CN113584225B

CN113584225B - Primer and probe combination for detecting HPV (human papillomavirus) virus, typing detection reagent and application thereof

Info

Publication number: CN113584225B
Application number: CN202110851585.8A
Authority: CN
Inventors: 赵飞; 马培志
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2022-06-28
Anticipated expiration: 2041-07-27
Also published as: CN113584225A

Abstract

The invention relates to the technical field of biology, in particular to a primer and probe combination for detecting HPV virus, a reagent for typing detection of the primer and probe combination, and application of the reagent. The sequence of the primer and probe combination is shown in SEQ ID No. 1-16. The invention combines the asymmetric fluorescence PCR technology and the melting curve technology to realize the typing detection of human papillomavirus types 16, 18, 31, 33, 45, 52 and 58 in a single-tube reaction, thereby improving the detection flux and reducing the detection cost while ensuring the detection sensitivity, specificity and accuracy.

Description

Primer and probe combination for detecting HPV (human papillomavirus) virus, typing detection reagent and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a primer and probe combination for detecting HPV virus, a detection reagent and application thereof.

Background

Human Papilloma Virus (HPV) belongs to the family of papillomaviruses, is a small-molecule, envelope-free, double-stranded circular DNA virus with a genome of about 8000bp in total length divided into 3 functional regions, namely an early transcribed region (E region), a late transcribed region (L region), and a non-transcribed region (LCR). Human is the only host of HPV virus, and more than 200 types of HPV are separated and identified, and are classified into high-risk types and low-risk types according to the difference of diseases. High-risk human papilloma virus (HR-HPV) persistent infection is related to cervical cancer, penile cancer, vulvar cancer, vaginal cancer, anal cancer and certain head and neck malignant tumors, and is particularly closely related to the occurrence of cervical cancer. The cervical cancer is one of the most common cancers in women, and researches prove that HPV DNA can be detected in 99.7 percent of cervical cancers, which indicates that the HPV infection and the correlation of the cervical cancer have universal significance.

The early symptoms of cervical cancer are not obvious, a long and reversible precancerous lesion stage exists in the development process, and 2/3 patients are in advanced cancer after the cervical cancer symptoms appear for three months. Clinical follow-up observation shows that about 10 years is required for the cervical cancer to develop from the common cervical cancer precursor lesion. From this point of view, cervical cancer is not feared, and it is a preventable and curable disease. The key point of prevention and treatment is as follows: gynecological examination is performed regularly, and the cervical cancer precancerous lesion is found and treated in time to stop the development of cervical cancer. If the prevention and treatment measures can be implemented, the cure rate of the cervical cancer is very high. Cervical cancer is also the only cancer whose cause is known with certainty, and this cause is HPV infection. Therefore, detection of HPV virus, especially high-risk HPV virus, can indicate the occurrence of cervical cancer. Research shows that

types

31, 33, 45, 52 and 58 are common in cervical cancer of Chinese women besides HPV16 and 18. The negative predictive value of HPV reaches 100 percent, namely, the cervical cancer can be basically excluded when no HPV infection exists.

There are two main categories of laboratory diagnostic methods currently used for early screening of cervical cancer: cytomorphological examination (aceto-leukosis test, pap smear, colposcopic biopsy, liquid-based thin-layer cytology, etc.) and molecular biological examination (sequencing, in situ hybridization, gene chip, hybrid capture, PCR, etc.). The cytomorphological detection is that abnormal change caused by HPV infection to an organism cannot be directly detected, certain time is needed for specific abnormal change caused by HPV infection to the organism, and the cytomorphological detection method has low sensitivity and specificity and has little significance for clinical early diagnosis. The sequencing and in situ hybridization techniques are complex, have high requirements on operators, are time-consuming and expensive, and are limited in clinical large-scale application. The gene chip has high price and high technical requirement, so that the large-scale application of the gene chip in clinic needs to be carried out for a while. The hybridization capture method has the characteristics of no internal standard, complex detection, long detection time, low sensitivity, high false positive rate and the like, and cannot meet the requirement of clinical high-throughput screening. The real-time fluorescence PCR has high sensitivity and strong specificity, but the common fluorescence PCR detection adopted at present can only detect one virus subtype at one time, the detection of all subtypes is realized by repeating for many times, and a multi-channel fluorescence detection single tube can only detect 3-5 subtypes at most, and can not meet the requirement of detecting multiple subtypes at one time. In addition, a common product is a type corresponding to a pair of type specific primers and a type specific probe, so that the background fluorescence signal is obviously increased when too many fluorescent probes are arranged in the system, and the detection cost is increased. Therefore, there is a need for a sensitive, specific, rapid, simple and convenient method for high-throughput detection of different types of HPV so as to meet the clinical need for rapid and accurate diagnosis.

