A human papillomavirus detection kit

Abstract

The present application relates to primer probe combination or kit for HPV detection or typing and its method, specifically, the primer probe combination comprises nucleic acid complex consisting of detection probe, signal probe and tag sequence. When the target nucleic acid exists, the signal probe is hydrolyzed under the action of DNA polymerase, so that its melting curve signal is low, thereby for judging whether HPV exists in the sample, and the HPV is typed according to the melting temperature. The method of the present application is simple in operation steps, low in cost and does not need other auxiliary equipment.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a human papillomavirus (HPV) detection kit. Background Technology

[0002] Cervical cancer is the most common malignant tumor among women, primarily occurring in multiparous women during early menopause. Approximately 190,000 women worldwide die from cervical cancer each year, with more than three-quarters of these deaths occurring in developing countries. Cervical cancer ranks seventh in incidence among all cancers, and third among women, after breast and colorectal cancer. In developing countries, less than 50% of women diagnosed with cervical cancer survive for more than five years, while in developed countries, the five-year survival rate is approximately 66%.

[0003] Existing cervical cancer screening technologies can be divided into two categories: 1. Morphology-based methods: identifying abnormal morphologies through examination at the cellular or tissue level; 2. Molecular biology-based methods: detecting biomarkers for cervical cancer, such as cervical epithelial tumors.

[0004] HPV screening: Researchers are currently comparing the advantages and disadvantages of HPV testing versus Pap testing as methods for cervical cancer screening. Because HPV is difficult to culture in vitro, and not all infected individuals have detectable antibody responses, HPV DNA testing is the best non-invasive method for detecting HPV infection. Currently, there are three main types of HPV testing methods:

[0005] The first category is direct probe binding, such as Southern blotting, dot blotting, and in situ hybridization filtering. These methods generally suffer from drawbacks such as low sensitivity and cumbersome operation.

[0006] The second category is signal amplification methods. Most research institutes use Digene's first and second generation Hybrid Capture (HC) systems, the only FDA-approved technology. Others use different PCR methods to detect HPV. Compared to the HC method, PCR has higher detection sensitivity, but the detection sensitivity of the second-generation HC2 has been greatly improved, approaching the level of PCR.

[0007] The third category is PCR-based amplification of sequence fragments. PCR methods use type-specific or universal primers to amplify the target fragment and hybridize it with a specific probe. Quantitative PCR (qPCR) uses various probes, including TaqMan probes, molecular beacons, and scorpions, which offer advantages such as high analytical sensitivity. However, due to current technological limitations, most instruments only offer four fluorescent dyes to choose from, thus allowing for the detection of a maximum of four different targets per tube. Flow cytometry, by adding different proportions of fluorescent dyes to microspheres, can detect up to 99 different targets. Therefore, commercial kits are available. However, its detection method relies on the PCR product, and the signal value reflects the concentration of the final PCR product, which is not highly correlated with the original virus concentration. Furthermore, because PCR products and probes require single-stranded hybridization, the high-temperature process required for melting hinders the application of automation technologies.

[0008] For the need for multiplex detection, in addition to the technologies mentioned above, there are many solutions based on fluorescence PCR. For example, patent CN 105087827 uses 8 reaction tubes, each labeled with a different color fluorescent group, to detect 16 types of HPV. However, an obvious problem with this method is the large number of reaction tubes and PCR reaction materials used. Furthermore, a 96-well fluorescence PCR instrument can only detect 12 samples at a time, which is not ideal in terms of detection speed and cost. In patent CN 106048081, primers are used in conjunction with SYBR Green dye for HPV genotyping. The detection speed and cost are controlled. However, because only one dye is used, it is necessary to place the sample at 70-90℃ for 15 different melting peaks. This means that the Tm values ​​of these different melting peaks differ by only 1℃, which places extremely high demands on reagent stability and instrument. The problem of false positives caused by the melting peak temperatures being too close is a significant drawback.

[0009] Based on the above, although many technologies have been developed for HPV detection, technical shortcomings still exist. Therefore, there is an urgent need in this field to develop a method that is simple to operate, accurate, and capable of detecting multiple HPV types. Summary of the Invention

[0010] The purpose of this invention is to provide a method for detecting 15 types of HPV using degenerate primers.

[0011] A first aspect of the present invention provides a primer-probe set for detecting human papillomavirus, the primer-probe set comprising:

[0012] (i) a detection probe, wherein the nucleotide sequence of the detection probe is selected from one or more of SEQ ID NO: 3 to 17;

[0013] (ii) a signal probe, wherein the nucleotide sequence of the signal probe is selected from one or more of SEQ ID NO: 18–32; and

[0014] (iii) A tag sequence, wherein the nucleotide sequence of the tag sequence is selected from one or more of SEQ ID NO: 33 to 47.

