Primer probe combination, kit and detection method for detecting human papilloma virus

Abstract

The invention discloses a primer probe combination, a kit and a detection method for detecting human papilloma virus. The primer probe combination comprises nucleotide series as shown in SEQ ID No. 1 to SEQ ID No. 45. Probes in the primer probe group are respectively marked with different fluorescent light-emitting groups. Specific primers and corresponding specific fluorescent probes are designed for E6 / E7 DNA of 14 high-risk HPVs (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68) and an internal reference HBB, so that 14 target genes are detected by one reaction tube, and moreover, a method of combining asymmetric PCR with a multicolor multiple melting curve has the advantages of high detection sensitivity, good specificity, high flux, simplicity in operation, low cost and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biological detection, and specifically relates to a primer-probe combination, a kit and a detection method for detecting human papillomavirus. Background Art

[0002] Cervical cancer is one of the common malignant tumors. It is now clear that human papillomavirus (HPV) is closely related to cervical lesions, and persistent infection with high-risk HPV is the main pathogenic factor of cervical precancerous lesions and cervical cancer. Human papillomavirus (HPV) is a spherical DNA virus that is widely present in nature. It has humans as its only host, is very tolerant to drying and can be stored for a long time. Currently, there are only more than 150 known HPV subtypes, which can be divided into low-risk and high-risk types according to the severity of the diseases they cause. Cervical cancer is mainly caused by persistent infection with high-risk HPV. High-risk HPV can be detected in 99.7% of cervical cancers; among them, HPV16 and HPV18 account for more than 70%. High-risk HPV can also cause cancers in the anus, vagina, vulva, penis, head and neck. Low-risk HPV can cause diseases such as anogenital warts and recurrent respiratory papilloma.

[0003] HPV infection is mainly confirmed through virus isolation and culture, histopathological examination, etc., and laboratory tests such as immunological examination can also be used to assist in the diagnosis of virus types. Clinically, virus isolation and culture, histopathological examination, and molecular biological testing are generally used as the main basis for diagnosing human papillomavirus infection. Both isolation and tissue culture have subjective differences, so the current mainstream clinical practice is mainly based on genetic testing.

[0004] At present, there are several methods for HPV genotyping detection. 1) In situ hybridization technology: nucleic acid probes (DNA or RNA) are used to perform in situ hybridization reactions with samples. The advantage of in situ hybridization is that suspicious cells can be located; the disadvantages are low sensitivity and high sample quality requirements. 2) PCR-reverse dot hybridization method: PCR in vitro amplification and DNA reverse dot hybridization are combined. It can perform full typing and can be used for auxiliary diagnosis of clinical HPV infection. The only disadvantage is that it is easy to be contaminated, the cycle is long and the cost is high. 3) Flow fluorescence hybridization method: This technology hybridizes the PCR amplification product with the cross-linked fluorescent marker probe on the microsphere, and finally detects the fluorescent signal on a multifunctional flow dot array. At present, commercial kits can detect and type 27 types of HPV at one time. The disadvantage is that the amplification product needs to be opened and is easy to be contaminated, and the detection takes 6 hours. 4) PCR amplification method: multi-tube amplification is performed. The biggest advantage of this method is that it is fast, but the disadvantage is obvious. It requires full typing and requires multi-tube operation, which will lead to many operation steps and confusing results. 5) Melting curve method: Melting curve is performed on the basis of amplification. The advantage is that amplification can be performed in one tube, but there are more primers and probes, which may lead to system nonspecificity and interference between adjacent melting peaks.

[0005] In the existing molecular technology PCR amplification or PCR-reverse dot hybridization technology, one PCR tube cannot perform multi-target typing and needs to be performed in multiple tubes, which will make the operation complicated and increase the cost. The PCR-reverse dot hybridization method has a long cycle, the hybridization process is prone to contamination, and the automated operation cycle is also long, which is different from the PCR amplification that completes the entire test in about an hour. In addition, both PCR and reverse dot hybridization have high costs. PCR multiple typing requires multi-tube operation, and the cost of consumables is too high. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the purpose of the present invention is to provide a primer-probe combination, a kit and a detection method for detecting human papillomavirus.