Disclosure of Invention

In view of this, the invention provides a primer and probe combination for detecting HPV virus, a reagent for typing detection thereof, and an application thereof. The primer and probe combination is combined with a real-time fluorescent PCR and melting curve analysis technology, so that the typing detection of

HPV types

16, 18, 31, 33, 45, 52 and 58 can be quickly realized, the detection sensitivity, specificity and accuracy are ensured, the single-tube detection flux is obviously improved, and the detection cost is reduced.

The invention provides a primer and probe combination, which comprises a primer with a sequence shown in SEQ ID No.1-11 and a probe with a sequence shown in SEQ ID No. 12-16.

In some embodiments, the primer and probe combination further comprises primers shown in SEQ ID No. 17-18 and a probe shown in SEQ ID No.19 for detecting an internal standard gene:

wherein, (1) primers and probes for detecting HPV16 types have base sequences of SEQ ID NO.1, 2 and 12 respectively; (2) primers and probes for detecting HPV18 types, wherein the base sequences are respectively SEQ ID NO.1, 2 and 13; (3) primers and probes for detecting HPV31 types, wherein the base sequences are respectively SEQ ID NO.2, 3 and 16; (4) primers and probes for detecting HPV33 types, wherein the base sequences are respectively SEQ ID NO.4, SEQ ID NO. 5 and SEQ ID NO. 16; (5) primers and probes for detecting HPV45 types, wherein the base sequences are SEQ ID NO.6, SEQ ID NO. 7 and SEQ ID NO. 15 respectively; (6) primers and probes for detecting HPV52 types, wherein the base sequences are SEQ ID NO.8, SEQ ID NO. 9 and SEQ ID NO. 14 respectively; (7) the base sequences of the primer and the probe for detecting HPV58 are SEQ ID NO.10, SEQ ID NO. 11 and SEQ ID NO. 16 respectively.

Specifically, the sequences of the primers and probes are as follows:

SEQ ID No.1：5′-CAACTATTTGTTACTGTGGTTGATACCAC-3′；

SEQ ID No.2：5′-GAAATATAAACTGCAAATCAAATTCCTC-3；

SEQ ID No.3：5′-ATTGGATGCAACGTGCTCAGGGA-3′；

SEQ ID No.4：5′-ATGGTTACTTCCGAATCTCAGTTA-3′；

SEQ ID No.5：5′-GTGCATAAAGTCATATTAGTACTGCGA-3′；

SEQ ID No.6：5′-CCACCTATATAGGTATTCATGGCACAC-3′；

SEQ ID No.7：5′-GCCACAGAAGGTGGTGGAAGA-3′；

SEQ ID No.8：5′-ACTACGTCGCAGGCGTAAACG-3′；

SEQ ID No.9：5′-AGACAGGTACAGGAGGCAGGTA-3′；

SEQ ID No.10：5′-GCCTTATTGGCTACAGCGTGCAC-3′；

SEQ ID No.11：5′-AGTGCTACGAGTGGTATCAACCACG-3′；

SEQ ID No.12：5′-TGCTGCCATATCTACTTCAGAAACTACATA-3′；

SEQ ID No.13：5′-TGCTTCTACACAGTCTCCTGTACCTGGG-3′；

SEQ ID No.14：5′-TCAGTGTGTCGACCTAGTGAAGCCACT-3′；

SEQ ID No.15：5′-CTTTGTGGTGGACTAGTAACAGTACTGT-3′

SEQ ID No.16：5′-CAACAATGGCATTTGCTGGGGCAATCAGT-3′

SEQ ID No.17：5′-GAATGATGGGTGGTGCGACAAC-3′；

SEQ ID No.18：5′-AATGTAAGCAATAGATGGCTCTGCC-3′；

SEQ ID No.19：5′-CAGGAGCCAGGGCTGGGCATAAAAG-3′。

in some embodiments, the 5' end of the probe is labeled with a fluorescent reporter group, specifically FAM, VIC, JOE, HEX, ROX, or CY 5; the 3' end is marked with a fluorescence quenching group, which can be BHQ1 or BHQ2 specifically; including but not limited to. In some specific embodiments, the probes shown in SEQ ID Nos. 12-15 are labeled with FAM at the 5 'end, BHQ1 at the 3' end, VIC at the 5 'end, BHQ2 at the 3' end, CY5 at the 5 'end and BHQ2 at the 3' end of the probe shown in SEQ ID No. 19.