[0015] In another preferred embodiment, the 3' end of the detection probe is phosphorylated.

[0016] In another preferred embodiment, the signal probe is modified with a fluorescent group and a quenching group.

[0017] In another preferred embodiment, the fluorescent group is selected from the group consisting of FAM, VIC, ROX, CY5, or combinations thereof.

[0018] In another preferred embodiment, the quenching group is selected from the group consisting of BHQ, TAMRA, DABCYL, or combinations thereof.

[0019] In another preferred embodiment, the primer-probe set is selected from the group consisting of:

[0020] (a) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 3, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 18, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 33;

[0021] (b) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 4, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 19, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 34;

[0022] (c) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 5, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 20, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 35;

[0023] (d) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 6, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 21, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 36;

[0024] (e) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 7, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 22, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 37;

[0025] (f) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 8, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 23, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 38;

[0026] (g) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 9, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 24, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 39;

[0027] (h) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 10, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 25, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 40;

[0028] (i) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 11, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 26, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 41;

[0029] (j) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 12, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 27, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 42;

[0030] (k) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 13, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 28, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 43;

[0031] (l) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 14, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 29, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 44;

[0032] (m) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 15, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 30, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 45;

[0033] (n) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 16, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 31, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 46;

[0034] (o) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 17, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 32, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 47; or a combination thereof.

[0035] In another preferred embodiment, signal probes having nucleotide sequences as shown in SEQ ID NO: 18, 20, 23 and 31 are modified with the same fluorescent group.

[0036] In another preferred embodiment, the signal probe having the nucleotide sequences shown in SEQ ID NO: 19, 28 and 30 is modified with the same fluorescent group.

[0037] In another preferred embodiment, a signal probe having nucleotide sequences as shown in SEQ ID NO: 24, 21, 32 and 26 is modified with the same fluorescent group.

[0038] In another preferred embodiment, the signal probe having the nucleotide sequences shown in SEQ ID NO: 25, 27, 22 and 29 is modified with the same fluorescent group.

[0039] In another preferred embodiment, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 18, 20, 23 or 31 modifies FAM and BHQ1.

[0040] In another preferred embodiment, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 1, 28 or 30 modifies VIC and BHQ1.

[0041] In another preferred embodiment, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 21, 24, 26 or 32 modifies ROX and BHQ2.

[0042] In another preferred embodiment, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 22, 25, 27 or 29 modifies CY5 and BHQ2.

[0043] In another preferred embodiment, the primer probe set further includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2.

[0044] In another preferred embodiment, the human papillomavirus is selected from the group consisting of: human papillomavirus 16, human papillomavirus 18, human papillomavirus 31, human papillomavirus 33, human papillomavirus 35, human papillomavirus 39, human papillomavirus 45, human papillomavirus 51, human papillomavirus 52, human papillomavirus 53, human papillomavirus 56, human papillomavirus 58, human papillomavirus 59, human papillomavirus 66, or human papillomavirus 68.

[0045] A second aspect of the present invention provides a kit comprising the primer and probe set as described in the first aspect of the present invention.

[0046] In another preferred embodiment, the kit further includes a PCR reaction solution for amplification.

[0047] In another preferred embodiment, the PCR reaction solution comprises PCR buffer, dNTPs, and MgCl2.

[0048] In another preferred embodiment, the kit further includes DNA polymerase.

[0049] In another preferred embodiment, the kit further includes a negative control, preferably purified water.

[0050] In another preferred embodiment, the concentration of the dNTPs in the PCR reaction solution is 0.01–10 mM, more preferably 0.05–5 mM, more preferably 0.1–3 mM, for example, about 0.2 mM.

[0051] In another preferred embodiment, the concentration of MgCl2 in the PCR reaction solution is 0.1–10 mM, more preferably 0.5–5 mM, more preferably 1–3 mM, for example, about 1.5 mM.

[0052] In another preferred embodiment, the kit is used to detect human papillomavirus.

[0053] In another preferred embodiment, the detection probe, signal probe, and tag sequence in the primer-probe set are mixed together.

[0054] A third aspect of the present invention provides a method for detecting human papillomavirus, the method comprising the steps of:

[0055] (s1) Using the primer and probe set as described in the first aspect of the present invention or the kit as described in the second aspect of the present invention, a nucleic acid amplification reaction is performed on the sample to be tested to obtain nucleic acid amplification products; and

[0056] (s2) The nucleic acid amplification product is subjected to melting curve analysis to obtain the detection results.

