative Negative HPV35 Negative 64.82 Negative Negative HPV39 Negative Negative 67.29 Negative HPV42 Negative Negative Negative 69.83 HPV43 Negative Negative Negative 47.05 HPV45 Negative Negative 44.57 Negative HPV51 60.66 Negative Negative Negative HPV52 Negative Negative 45.92 Negative HPV53 Negative 57.41 Negative Negative HPV56 Negative Negative Negative 59.13 HPV58 Negative Negative 51.86 Negative HPV59 Negative Negative 67.92 Negative HPV66 62.13 Negative Negative Negative HPV68 Negative 43.01 Negative Negative HPV73 Negative 55.03 Negative Negative HPV81 Negative Negative 61.83 Negative HPV82 Negative Negative 59.03 Negative

[0204] 2. Specificity detection: The HPV negative reference materials NC1 to NC5 in the second generation human papillomavirus whole genome typing national reference materials were tested, which are CMV, HSV-1, CT, GBS, and UU. The results are shown in Table 8, indicating that there is no cross reaction with other pathogens and the specificity is good.

[0205] Table 8 Second-generation human papillomavirus whole genome typing national negative reference

[0206] Type FAM HEX ROX CY5 CMV Negative Negative Negative Negative HSV-1 Negative Negative Negative Negative CT Negative Negative Negative Negative GBS Negative Negative Negative Negative UU Negative Negative Negative Negative

[0207] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A HPV typing primer probe composition, characterized in that: Includes at least one of the following primer pairs: HPV6 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 18; HPV53 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.32; HPV58 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 1 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 34; HPV66 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.36; HPV33 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.24; HPV56 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.33; HPV45 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.1 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.29; HPV11 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 8, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 19; HPV35 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 25; HPV18 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 21; HPV31 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 23; HPV52 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 31; HPV39 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 26; HPV42 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 2 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 27; HPV16 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 9, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.20; HPV26 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.22; HPV82 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 10, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.40; HPV51 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 12, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.30; HPV68 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.3 and 15, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.37; HPV81 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and 7, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 39; HPV73 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO. 4 and 13, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO. 38; HPV59 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.5 and 14, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.35; and, HPV43 typing primer pair: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.6 and 11, respectively, and the nucleotide sequence of the probe is shown in SEQ ID NO.

28.

2. The HPV typing primer-probe composition according to claim 1, characterized in that: Each probe is independently modified with a luminescent group and a quenching group; Optionally, the luminescent group includes one or more of quantum dots, FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX and Texas Red; Optionally, the luminescent group is modified at the 5' end of the probe; Optionally, the quenching group includes one or more of BHQ1, BHQ2, BHQ3 and MGB; Optionally, the quencher group is modified at the 3' end of the probe.

3. The HPV typing primer-probe composition according to claim 1, characterized in that: At least one probe in at least one primer pair contains at least one modified deoxynucleotide; Optionally, the modification comprises replacing at least one deoxynucleotide in the probe with a locked nucleic acid; Optionally, the 10th G of the probe with a nucleotide sequence as shown in SEQ ID NO.22 is a locked nucleic acid; and / or, The nucleotide sequence of the probe shown in SEQ ID NO.23 has a locked nucleic acid at position 9 G and a locked nucleic acid at position 12 A; and / or, The nucleotide sequence of the probe shown in SEQ ID NO.25 is a locked nucleic acid at position 5 T and a locked nucleic acid at position 12 G; and / or, The nucleotide sequence of the probe shown in SEQ ID NO.26, the 12th C is a locked nucleic acid; and / or, The nucleotide sequence of the probe shown in SEQ ID NO.32, G at position 11 is a locked nucleic acid; and / or, The nucleotide sequence of the probe shown in SEQ ID NO.33, position 10 A is a locked nucleic acid; and / or, The 9th T of the probe with the nucleotide sequence as shown in SEQ ID NO.35 is a locked nucleic acid; and / or, The 12th C of the probe with the nucleotide sequence shown in SEQ ID NO.36 is a locked nucleic acid.