The invention also provides application of the primer and the probe combination in preparing a detection reagent for detecting HPV virus and/or HPV virus typing.

The HPV virus comprises one or more of HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV45 type, HPV52 type and HPV58 type.

The invention also provides a reagent for typing and detecting HPV virus, which comprises the primer and the probe combination.

In the combination of the primers and the probes, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 and SEQ ID No.17 are limiting primers, and the concentration of the limiting primers is 0.01-0.1 uM; SEQ ID No.2, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No.18 are excess primers, and the concentration of the excess primers is 0.1-1 uM; the concentration of the probe is 0.1-0.2 uM.

In some embodiments, the reagent of the present invention comprises PCR reaction solution A and PCR reaction solution B.

The reaction solution A comprises Tricine (pH8.3), KOAc, DMSO and NaN₃UNG enzyme, rTth DNA polymerase and dNTP; the reaction solution B contains Mn (OAc)₂And NaN₃. In some embodiments, the reaction solution a includes: tricine (pH8.3)50mM, KOAc 120mM, DMSO 4.3%, NaN₃0.01%, 0.2. mu.L UNG enzyme at a concentration of 1U/. mu.L, 0.5. mu.L rTth enzyme at a concentration of 5U/. mu.L, 0.4mM each of dATP, dGTP, dCTP and dUTP, each of primers and probes (final concentration of each of the primers and probes: 0.01 to 0.1uM for the limiting primer, 0.1 to 1uM for the excess primer, and 0.1 to 0.2uM for the specific probe), and the balance being water; the reaction solution B contains Mn (OAc)₂0.5mM, NaN30.01%, and the balance of water.

In some embodiments, the reagents of the invention further comprise a negative control, a critical positive control, and a strong positive control variety; the positive control product is a plasmid solution containing HPV16, 18, 31, 33, 45, 52 and 58 type specific target sequences; the strong positive control is a plasmid solution containing HPV16, 45 and 58 type specific target sequences; the negative control is normal saline without HPV virus.

The invention also provides a method for detecting human papilloma virus, which comprises the following steps:

the kit of the invention is used for carrying out real-time fluorescence quantitative PCR detection on a nucleic acid sample to be detected, and the Ct value of an amplification curve is less than or equal to 36, and the amplification curve has a typical S-shaped amplification curve and is positive.

In a specific embodiment of the invention, FAM is marked at the 5 'end of the probe shown in SEQ ID Nos. 12-15, BHQ1 is marked at the 3' end, VIC is marked at the 5 'end of the probe shown in SEQ ID No.16, BHQ2 is marked at the 3' end, CY5 is marked at the 5 'end of the probe shown in SEQ ID No.19, and BHQ2 is marked at the 3' end. The method for judging the result specifically comprises the following steps:

(1) firstly, whether an amplification curve is detected by an internal standard in a CY5 channel or not is analyzed, and Ct is less than or equal to 36, if yes, the detection is effective, and whether the following requirements are met by continuing negative quality control and positive quality control or not is analyzed:

negative quality control: FAM and VIC channels have no amplification curve or amplification curve but Ct is more than 36;

positive quality control: the Ct values of VIC channel amplification are all less than or equal to 30, and the melting curve typing results are HPV16, HPV58 and HPV 45.