[0057] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0058] In another preferred embodiment, the method is in vitro.

[0059] In another preferred embodiment, step (s2) includes heating the nucleic acid amplification product at 95°C for 1–5 min, then cooling it to 15–20°C, then heating it to 65–80°C at a rate of 0.03–0.1°C / s, and detecting the fluorescence signal to obtain the detection result.

[0060] In another preferred embodiment, the amount of DNA polymerase in the nucleic acid amplification reaction is 0.1–5 U, more preferably 0.5–3 U, and even more preferably 0.5–2 U.

[0061] In another preferred embodiment, in the nucleic acid amplification reaction, the reaction concentrations of the upstream primer and the downstream primer are each independently 50–500 nM, more preferably 100–400 nM, more preferably 100–300 nM, for example, about 200 nM.

[0062] In another preferred embodiment, the nucleic acid amplification reaction includes heating at 95°C for 10 min; denaturation at 95°C for 15 s, extension at 60°C for 20 s, and annealing at 72°C for 15 s (40-50 cycles).

[0063] In another preferred embodiment, the amount of the sample to be tested is 1 to 100 μL, more preferably 1 to 50 μL, more preferably 1 to 10 μL, for example, about 5 μL.

[0064] In another preferred embodiment, the reaction concentrations of the detection probe and the tag sequence are each 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0065] In another preferred embodiment, the reaction concentration of the signal probe is 1 nM to 300 mM, more preferably 10 nM to 50 mM, and even more preferably 20 to 500 nM, for example about 50 nM.

[0066] In another preferred embodiment, the molar ratio of the detection probe, signal probe and tag sequence is 1-10:1-5:1-10, more preferably 1-8:1-3:1-8, more preferably 1-5:1-3:1-5, for example, 2:1:2.

[0067] In another preferred embodiment, the criterion for judgment of the method is:

[0068] If the melting curve height H1 of the test sample decreases by more than 10% compared to the melting curve height H0 of the negative control, i.e. (H0-H1) / H0>10%, it indicates the presence of human papillomavirus in the test sample.

[0069] In another preferred embodiment, the method is also used for typing human papillomavirus.

[0070] In another preferred embodiment, the Tm values ​​of the signal probes are different within the same system. In yet another preferred embodiment, the Tm values ​​of the melting curves of the method vary considerably; when designing the primer probes, detection probes, and tag sequences, the Tm values ​​of their melting curves can be selected between 20-70°C, preferably 30-70°C.

[0071] In another preferred embodiment, the signal probes of the same fluorescence channel have different Tm values. The Tm value interval of the melting curves between different types is ≥5℃, preferably ≥6℃, 7℃, 8℃, 9℃, or even 10℃ and above.

[0072] In another preferred embodiment, the melting temperatures of signal probes corresponding to different targets are different in the same reaction system.

[0073] In another preferred embodiment, in the same reaction system, the Tm value interval of the melting curves between signal probes corresponding to different targets is ≥5℃, preferably ≥6℃, ≥7℃, ≥8℃, or ≥9℃, more preferably ≥10℃.

[0074] In another preferred embodiment, the criterion for judgment of the method is:

[0075] To determine the type of human papillomavirus (HPV), the melting curve height H1 of the test sample must decrease by more than 10% compared to the melting curve height H0 of the negative control, i.e., (H0-H1) / H0 > 10%, and the melting temperature must be used to determine the type of HPV.

[0076] In another preferred embodiment, the criterion for judgment of the method is:

[0077] In the same fluorescence channel, when Tm is 50.5±1.0℃, the sample to be tested is HPV16;

[0078] When Tm is 57.0±1.0℃, the sample to be tested is HPV31;

[0079] When Tm is 33.5±1.0℃, the sample to be tested is HPV39;

[0080] When Tm is 42.0±1.0℃, the sample to be tested is HPV66.

[0081] In the same fluorescence channel, when Tm is 38.5±1.0℃, the sample to be tested is HPV18;

[0082] When Tm is 57.0±1.0℃, the sample to be tested is HPV56;

[0083] When Tm is 50.0±1.0℃, the sample to be tested is HPV59.

[0084] In another preferred embodiment, the criterion for judgment of the method is:

[0085] In the same fluorescence channel, when Tm is 30.0±1.0℃, the sample to be tested is HPV45;

[0086] When Tm is 41.0±1.0℃, the sample to be tested is HPV33;

[0087] When Tm is 47.0±1.0℃, the sample to be tested is HPV68;

[0088] When Tm is 56.5±1.0℃, the sample to be tested is HPV52.