4. The HPV typing primer-probe composition according to claim 1, characterized in that: Also included are primer pairs for detecting the internal standard; Optionally, the target gene of the internal standard includes β-globin; Optionally, the nucleotide sequences of the upstream primer and the downstream primer of the primer pair for detecting the internal standard are shown as SEQ ID NO.16 and 17 respectively, and the nucleotide sequence of the probe is shown as SEQ ID NO.41; Optionally, the 10th C of the probe with a nucleotide sequence as shown in SEQ ID NO.41 is a locked nucleic acid.

5. The HPV typing primer-probe composition according to claim 1, characterized in that: The upstream primer is a limiting primer, and the downstream primer is a non-limiting primer; Optionally, the working concentration of the upstream primer in each primer pair is independently 0.01 to 0.05 μM; Optionally, the working concentration of the downstream primer in each primer pair is independently 0.05 to 0.5 μM; Optionally, the working concentration of the probe in each primer pair is independently 0.05 to 0.3 μM, preferably 0.18 μM; Optionally, the working concentration of the upstream primer in the HPV18, HPV66, HPV53, HPV35, HPV26, HPV52, HPV11, HPV81, HPV59, HPV43 and HPV16 typing primer pairs is 0.02 μM, and the working concentration of the downstream primer is 0.25 μM; Optionally, the working concentration of the upstream primer in the HPV33, HPV51, HPV68, HPV73, HPV31, HPV45, HPV58, HPV82, HPV39, HPV6, HPV56 and HPV42 typing primer pairs is 0.015 μM, and the working concentration of the downstream primer is 0.35 μM.

6. The HPV typing primer-probe composition according to any one of claims 1 to 5, characterized in that: comprising a first primer-probe combination and / or a second primer-probe combination; The first primer-probe composition includes HPV18, HPV66, HPV53, HPV35, HPV26, HPV52, HPV11, HPV81, HPV59, HPV43 and HPV16 typing primer pairs; The second primer-probe composition includes HPV33, HPV51, HPV68, HPV73, HPV31, HPV45, HPV58, HPV82, HPV39, HPV6, HPV56 and HPV42 typing primer pairs; Optionally, the first primer-probe composition further comprises a primer pair for detecting an internal standard; and / or, the second primer-probe composition further comprises a primer pair for detecting an internal standard; Optionally, the probes in the HPV18 and HPV66 typing primer pairs are all labeled with a first luminescent group, the probes in the HPV53, HPV35 and HPV26 typing primer pairs are all labeled with a second luminescent group, the probes in the HPV52, HPV11, HPV81 and HPV59 typing primer pairs are all labeled with a third luminescent group, and the probes in the HPV43 and HPV16 typing primer pairs are all labeled with a fourth luminescent group; and the first luminescent group, the second luminescent group, the third luminescent group and the fourth luminescent group are different from each other; And / or, the probes in the HPV33 and HPV51 typing primer pairs are all labeled with a fifth luminescent group, the probes in the HPV68, HPV73 and HPV31 typing primer pairs are all labeled with a sixth luminescent group, the probes in the HPV45, HPV58, HPV82 and HPV39 typing primer pairs are all labeled with a seventh luminescent group, and the probes in the HPV6, HPV56 and HPV42 typing primer pairs are all labeled with an eighth luminescent group; and the fifth luminescent group, the sixth luminescent group, the seventh luminescent group and the eighth luminescent group are different from each other; Optionally, the first luminescent group is FAM, the second luminescent group is HEX, the third luminescent group is ROX and the fourth luminescent group is CY5; Optionally, the fifth luminescent group is FAM, the sixth luminescent group is HEX, the seventh luminescent group is ROX and the eighth luminescent group is CY5.

7. Use of the HPV typing primer-probe combination according to any one of claims 1 to 6 in the preparation of HPV typing detection products or HPV typing for non-diagnostic and treatment purposes.

8. A kit for HPV typing detection, characterized in that: A composition comprising the HPV typing primer and probe according to any one of claims 1 to 6; Optionally, the kit further comprises one or more of a buffer, DMSO, a preservative, a salt, dATP, dCTP, dGTP, dTTP, dUTP, a DNA polymerase, a negative control, and a positive control; Optionally, the kit includes a reaction solution A, which includes: Tris-HCl, DMSO, magnesium ions and dNTPs and the HPV typing primer probe composition; Optionally, the reaction solution A includes reaction solution A1 and reaction solution A2, wherein the reaction solution A1 contains the first primer-probe combination described in claim 6; and the reaction solution A2 contains the second primer-probe combination described in claim 6; Optionally, the kit includes a reaction solution B, wherein the reaction solution B includes a DNA polymerase and an enzyme for preventing false positives; Optionally, the reaction solution B includes Tris-HCl, KCl, an enzyme for preventing false positives and a DNA polymerase; the DNA polymerase includes Taq DNA polymerase, and the enzyme for preventing false positives includes UNG enzyme.