(2) And after the negative quality control and the positive quality control meet the requirements, carrying out the following specific analysis on the detection result of the sample:

a) the sample has an amplification signal in the FAM channel, the Ct value of the amplification is less than or equal to 36, the sample can be judged to be positive, if the FAM channel detects a characteristic peak of Tm (71.0 +/-2.0 ℃), the HPV16 type detection result is positive; if the FAM channel detects a Tm (66.0 +/-2.0 ℃) characteristic peak, the detection result of the HPV 18 type is positive; if the FAM channel detects a Tm (62.0 +/-2.0 ℃) characteristic peak, the HPV45 type detection result is positive; if the FAM channel detects a Tm (56.0 +/-2.0 ℃) characteristic peak, the HPV 52 type detection result is positive;

b) The sample has an amplification signal in the VIC channel, the Ct value of the amplification is less than or equal to 36, the sample can be judged to be positive, if the characteristic peak of Tm (69.0 +/-2.0 ℃) is detected in the VIC channel, the HPV 31 type detection result is positive; if the characteristic peak of Tm (61.0 +/-2.0 ℃) is detected by the VIC channel, the HPV 33 type detection result is positive; if a characteristic peak of Tm (56.0 +/-2.0 ℃) is detected in the VIC channel, the HPV 58 type detection result is positive;

c) if the internal standard does not detect Ct or Ct > 36 in the CY5 channel, the result is invalid.

The primer probe combination of the invention is utilized to perform typing detection on

types

16, 18, 31, 33, 45, 52 and 58 of human papillomavirus, the result is analyzed through parameters of two dimensions of Ct value and Tm value, the negative and positive of the detection result are judged according to the Ct value of the melting curve, the Tm value of the melting curve is analyzed to determine different types of HPV, the single-tube detection flux is obviously improved while the detection sensitivity, specificity and accuracy are ensured, the rapid typing and high-flux detection of

types

16, 18, 31, 33, 45, 52 and 58 of human papillomavirus are realized in a single-tube reaction, the time is greatly saved, and the detection cost is reduced.

The invention adopts asymmetric fluorescence quantitative PCR to detect a nucleic acid sample to be detected, and in an amplification system, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 and SEQ ID No.17 are restrictive primers, and the concentration of each primer is 0.01-0.1 uM; SEQ ID No.2, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, and SEQ ID No.18 are excess primers, each at a concentration of 0.1 to 1 uM; the concentration of each probe is 0.1-0.2 uM.

Based on the homologous relation among different types of HPV, when the HPV primer probe is designed, the invention ensures that partial types share the primer or the probe, thereby reducing the using amount of the primer and the probe in the system, obviously reducing the background fluorescence value and reducing the detection cost while ensuring the detection accuracy, sensitivity and specificity. In addition, the invention adopts a method of combining real-time fluorescence PCR and melting curve analysis, thereby obviously improving the single-tube detection flux and breaking through the restriction of a common real-time fluorescence PCR fluorescence channel.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the melting curve of HPV 16 type in the FAM channel;

FIG. 2 shows the melting curve of HPV 18 type in the FAM channel;

FIG. 3 shows the melting curve of HPV type 31 in the VIC channel;

FIG. 4 shows the melting curve of HPV 33 type in the VIC channel;

FIG. 5 shows the melting curve of HPV type 45 in the FAM channel;

FIG. 6 shows the melting curve of HPV type 52 in the FAM channel;

FIG. 7 shows the melting curve of HPV type 58 in the VIC channel;

FIG. 8 shows the melting curve of the HPV internal parameters in the CY5 channel;

FIG. 9 shows the kit detecting HPV 16 and 45 mixed positive reference in FAM channel melting curves;

FIG. 10 shows the melting curve of the kit for detecting HPV 18 and 52 types of mixed positive reference in the FAM channel;

FIG. 11 shows the melting curve of the kit for detecting HPV31, 33 and 58 types mixed positive reference in CIV channel.

Detailed Description

The invention discloses a primer and probe combination for detecting HPV virus, a reagent for typing detection of the HPV virus and application of the reagent. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention is further illustrated by the following examples:

example 1

1. Primer Probe Synthesis

The England Weiji (Shanghai) trade company Limited was entrusted to synthesize a primer probe, and the sequence of the synthesized primer probe was specifically as follows:

TABLE 1

The above sequences were synthesized according to Table 1, and dissolved in sterile double distilled water to a concentration of 200. mu.M and stored at-20 ℃.

2. Extracting nucleic acid of a sample: HPV clinical positive samples, negative controls, positive controls and critical positive controls are extracted by adopting a magnetic bead method nucleic acid extraction reagent. After the extraction of the nucleic acid is finished, the nucleic acid extracting solution is used for PCR detection, or the nucleic acid extracting solution is transferred into a centrifugal tube to be stored at the temperature of-20 ℃.