[0089] In another preferred embodiment, the criterion for judgment of the method is:

[0090] In the same fluorescence channel, when Tm is 29.0±1.0℃, the sample to be tested is HPV51;

[0091] When Tm is 39.0±1.0℃, the sample to be tested is HPV53;

[0092] When Tm is 48.0±1.0℃, the sample to be tested is HPV35;

[0093] When Tm is 58.0±1.0℃, the sample to be tested is HPV58.

[0094] In another preferred embodiment, the detection limit of the method is ≤100 copies, more preferably ≤50 copies, and even more preferably ≤25 copies.

[0095] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0096] Figure 1 The results of detecting channel 1 (corresponding to FAM fluorescent groups) of 15 types of HPV using the method of the present invention are shown.

[0097] Figure 2 The results of detecting channel 2 (corresponding to VIC fluorescent groups) of 15 types of HPV using the method of the present invention are shown.

[0098] Figure 3 The results of detecting channel 3 (corresponding to ROX fluorescent groups) of 15 types of HPV using the method of the present invention are shown.

[0099] Figure 4 The results of detecting channel 4 (corresponding to CY5 fluorescent group) of 15 types of HPV using the method of the present invention are shown. Detailed Implementation

[0100] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a primer-probe combination, kit, and method for simultaneously detecting and genotyping 15 types of HPV. The primer-probe combination comprises a nucleic acid complex consisting of a detection probe, a signal probe, and a tag sequence. When the target nucleic acid is present, the signal probe is hydrolyzed by DNA polymerase, resulting in a low melting curve signal, which is then used to determine the presence of HPV in the sample. This invention was completed based on this discovery.

[0101] the term

[0102] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0103] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0104] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0105] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0106] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0107] As used in this article, the terms “deconstruction temperature” and “Tm value” are used interchangeably.

[0108] As used herein, the term "Rm" refers to the fluorescence value variable corresponding to different melting peak values. This fluorescence value variable is automatically output by the device and is obtained by subtracting the sample's background (automatically removed by the device) from the absolute value of the melting peak value displayed in the melting curve spectrum.

[0109] Human papillomavirus

[0110] Human papillomavirus (HPV) is a genus of papillomavirus A belonging to the family Papillomaviridae. It is a spherical DNA virus that causes proliferative lesions on human skin and mucous membranes. Based on their pathogenicity, it can be broadly classified into high-risk and low-risk types. High-risk HPV types, such as HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, are closely related to the development of malignant tumors such as cervical cancer. Persistent infection with high-risk HPV is a major risk factor for cervical cancer. More than 90% of cervical cancers are associated with high-risk HPV infection.

[0111] The primer probe set of the present invention

[0112] The primer and probe set of the present invention is used for detecting or typing human papillomavirus, including:

[0113] (a) A detection probe, wherein the nucleotide sequence of the detection probe is selected from one or more of SEQ ID NO: 3 to 17;

[0114] (b) a signal probe, wherein the nucleotide sequence of the signal probe is selected from one or more of SEQ ID NO: 18–32; and

[0115] (c) A tag sequence, wherein the nucleotide sequence of the tag sequence is selected from one or more of SEQ ID NO: 33 to 47.

[0116] In a preferred embodiment, the 3' end of the detection probe is phosphorylated.

[0117] In a preferred embodiment, the signal probe is modified with a fluorescent group and a quenching group.

[0118] In a preferred embodiment, the primer-probe set is selected from the group consisting of:

[0119] (a) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 3, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 18, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 33;

[0120] (b) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 4, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 19, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 34;

[0121] (c) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 5, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 20, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 35;

[0122] (d) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 6, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 21, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 36;

[0123] (e) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 7, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 22, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 37;

[0124] (f) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 8, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 23, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 38;

[0125] (g) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 9, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 24, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 39;

[0126] (h) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 10, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 25, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 40;

[0127] (i) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 11, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 26, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 41;

[0128] (j) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 12, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 27, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 42;

[0129] (k) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 13, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 28, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 43;

[0130] (l) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 14, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 29, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 44;

[0131] (m) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 15, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 30, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 45;

[0132] (n) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 16, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 31, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 46;

[0133] (o) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 17, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 32, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 47; or a combination thereof.

[0134] In a preferred embodiment, signal probes having nucleotide sequences as shown in SEQ ID NO: 18, 20, 23, and 31 are modified with different fluorescent groups; signal probes having nucleotide sequences as shown in SEQ ID NO: 19, 28, and 30 are modified with different fluorescent groups; signal probes having nucleotide sequences as shown in SEQ ID NO: 24, 21, 32, and 26 are modified with different fluorescent groups; and signal probes having nucleotide sequences as shown in SEQ ID NO: 25, 27, 22, and 29 are modified with different fluorescent groups. The purpose of modifying the signal probes with different fluorescent groups is to detect multiple targets in a single tube based on the different fluorescence signals.