[0007] To achieve the above object, according to the first aspect of the present invention, the primer-probe combination for detecting human papillomavirus provided by the embodiment of the present invention comprises the following 14 primer-probe groups:

[0008] Primers and probes for detecting HPV16 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.1 and SEQ ID No.2 respectively; the probe sequence is shown in SEQ ID No.3;

[0009] Primers and probes for detecting HPV18 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.4 and SEQ ID No.5 respectively; the probe sequence is shown in SEQ ID No.6;

[0010] Primers and probes for detecting HPV31 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the probe sequence is shown in SEQ ID No.9;

[0011] Primers and probes for detecting HPV33 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.10 and SEQ ID No.11 respectively; the probe sequence is shown in SEQ ID No.12;

[0012] Primers and probes for detecting HPV35 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.13 and SEQ ID No.14 respectively; the probe sequence is shown in SEQ ID No.15;

[0013] Primers and probes for detecting HPV39 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.16 and SEQ ID No.17 respectively; the probe sequence is shown in SEQ ID No.18;

[0014] Primers and probes for detecting HPV45 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.19 and SEQ ID No.20 respectively; the probe sequence is shown in SEQ ID No.21;

[0015] Primers and probes for detecting HPV51 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.22 and SEQ ID No.23 respectively; the probe sequence is shown in SEQ ID No.24;

[0016] Primers and probes for detecting HPV52 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.25 and SEQ ID No.26 respectively; the probe sequence is shown in SEQ ID No.27;

[0017] Primers and probes for detecting HPV56 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.28 and SEQ ID No.29 respectively; the probe sequence is shown in SEQ ID No.30;

[0018] Primers and probes for detecting HPV58 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.31 and SEQ ID No.32 respectively; the probe sequence is shown in SEQ ID No.33;

[0019] Primers and probes for detecting HPV59 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.34 and SEQ ID No.35 respectively; the probe sequence is shown in SEQ ID No.36;

[0020] Primers and probes for detecting HPV66 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.37 and SEQ ID No.38 respectively; the probe sequence is shown in SEQ ID No.39;

[0021] Primers and probes for detecting HPV68 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.40 and SEQ ID No.41 respectively; the probe sequence is shown in SEQ ID No.42;

[0022] Primers and probes for detecting internal reference HBB: wherein the upstream and downstream primer sequences are shown as SEQ ID No.43 and SEQ ID No.44 respectively; the probe sequence is shown as SEQ ID No.45.

[0023] According to an embodiment of the present invention, the probes in the primer probe set are respectively labeled with different fluorescent light-emitting groups.

[0024] According to the second aspect of the present invention, an embodiment of the present invention provides the use of the primer-probe combination as described above in the preparation of a kit, the function of the kit is to use the melting curve method to perform typing and qualitative analysis on 14 different human papillomaviruses, and the 14 human papillomaviruses are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68.

[0025] According to the third aspect of the present invention, the kit provided by the embodiment of the present invention comprises the primer-probe combination as described above.

[0026] According to one embodiment of the present invention, it also includes a nucleic acid reaction solution, a positive control and a negative control.

[0027] According to one embodiment of the present invention, the nucleic acid reaction solution includes PCR buffer, MgCl2, dNTPs, DNA polymerase, UNG enzyme and DNase-free water.

[0028] According to one embodiment of the present invention, the PCR buffer is a 10×PCR buffer, which includes Tris-HCL, KCl, Tween 20, Brij35, PEG-6000, BSA and water;

[0029] Among them, the pH value of Tris-HCL is 8.5-9.0, and the final concentration is 50-200 mM; the final concentration of KCl is 200-500 mM, the final concentration of Briji35 is 0.01%-0.2% (mass volume ratio); the final concentration of Tween 20 is 0.01%-0.1% (mass volume ratio), the final concentration of BSA is 0.2 mg / ml-1 mg / ml, and the final concentration of PEG-6000 is 1%-8% (mass volume ratio).