3. Preparing a reaction system: the reaction system was 35. mu.L. Adding 20 mu LRT-PCR reaction solution A, 10 mu L RT-PCR reaction solution B and 5 mu L nucleic acid extract into the amplification tube in sequence, covering the reaction tube tightly, marking, centrifuging for 10s, and transferring to a PCR amplification area for PCR detection.

4. And (3) computer detection: the macro stone SLAN 96 instrument is used for detection. Item type selection "standard melt curve" and simultaneously FAM, HEX, ROX and Cy5 channels; the melting curve parameter selects "Step Mode", then the "scan interval" is set to 0.5 ℃ and the "constant temperature" is set to 8 s. In the multiplex PCR reaction step, the PCR amplification procedure is as follows: 2min at 50 ℃; 3min at 95 ℃; carrying out 50 cycles at 95 ℃ for 15s, 55 ℃ for 30s and 72 ℃ for 30s, and simultaneously collecting fluorescent signals of four channels including FAM, HEX, ROX and CY5 at the annealing stage at 55 ℃; the program of the analysis step of the product melting curve is as follows: 1min at 95 ℃; 1min at 45 ℃; 1min at 25 ℃; 1min at 30 ℃; the temperature was gradually increased to 85 ℃ and fluorescence signals were collected 1 time per 0.5 ℃ rise during this process.

And (4) analyzing results:

(1) the target detection signal is FAM, HEX (or VIC), and the internal reference detection signal is CY 5;

(2) determining a baseline and a threshold value respectively for each channel: baseline is generally set to be 3-15 cycles, and can be adjusted according to actual conditions. Setting Threshold: the rule is set to have the threshold line just exceed the highest point of the normal negative control.

(3) Firstly, whether an amplification curve is detected by an internal standard in a CY5 channel or not is analyzed, and Ct is less than or equal to 36, if yes, the detection is effective, and whether the following requirements are met by continuing negative quality control and positive quality control or not is analyzed:

positive quality control: the Ct values of FAM and VIC channel amplification are both less than or equal to 30, and the melting curve typing results are HPV16, HPV58 and HPV 45.

(4) And (3) analyzing the detection result of the sample after the negative quality control and the positive quality control meet the requirements:

b) The sample has an amplification signal in the VIC channel, and the Ct value of the amplification is less than or equal to 36, the sample can be judged to be positive, if the characteristic peak of Tm (69.0 +/-2.0 ℃) is detected in the VIC channel, the HPV31 type detection result is positive; if the characteristic peak of Tm (61.0 +/-2.0 ℃) is detected in the VIC channel, the HPV33 type detection result is positive; if a characteristic peak of Tm (56.0 +/-2.0 ℃) is detected in the VIC channel, the HPV58 type detection result is positive;

c) if the internal standard does not detect Ct or Ct > 36 in the CY5 channel, it indicates that the concentration of the detected sample is too low or an interfering substance inhibits the reaction, and the experiment needs to be prepared again.

Example 2 typing assay

Taking a positive sample determined to contain at least one of HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 as a positive standard, and detecting according to the method described in example 1. The results are shown in FIGS. 1 to 11.

According to the result, the method can accurately detect the HPV virus and accurately classify related types.

Example 3 specificity assay

Pathogens (CMV, HSV-1, CT, GBS and UU) having homology to nucleic acid sequences and being liable to cause the same or similar clinical symptoms and other types of HPV (HPV35, 39, 51, 56, 59, 6, 68, etc.) are detected by the method described in example 1, and whether melting peaks are formed only at specific Tm values of corresponding channels in a multiplex PCR reaction system or not and whether cross-reactions occur between different viruses are observed, thereby verifying the specificity of the reaction system. The results show that the method has no cross reaction with other pathogens and other types of HPV, and the composition has good specificity.

Example 4 sensitivity assay

Taking different types of positive plasmids respectively, and adopting normal saline as diluent to carry out gradient dilution in turn to obtain detection limit reference substances with different concentrations such as 4000copies/ml, 2000copies/ml, 1000copies/ml, 500copies/ml, 250copies/ml and the like. The test results of the reference samples with different detection limits were obtained by using the kit and the test method of example 1 of the present invention, human papillomavirus (23 types) nucleic acid typing test kit (fluorescence PCR method) of kaypu chemical limited, Chaozhou, as a contrast reagent 1, and human papillomavirus genotyping (23 types) test kit (PCR-reverse dot hybridization method) of Shenzhen Biotechnology (Shenzhen) Limited, as a contrast reagent 2, respectively, and are shown in table 2.