[0135] In a preferred embodiment, the primer probe set further includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2.

[0136] In a preferred embodiment, the primer-probe set includes the following:

[0137]

[0138]

[0139] The reagent kit of the present invention

[0140] The kit of the present invention is used for the detection or typing of human papillomavirus and contains the primer and probe set of the present invention.

[0141] In a preferred embodiment, the kit further includes a PCR reaction solution for amplification; the PCR reaction solution comprises PCR buffer, dNTPs, and MgCl2.

[0142] In a preferred embodiment, the kit further includes DNA polymerase.

[0143] In a preferred embodiment, the kit further includes a negative control, preferably purified water.

[0144] In a preferred embodiment, the concentration of the dNTPs in the PCR reaction solution is 0.01–10 mM, more preferably 0.05–5 mM, more preferably 0.1–3 mM, for example, about 0.2 mM. In a preferred embodiment, the concentration of the MgCl2 in the PCR reaction solution is 0.1–10 mM, more preferably 0.5–5 mM, more preferably 1–3 mM, for example, about 1.5 mM.

[0145] The method of the present invention

[0146] As used herein, the terms "method of the present invention," "method for detecting human papillomavirus," and "method for typing human papillomavirus" are used interchangeably, including the following steps:

[0147] (s1) Using the primer and probe set of the present invention or the kit of the present invention, a nucleic acid amplification reaction is performed on the sample to be tested to obtain nucleic acid amplification products; and

[0148] (s2) The nucleic acid amplification product is subjected to melting curve analysis to obtain the detection results.

[0149] In a preferred embodiment, step (s2) includes heating the nucleic acid amplification product at 95°C for 1–5 min, then cooling it to 15–20°C, and then heating it to 65–80°C at a rate of 0.03–0.1°C / s, and detecting the fluorescence signal to obtain the detection result.

[0150] In a preferred embodiment, the criterion for judgment of the method is:

[0151] If the melting curve height H1 of the test sample decreases by more than 90% compared to the melting curve height H0 of the negative control ((H0-H1) / H0>90%), it indicates the presence of human papillomavirus in the test sample.

[0152] In a preferred embodiment, the amount of DNA polymerase in the nucleic acid amplification reaction is 0.1–5 U, more preferably 0.5–3 U, and even more preferably 0.5–2 U. In a preferred embodiment, the reaction concentrations of the upstream and downstream primers in the nucleic acid amplification reaction are each independently 50–500 nM, more preferably 100–400 nM, and even more preferably 100–300 nM, for example, about 200 nM. In a preferred embodiment, the nucleic acid amplification reaction includes heating at 95°C for 10 min; denaturation at 95°C for 15 s, extension at 60°C for 20 s, and annealing at 72°C for 15 s (40–50 cycles).

[0153] In a preferred embodiment, the molar ratio of the detection probe, signal probe, and tag sequence is 1–10:1–5:1–10, more preferably 1–8:1–3:1–8, more preferably 1–5:1–3:1–5, for example, 2:1:2. In a preferred embodiment, the reaction concentrations of the detection probe and tag sequence are each 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example, about 100 nM. In a preferred embodiment, the reaction concentration of the signal probe is 1 nM to 300 mM, more preferably 10 nM to 50 mM, more preferably 20 to 500 nM, for example, about 50 nM.

[0154] In a preferred embodiment, the method of the present invention includes a PCR detection method using degenerate primers to quantitatively detect 15 HPV types in a single tube and to genotype the samples by combining melting curve analysis. The method of the present invention can genotype 15 HPV types, including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, or 68.

[0155] The method of this invention includes a nucleic acid complex formed by base complementarity of a detection probe, a signal probe, and a tag sequence. By introducing a detection probe with 3' phosphorylation modification, during amplification, the 3' phosphorylation-modified detection probe is hydrolyzed by Taq polymerase, releasing the detection probe. The detection probe acts as a primer to extend the tag sequence, and the signal probe is hydrolyzed during the extension process. After PCR, the amount of signal probe decreases. By observing the signal change of the melting peak corresponding to the fluorescence channel and the corresponding melting temperature, the purpose of genotyping 15 HPV types can be achieved.