[0030] According to one embodiment of the present invention, the final concentration of MgCl2 is 1 to 5 mM, the dNTPs include dATP, dTTP, dCTP, dGTP and dUTP, and the ratios are 1:0.5:1:1:0.5, respectively, and the final concentration is 0.1 mM to 0.7 mM; the DNA polymerase is a high-tolerance fast Tag enzyme with a final concentration of 2 to 5 U; the final concentration of the UNG enzyme is 0.05 U to 0.3 U at a temperature of 50°C.

[0031] According to one embodiment of the present invention, the positive control comprises artificially synthesized HPV gene plasmids of different concentrations and contains at least one HPV typing in each fluorescent channel.

[0032] According to a fourth aspect of the present invention, an embodiment of the present invention provides a method for detecting human papillomavirus for the purpose of non-disease diagnosis and / or treatment, comprising:

[0033] 1) Designing HPV primer probes based on the conserved sequences of E6 / E7 of 14 human papillomaviruses; the 14 human papillomaviruses are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68;

[0034] 2) Designing primers and probes for the internal reference gene HBB based on the conserved sequence of the internal reference gene HBB;

[0035] 3) 14 human papillomavirus and internal reference gene primer probes are mixed according to the proportion to form primer probe MIX;

[0036] 4) Mix the primer probe MIX with the reaction solution MIX, and add the template to perform PCR amplification;

[0037] 5) Determine whether the sample is positive based on the fluorescence amplification signals of different channels, and perform typing of the positive nucleic acid using the melting curve analysis results in the corresponding fluorescence channel;

[0038] The HPV types corresponding to the melting curve analysis of the FAM channel were HPV31, HPV52, HPV68, and HPV45;

[0039] The HPV types corresponding to the melting curve analysis of the HEX channel were HPV33, HPV56, HPV16, and HPV66;

[0040] The HPV types corresponding to the melting curve analysis of the ROX channel were HPV35, HPV58, and HPV18;

[0041] The HPV types corresponding to the melting curve analysis of the Cy5 channel were HPV39, HPV59, HPV51, and the internal reference;

[0042] Wherein, the HPV primer probe and the internal reference gene HBB primer probe are as follows:

[0043] Primers and probes for detecting HPV16 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.1 and SEQ ID No.2 respectively; the probe sequence is shown in SEQ ID No.3;

[0044] Primers and probes for detecting HPV18 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.4 and SEQ ID No.5 respectively; the probe sequence is shown in SEQ ID No.6;

[0045] Primers and probes for detecting HPV31 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the probe sequence is shown in SEQ ID No.9;

[0046] Primers and probes for detecting HPV33 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.10 and SEQ ID No.11 respectively; the probe sequence is shown in SEQ ID No.12;

[0047] Primers and probes for detecting HPV35 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.13 and SEQ ID No.14 respectively; the probe sequence is shown in SEQ ID No.15;

[0048] Primers and probes for detecting HPV39 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.16 and SEQ ID No.17 respectively; the probe sequence is shown in SEQ ID No.18;

[0049] Primers and probes for detecting HPV45 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.19 and SEQ ID No.20 respectively; the probe sequence is shown in SEQ ID No.21;

[0050] Primers and probes for detecting HPV51 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.22 and SEQ ID No.23 respectively; the probe sequence is shown in SEQ ID No.24;

[0051] Primers and probes for detecting HPV52 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.25 and SEQ ID No.26 respectively; the probe sequence is shown in SEQ ID No.27;

[0052] Primers and probes for detecting HPV56 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.28 and SEQ ID No.29 respectively; the probe sequence is shown in SEQ ID No.30;

[0053] Primers and probes for detecting HPV58 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.31 and SEQ ID No.32 respectively; the probe sequence is shown in SEQ ID No.33;