The results show that the method can detect the target corresponding to the concentration of 500copies/ml, the detection sensitivity is 500copies/ml, the detection sensitivity for partial types can reach 250copies/ml, the contrast reagent 1-2 can only detect the target corresponding to the concentration of 1000copies/ml, and the target corresponding to 500copies/ml can be detected for partial types. In addition, the kit has the advantages of few operation steps, short detection time and large single-tube flux.

TABLE 2 detection of different concentration detection limit reference

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

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Claims

1. A primer and probe combination is characterized by comprising a primer with a sequence shown as SEQ ID No.1-11 and a probe with a sequence shown as SEQ ID No. 12-16.

2. The primer and probe combination of claim 1, further comprising primers shown in SEQ ID Nos. 17-18 and a probe shown in SEQ ID No.19 for detecting an internal standard gene.

3. Use of the primer and probe combination of claim 1 or 2 for the preparation of a reagent for the detection of HPV virus and/or HPV viral typing; the HPV virus comprises one or more of HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV45 type, HPV52 type and HPV58 type.

4. An agent for typing HPV virus, comprising the primer and probe combination of claim 1 or 2 and a detection field acceptable agent.

5. The reagent of claim 4, further comprising at least one of a negative control, a cutoff positive control, and a strong positive control variety; the positive control product is a plasmid solution containing HPV16, 18, 31, 33, 45, 52 and 58 type specific target sequences; the strong positive control is a plasmid solution containing HPV16, 45 and 58 type specific target sequences; the negative control is normal saline without HPV virus.

6. The reagent according to claim 4, comprising a PCR reaction solution A and a PCR reaction solution B;

the reaction solution A comprises the primer, the probe, Tricine, KOAc, DMSO, NaN3, UNG enzyme, rTth DNA polymerase, dNTP and water in the combination of the primer and the probe according to claim 1 or 2;

the reaction solution B comprises Mn (OAc)2, NaN3 and water.

7. The reagent according to any one of claims 4 to 6, wherein in the combination of the primer and the probe, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 and SEQ ID No.17 are limiting primers and the concentration is 0.01 to 0.1 uM; SEQ ID No.2, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, and SEQ ID No.18 are excess primers, each at a concentration of 0.1 to 1 uM; the concentration of the probe is 0.1-0.2 uM.

8. A method for typing human papillomavirus for non-diagnostic purposes, comprising the steps of:

the reagent according to any one of claims 4 to 7, wherein the nucleic acid sample to be tested is subjected to real-time fluorescent quantitative PCR detection, and the HPV type is determined from the amplification curve.

9. The method according to claim 8, wherein the determination method is:

a) The sample has an amplification signal in the FAM channel, the Ct value of the amplification is less than or equal to 36, the sample can be judged to be positive, if the FAM channel detects a characteristic peak of Tm (71.0 +/-2.0 ℃), the HPV 16 type detection result is positive; if the FAM channel detects a Tm (66.0 +/-2.0 ℃) characteristic peak, the detection result of the HPV 18 type is positive; if the FAM channel detects a Tm (62.0 +/-2.0 ℃) characteristic peak, the HPV 45 type detection result is positive; if the FAM channel detects a Tm (56.0 +/-2.0 ℃) characteristic peak, the HPV 52 type detection result is positive;

b) the sample has an amplification signal in the VIC channel, the Ct value of the amplification is less than or equal to 36, the sample can be judged to be positive, if the characteristic peak of Tm (69.0 +/-2.0 ℃) is detected in the VIC channel, the HPV 31 type detection result is positive; if the characteristic peak of Tm (61.0 +/-2.0 ℃) is detected by the VIC channel, the HPV 33 type detection result is positive; if the characteristic peak of Tm (56.0 +/-2.0 ℃) is detected by the VIC channel, the HPV 58 type detection result is positive;

10. The method according to claim 8, wherein the fluorescent quantitative PCR is asymmetric fluorescent quantitative PCR, and the concentration of SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10 and SEQ ID No.17 in an amplification system is 0.01-0.1 uM; the concentration of SEQ ID No.2, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11 and SEQ ID No.18 is 0.1-1 uM, and the concentration of each probe is 0.1-0.2 uM.