9. A method for HPV typing for non-diagnostic and therapeutic purposes, characterized in that The HPV typing includes distinguishing the following types of HPV: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV42, HPV43, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV81 and HPV82, and the HPV typing method includes: (A) obtaining the temperature range of the Tm value for amplifying the corresponding HPV type by the primer pair described in any one of claims 1 to 6; (B) using the primer-probe combination according to any one of claims 1 to 6 or the kit according to claim 8 to amplify the sample to be tested, and the primer pairs with overlapping Tm value ranges of corresponding types are assigned to different fluorescent channels; (C) obtaining a melting curve of the sample to be tested in each fluorescent channel, and comparing it with the Tm value range of step (A); if a primer pair of a fluorescent channel has a melting peak within the Tm value range of the corresponding type, it is determined that the HPV type corresponding to the primer pair exists in the sample to be tested; Optionally, the step (B) further comprises amplifying a negative control and a positive control, and step (B) is performed when the melting curve data of the negative control and the positive control are correct; Optionally, each reaction system contains at least one primer pair for detecting an internal standard, and step (B) is performed on the premise that the melting curve data of the internal standard is correct; Optionally, the step (B) comprises two reaction systems, and the two reaction systems respectively use the first primer-probe combination and the second primer-probe combination described in claim 6 for amplification.

10. The HPV typing method according to claim 9, characterized in that: The Tm value range corresponding to the HPV18 typing primer pair is 54.5-57.5°C; and / or, the Tm value range corresponding to the HPV66 typing primer pair is 60.5-63.5°C; and / or, the Tm value range corresponding to the HPV53 typing primer pair is 56.5-59.5°C; and / or, the Tm value range corresponding to the HPV35 typing primer pair is 62.5-65.5°C; and / or, the Tm value range corresponding to the HPV26 typing primer pair is 67.5-70.5°C; and / or, the Tm value range corresponding to the HPV52 typing primer pair is 44.5- 47.5°C; and / or, the Tm value range of the HPV11 typing primer pair is 52.5-55.5°C; and / or, the Tm value range of the HPV81 typing primer pair is 60.5-63.5°C; and / or, the Tm value range of the HPV59 typing primer pair is 66.5-69.5°C; and / or, the Tm value range of the HPV43 typing primer pair is 43.5-48.5°C; and / or, the Tm value range of the HPV16 typing primer pair is 64.0-70.0°C; and / or, the Tm value range of the HPV33 typing primer pair is The Tm value range of the HPV51 typing primer pair is 58.5-61.5°C; and / or, the Tm value range of the HPV68 typing primer pair is 41.5-44.5°C; and / or, the Tm value range of the HPV73 typing primer pair is 53.5-56.5°C; and / or, the Tm value range of the HPV31 typing primer pair is 62.5-67.5°C; and / or, the Tm value range of the HPV45 typing primer pair is 43.5-46.5°C; and / or, the Tm value range of the HPV51 typing primer pair is 58.5-61.5°C; and / or, the Tm value range of the HPV68 typing primer pair is 41.5-44.5°C; and / or, the Tm value range of the HPV73 typing primer pair is 53.5-56.5°C; and / or, the Tm value range of the HPV31 typing primer pair is 62.5-67.5°C; and / or, the Tm value range of the HPV45 typing primer pair is 43.5-46.5°C; and / or, The Tm value range corresponding to the HPV8 typing primer pair is 50.5-53.5°C; and / or, the Tm value range corresponding to the HPV82 typing primer pair is 57.5-60.5°C; and / or, the Tm value range corresponding to the HPV39 typing primer pair is 64.5-69.5°C; and / or, the Tm value range corresponding to the HPV6 typing primer pair is 51.5-56.5°C; and / or, the Tm value range corresponding to the HPV56 typing primer pair is 58.5-61.5°C; and / or, the Tm value range corresponding to the HPV42 typing primer pair is 67.5-72.5°C.

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