[0054] Primers and probes for detecting HPV59 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.34 and SEQ ID No.35 respectively; the probe sequence is shown in SEQ ID No.36;

[0055] Primers and probes for detecting HPV66 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.37 and SEQ ID No.38 respectively; the probe sequence is shown in SEQ ID No.39;

[0056] Primers and probes for detecting HPV68 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.40 and SEQ ID No.41 respectively; the probe sequence is shown in SEQ ID No.42;

[0057] Primers and probes for detecting internal reference HBB: wherein the upstream and downstream primer sequences are shown as SEQ ID No.43 and SEQ ID No.44 respectively; the probe sequence is shown as SEQ ID No.45.

[0058] Through the above technical scheme, the primer-probe combination, kit and detection method for detecting human papillomavirus of the present invention, by designing specific primers and corresponding specific fluorescent probes for E6 / E7 DNA and internal reference HBB of 14 high-risk HPVs (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68), realizes the detection of 14 target genes in one reaction tube, and utilizes asymmetric PCR combined with a multi-color multiple melting curve method, which has the advantages of high detection sensitivity, good specificity, high throughput, simple operation and low cost.

[0059] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is the test clinical sample result 1;

[0061] Figure 2 It is the test clinical sample result 2;

[0062] Figure 3 It is the test clinical sample result 3. DETAILED DESCRIPTION

[0063] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. Preferred embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0065] The embodiment of the present invention provides an application of an asymmetric PCR melting curve method in the typing and qualitative analysis of 14 high-risk human papillomaviruses, so as to solve the shortcomings of the existing detection technology, such as the need for PCR multi-tube detection for full typing; insufficient typing and unclear typing in single-tube detection, long reverse dot hybridization time and easy contamination. The present invention utilizes the technical principle of asymmetric PCR amplification combined with melting curve analysis, and uses the HPV E6 / E7 region specific sequences of 16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 66 / 68 to design primer probes. The specific scheme is as follows:

[0066] The primer-probe combination for detecting human papillomavirus provided in the embodiment of the present invention includes the following 14 primer-probe groups:

[0067] Primers and probes for detecting HPV16 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.1 and SEQ ID No.2 respectively; the probe sequence is shown in SEQ ID No.3;

[0068] Primers and probes for detecting HPV18 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.4 and SEQ ID No.5 respectively; the probe sequence is shown in SEQ ID No.6;

[0069] Primers and probes for detecting HPV31 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the probe sequence is shown in SEQ ID No.9;

[0070] Primers and probes for detecting HPV33 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.10 and SEQ ID No.11 respectively; the probe sequence is shown in SEQ ID No.12;

[0071] Primers and probes for detecting HPV35 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.13 and SEQ ID No.14 respectively; the probe sequence is shown in SEQ ID No.15;

[0072] Primers and probes for detecting HPV39 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.16 and SEQ ID No.17 respectively; the probe sequence is shown in SEQ ID No.18;

[0073] Primers and probes for detecting HPV45 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.19 and SEQ ID No.20 respectively; the probe sequence is shown in SEQ ID No.21;

[0074] Primers and probes for detecting HPV51 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.22 and SEQ ID No.23 respectively; the probe sequence is shown in SEQ ID No.24;

[0075] Primers and probes for detecting HPV52 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.25 and SEQ ID No.26 respectively; the probe sequence is shown in SEQ ID No.27;

[0076] Primers and probes for detecting HPV56 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.28 and SEQ ID No.29 respectively; the probe sequence is shown in SEQ ID No.30;

[0077] Primers and probes for detecting HPV58 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.31 and SEQ ID No.32 respectively; the probe sequence is shown in SEQ ID No.33;

[0078] Primers and probes for detecting HPV59 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.34 and SEQ ID No.35 respectively; the probe sequence is shown in SEQ ID No.36;

[0079] Primers and probes for detecting HPV66 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.37 and SEQ ID No.38 respectively; the probe sequence is shown in SEQ ID No.39;

[0080] Primers and probes for detecting HPV68 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.40 and SEQ ID No.41 respectively; the probe sequence is shown in SEQ ID No.42;

[0081] Primers and probes for detecting internal reference HBB: wherein the upstream and downstream primer sequences are shown as SEQ ID No.43 and SEQ ID No.44 respectively; the probe sequence is shown as SEQ ID No.45.