[0156] In a preferred embodiment, the method of the present invention includes the following steps:

[0157] a) Synthesize a pair of degenerate primers based on the specific nucleic acid sequence of the HPV gene to be detected, with a distance of 160-200 bases between the upstream and downstream primers; design and synthesize 3' phosphorylation modified detection probes based on the sequence located between the two primers in the specific nucleic acid sequence of each type of HPV gene; and design a set of signal probes that can generate different Tm values ​​in different fluorescence channels and a tag sequence in some regions that can be complementary to the signal probes.

[0158] b) A fluorescent reaction system is composed of primers, fluorescent probes, signal probes and tag sequences, wherein the concentration of magnesium ions is 5-10 mM, Taq polymerase is 0.5-2 U, and the concentration of various primers and probes is 100-200 nM.

[0159] c) Extract DNA from the clinical specimen to be tested, add an appropriate amount to the reaction system in step b), mix by centrifugation, and then place the reaction tube into a real-time quantitative PCR instrument for cyclic amplification reaction;

[0160] d) After the reaction is complete, perform melting curve analysis to detect the fluorescence value in the reaction tube;

[0161] e) Compare the melting curves of each type in the negative control with the melting curves of each type in each reaction tube, and determine the presence of a certain type of HPV nucleic acid based on the change in melting curve height.

[0162] The main advantages of this invention include:

[0163] 1. The method of the present invention can detect 15 HPV types simultaneously in the same reaction tube, with good specificity.

[0164] 2. The melting peak of the method of the present invention comes from the probe, which solves the problem that there are still variations in different HPV types.

[0165] 3. The method of the present invention uses a common DNA polymerase with exonuclease activity, which reduces the difficulty of obtaining raw materials.

[0166] 4. The method of the present invention realizes the digital interpretation of results, and achieves fully automated software interpretation of HPV detection.

[0167] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0168] Example 1

[0169] 1. Primers and detection probes

[0170] Different HPV types share some homology with some specificity in their genomes. Degenerate primers are designed based on homologous sequences. The primer and probe design principle for detecting 15 HPV types in this invention is as follows: forward and reverse primers are designed based on sequences with high homology for each HPV type genome, and probe sequences are designed based on sequences with high specificity. PCR is performed in the same tube, and detection probes are used to detect all 15 HPV types simultaneously. The primer and detection probe sequences used in this embodiment are shown in Table 1.

[0171] Table 1 Primer and detection probe sequences

[0172]

[0173]

[0174] 2. Signal probe and tag sequence

[0175] To simultaneously detect 15 HPV types, signal probes and tag sequences need to be added. The signal probes and tag sequences are shown in Tables 2a and 2b.

[0176] Table 2a Signal Probes

[0177] SEQ ID NO: sequence name nucleotide sequence 5'-3' 18 Signal probe 1 FAM-TGGTAGGGAACTCGTTT-BHQ1 19 Signal probe 2 VIC-ATCGGTTGTTGTTCTTTT-BHQ1 20 Signal probe 3 FAM-AGCTCCTATTGCCAACGT-BHQ1 21 Signal probe 4 ROX-TACCTTGATGGTCAGCAA-BHQ1 22 Signal probe 5 CY5-TACTAACATTAAGTTGAGG-BHQ1 23 Signal probe 6 FAM-ACACAGCACTCGTCTTCAA-BHQ1 24 Signal probe 7 ROX-TCGCTTAAGATGGCCGATCC-BHQ1 25 Signal probe 8 CY5-CGCTGTTCTCGTTCCTCACT-BHQ1 26 Signal probe 9 ROX-TCCACAAGCTCCAGCAGCA-BHQ2 27 Signal probe 10 CY5-ACCACAAGCACCTGCTACG-BHQ2 28 Signal probe 11 VIC-AGGTCACAGGACACGCAAC-BHQ2 29 Signal probe 12 CY5-AGTCCGCCTATACGCCTGCT-BHQ2 30 Signal probe 13 VIC-CGCCGATATACCTAGCAAGC-BHQ2 31 Signal probe 14 FAM-TGTTGCTTTTGCTGATTCAC-BHQ2 32 Signal probe 15 ROX-CCGGGCTCAGGTACTCCGA-BHQ2