[0082] In one embodiment of the present invention, the probes in the primer probe set are respectively labeled with different fluorescent luminescent groups. That is, to achieve multiple typing detection, the 14 probe sequences in this embodiment are respectively labeled with different fluorescent luminescent groups (fluorophores). These luminescent groups emit different fluorescence wavelengths, which can be distinguished by different fluorescence channels during the melting curve analysis process.

[0083] By labeling all probes with different luminescent groups and combining asymmetric PCR amplification with high-resolution melting curve analysis, 14 high-risk HPV typings can be simultaneously identified in a single-tube reaction system, avoiding the defects of traditional methods that require multiple typing or dot hybridization, greatly simplifying the operation process, and improving the efficiency and specificity of detection.

[0084] An embodiment of the present invention also provides the use of the primer-probe combination as described above in the preparation of a kit, the function of the kit is to use the melting curve method to perform typing and qualitative analysis on 14 different human papillomaviruses, and the 14 human papillomaviruses are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68.

[0085] An embodiment of the present invention further provides a kit, comprising the primer-probe combination as described above.

[0086] By using the kit of this embodiment to carry out an asymmetric PCR amplification reaction and combining it with a melting curve analyzer, it is possible to detect whether 14 types of high-risk HPV and specific typing information are present in a patient sample at one time in a single tube system.

[0087] In one embodiment of the present invention, the kit further includes a nucleic acid reaction solution, a positive control and a negative control. The positive control is a positive quality control product containing a known concentration, which contains 14 high-risk HPV typings and an internal reference sequence. The positive control must be tested at the same time during each test to assess whether there are problems such as abnormalities in the reaction system as part of the quality control of the entire experimental process. The negative control is a blank control that does not contain any target sequence. By testing the negative control, it can be determined whether there is exogenous contamination or non-specific amplification, thereby ensuring the specificity of the results.

[0088] In one embodiment of the present invention, the positive control comprises artificially synthesized HPV gene plasmids of different concentrations, and contains at least one HPV typing in each fluorescent channel. Preferably, FAM is HPV31 typing, HEX is HPV16 typing, ROX fluorescent channel HPV18 typing and CY5 fluorescent channel HPV39 typing, mixed into one tube.

[0089] In one embodiment of the present invention, the nucleic acid reaction solution includes PCR buffer, MgCl2, dNTPs, DNA polymerase, UNG enzyme and DNase-free water.

[0090] In one embodiment of the present invention, the PCR buffer is 10×PCR buffer, which includes Tris-HCL, KCl, Tween 20, Brij35, PEG-6000, BSA and water.

[0091] Among them, the pH value of Tris-HCL is 8.5-9.0, and the final concentration is 50-200 mM; the final concentration of KCl is 200-500 mM, the final concentration of Briji35 is 0.01%-0.2% (mass volume ratio); the final concentration of Tween 20 is 0.01%-0.1% (mass volume ratio), the final concentration of BSA is 0.2 mg / ml-1 mg / ml, and the final concentration of PEG-6000 is 1%-8% (mass volume ratio).

[0092] In this embodiment, Tris-HCl maintains a suitable pH value, KCl provides ionic strength, Tween 20 and Brij-35 are non-ionic surfactants to increase the stability of the reaction system, PEG-6000 is a polyethylene glycol that improves the reaction specificity, and BSA is bovine serum albumin to prevent enzyme inactivation. These components are used in the above concentration range to provide an optimal reaction environment for DNA polymerase.