[0178] Table 2b Label Sequence

[0179] SEQ ID NO: sequence name nucleotide sequence 5'-3' 33 Tag Sequence 1 AAACGAGTTCCCTACCA-TGTAAAACCCACAGGGTC 34 Tag Sequence 2 AAAAGAACAACAACCGAT-AGTCAGCTGATGCACAATC 35 Tag sequence 3 ACGTTGGCAATAGGAGCT-TGTGTATTCAAGGCTCCC 36 Tag sequence 4 TTGCTGACCATCAAGGTA-CAATGCTGCAATCGTGC 37 Tag sequence 5 CCTCAACTTAATGTTAGTA-TAGACGTTGTCCAAATG 38 Tag sequence 6 TTGAAGACGAGTGCTGTGT-CTTCGGAGCTATTGCTG 39 Tag sequence 7 GGATCGGCCATCTTAAGCGA-TGTTCACGTATGTTTTCC 40 Tag sequence 8 AGTGAGGAACGAGAACAGCG-TGCATCACTACAATATGG 41 Tag sequence 9 TGCTGCTGGAGCTTGTGGA-GATTCTGTTGAACTCTC 42 Tag sequence 10 CGTAGCAGGTGCTTGTGGT-GTTCCAGACGCGGCTGT 43 Tag sequence 11 GTTGCGTGTCCTGTGACCT-AACTTAGCGCCAGCAAC 44 Tag sequence 12 AGCAGGCGTATAGGCGGACT-ATACCGCTACCGTACCT 45 Tag sequence 13 GCTTGCTAGGTATATCGGCG-TTAAGTGTAAAACCCAC 46 Tag sequence 14 GTGAATCAGCAAAAGCAACA-GATGTGCATGTAAGACC 47 Tag sequence 15 TCGGAGTACCTGAGCCCGG-AGTTTAGAAACCCCAC

[0180] 3. PCR reaction

[0181] This embodiment uses a non-ionic detergent in conjunction with a high-temperature environment to lyse the HPV outer membrane proteins in the sample, thereby releasing DNA. Simultaneously, Chelex-100 is used to remove impurities such as metal ions that inhibit PCR, thus improving detection efficiency. To simultaneously detect 15 HPV types in the same reaction tube, the primer and probe concentrations and reaction system must be optimized (see Example 2) to avoid interference between primers and probes.

[0182] The optimized PCR system is shown in Table 3a, and the PCR reaction procedure is shown in Table 3b.

[0183] Table 3a PCR system

[0184]

[0185] Table 3b PCR reaction procedure

[0186]

[0187] 4. Melting curve analysis

[0188] To enable simultaneous detection of 15 HPV types in the same reaction tube, the melting curves of the signal probes were analyzed using a real-time PCR instrument. The reaction procedure for melting curve analysis is shown in Table 4.

[0189] Table 4 Reaction Procedure for Melting Curve Analysis

[0190]

[0191] 5. Results

[0192] The melting curve height in each result is compared with the melting curve height in the negative control. A melting curve height decrease of more than 90% is considered positive.

[0193] The results are as follows Figure 1-4 As shown, the Rm values ​​at the melting peaks of all 15 HPV types obtained using the method of this invention showed significant changes. By comparing these Rm values ​​with those from the NTC results, the type information corresponding to the sample can be determined. The Rm results for different samples are shown in Table 5.

[0194] Table 5

[0195]

[0196] Example 2: Optimization of PCR Reaction System

[0197] The PCR system used in this embodiment can be optimized and adjusted with reference to conventional multiplex fluorescent PCR concentrations. However, there is no publicly available technical information, such as literature, regarding the concentration patterns of signal probes and tag sequences. This embodiment will optimize the concentrations of signal probes and tag sequences.

[0198] Table 6a Different PCR systems

[0199]

[0200]

[0201] Table 6b PCR reaction procedure

[0202]

[0203] 4. Melting curve analysis

[0204] To enable simultaneous detection of 15 HPV types in the same reaction tube, the melting curves of the signal probes were analyzed using a real-time PCR instrument. The reaction procedure for melting curve analysis is shown in Table 4.

[0205] Table 7 Reaction Procedure for Melting Curve Analysis

[0206]

[0207] 5. Nucleic acid from 50 clinically collected cervical swab samples was tested using different systems.

[0208] Table 8

[0209]

[0210]

[0211] The results are shown in Table 8. The detection results of systems 1-4 are basically consistent with the results of the sequencing method and can be used for human papillomavirus typing. The results show that the concentrations of signal probes and tag sequences in system 2 of this embodiment have the highest consistency with the sequencing method (gold standard) in the detection results of clinical samples.

[0212] Example 3: Clinical Sample Testing

[0213] Fifty clinically collected cervical swab samples were collected, and DNA was extracted from the samples using a magnetic bead DNA extraction kit (Shanghai TransGen Diagnostics Technology Co., Ltd.). The human papillomavirus nucleic acid in the samples was detected using a commercial human papillomavirus genotyping (23 types) detection kit and the method described in Example 1.

[0214] Table 9

[0215]

[0216]

[0217] The results are shown in Table 5. The results indicate that the method described in this invention achieves sensitivity and specificity for clinical samples that are closer to the gold standard, effectively avoiding the sensitivity issues caused by asymmetric PCR required by current commercial kits. It also offers advantages such as low cost, fewer operational steps, and less equipment usage, demonstrating significant development potential. Furthermore, this method can be used on most current quantitative PCR instruments, achieving multiplexing capabilities without the need for additional instruments, resulting in superior economic benefits.

[0218] Compared with existing technologies, the method of the present invention is a closed-tube detection, which eliminates the need for steps such as opening the tube for hybridization and color development after PCR, significantly shortening the operation time, reducing the use of supporting equipment and consumables, and reducing the pollution problem caused by high concentration PCR products.

[0219] Example 4 Sensitivity Test

[0220] HPV16, HPV31, HPV39, and HPV66 with copy numbers of 10000, 1000, 100, 50, and 25, respectively, were detected according to the method described in Example 1, using the FAM channel as an example. The results are shown in Tables 10 and 11.

[0221] Table 10

[0222]

[0223]

[0224] As shown in Table 10, the method of the present invention can detect HPV as low as 25 copies, and performs excellently in detecting HPV up to 50 copies, as shown in Table 11.

[0225] Table 11

[0226]

[0227]

[0228] As shown in Table 11, the Rm values ​​of Tm corresponding to the melting peaks of each target show that the percentage decrease in melting curve height tends to decrease with decreasing concentration. At high concentrations (10,000 copies), the decrease can reach up to 95.08%, i.e., (216.03-10.62) / 216.03 = 95.08%; at low concentrations (25 copies), the decrease can reach as low as 10%, i.e., (146.54-131.82) / 146.54 = 10.04%. This allows for maintaining sensitivity within a good range while accommodating various types. Therefore, a decrease in melting curve height of at least 10% will result in better sensitivity and discrimination.

[0229] Table 12 Detection probes, signal probes, and tag sequences and their corresponding targets

[0230]

[0231]

[0232] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer and probe set for detecting human papillomavirus, characterized in that, The primer-probe set includes: (i) a detection probe, wherein the nucleotide sequence of the detection probe is selected from one or more of SEQ ID NO: 3 to 17; (ii) a signal probe, wherein the nucleotide sequence of the signal probe is selected from one or more of SEQ ID NO: 18–32; and (iii) A tag sequence, wherein the nucleotide sequence of the tag sequence is selected from one or more of SEQ ID NO: 33 to 47.

2. The primer-probe set as described in claim 1, characterized in that, The 3' end of the detection probe is phosphorylated.

3. The primer-probe set as described in claim 1, characterized in that, The signal probe is modified with fluorescent groups and quenching groups.

4. The primer-probe set as described in claim 1, characterized in that, The primer-probe set is selected from the following group: (a) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 3, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 18, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 33; (b) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 4, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 19, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 34; (c) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 5, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 20, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 35; (d) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 6, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 21, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 36; (e) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 7, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 22, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 37; (f) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 8, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 23, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 38; (g) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 9, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 24, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 39; (h) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 10, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 25, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 40; (i) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 11, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 26, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 41; (j) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 12, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 27, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 42; (k) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 13, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 28, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 43; (l) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 14, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 29, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 44; (m) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 15, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 30, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 45; (n) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 16, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 31, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 46; (o) A detection probe having a nucleotide sequence as shown in SEQ ID NO: 17, a signal probe having a nucleotide sequence as shown in SEQ ID NO: 32, and a tag sequence having a nucleotide sequence as shown in SEQ ID NO: 47; or a combination thereof.

5. The primer-probe set as described in claim 1, characterized in that, The primer-probe set further includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO:

2.

6. A reagent kit, characterized in that, The kit contains the primer and probe set as described in claim 1.

7. The kit according to claim 6, characterized in that, The kit also includes PCR reaction solution for amplification.

8. A method for detecting human papillomavirus, characterized in that, The method includes the following steps: (s1) Using the primer and probe set as described in claim 1 or the kit as described in claim 5, perform a nucleic acid amplification reaction on the sample to be tested to obtain nucleic acid amplification products; and (s2) The nucleic acid amplification product is subjected to melting curve analysis to obtain the detection results.

9. The method as described in claim 8, characterized in that, Step (s2) includes heating the nucleic acid amplification product at 95°C for 1–5 min, then cooling it to 15–20°C, and then heating it to 65–80°C at a rate of 0.03–0.1°C / s, while detecting the fluorescence signal to obtain the detection result.

10. The method as described in claim 8, characterized in that, The judgment criteria for the method are as follows: If the melting curve height of the test sample decreases by more than 10% compared to that of the negative control, it indicates the presence of human papillomavirus in the test sample.

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