[0093] In one embodiment of the present invention, the final concentration of MgCl2 is 1 to 5 mM, the dNTPs include dATP, dTTP, dCTP, dGTP and dUTP, and the ratios are 1:0.5:1:1:0.5, respectively, and the final concentration is 0.1 mM to 0.7 mM; the DNA polymerase is a high-tolerance fast Tag enzyme with a final concentration of 2 to 5 U; the final concentration of the UNG enzyme is 0.05 U to 0.3 U at a temperature of 50°C.

[0094] In this embodiment, Mg 2+ It is an auxiliary agent necessary for DNA polymerase activity. Appropriate concentration can improve amplification efficiency. The introduction of dUTP is used to eliminate contamination of UNG enzyme. DNA polymerase uses high-tolerance fast Taq enzyme, which can effectively resist reaction inhibitors. UNG enzyme can digest residual contaminating DNA before PCR to improve the specificity of the results.

[0095] One embodiment of the present invention further provides a method for detecting human papillomavirus for the purpose of non-disease diagnosis and / or treatment, comprising:

[0096] 1) Designing HPV primer probes based on the conserved sequences of E6 / E7 of 14 human papillomaviruses; the 14 human papillomaviruses are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68;

[0097] 2) Designing primers and probes for the internal reference gene HBB based on the conserved sequence of the internal reference gene HBB;

[0098] 3) 14 human papillomavirus and internal reference gene primer probes are mixed according to the proportion to form primer probe MIX;

[0099] 4) Mix the primer probe MIX with the reaction solution MIX, and add the template amount to perform PCR amplification. That is, mix the primer probe MIX with the pre-prepared reaction solution MIX (including buffer, Mg2+, dNTPs, DNA polymerase, UNG enzyme, etc.), then add the extracted sample nucleic acid, construct a PCR amplification reaction system, perform asymmetric PCR cycles, and amplify the target sequence.

[0100] 5) Determine whether the sample is positive based on the fluorescence amplification signals of different channels, and type the positive nucleic acid using the melting curve analysis results in the corresponding fluorescence channel. In other words, use the real-time fluorescence amplification curve to determine whether the sample is HPV positive. Further melting curve analysis is performed on positive samples, and the specific HPV typing can be identified based on the position of the melting peaks in different channels (FAM / HEX / ROX / Cy5).

[0101] The HPV types corresponding to the melting curve analysis of the FAM channel were HPV31, HPV52, HPV68, and HPV45;

[0102] The HPV types corresponding to the melting curve analysis of the HEX channel were HPV33, HPV56, HPV16, and HPV66;

[0103] The HPV types corresponding to the melting curve analysis of the ROX channel were HPV35, HPV58, and HPV18;

[0104] The HPV types corresponding to the melting curve analysis of the Cy5 channel were HPV39, HPV59, HPV51, and the internal reference;

[0105] Wherein, the HPV primer probe and the internal reference gene HBB primer probe are as follows:

[0106] Primers and probes for detecting HPV16 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.1 and SEQ ID No.2 respectively; the probe sequence is shown in SEQ ID No.3;

[0107] Primers and probes for detecting HPV18 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.4 and SEQ ID No.5 respectively; the probe sequence is shown in SEQ ID No.6;

[0108] Primers and probes for detecting HPV31 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the probe sequence is shown in SEQ ID No.9;

[0109] Primers and probes for detecting HPV33 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.10 and SEQ ID No.11 respectively; the probe sequence is shown in SEQ ID No.12;

[0110] Primers and probes for detecting HPV35 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.13 and SEQ ID No.14 respectively; the probe sequence is shown in SEQ ID No.15;

[0111] Primers and probes for detecting HPV39 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.16 and SEQ ID No.17 respectively; the probe sequence is shown in SEQ ID No.18;

[0112] Primers and probes for detecting HPV45 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.19 and SEQ ID No.20 respectively; the probe sequence is shown in SEQ ID No.21;

[0113] Primers and probes for detecting HPV51 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.22 and SEQ ID No.23 respectively; the probe sequence is shown in SEQ ID No.24;

[0114] Primers and probes for detecting HPV52 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.25 and SEQ ID No.26 respectively; the probe sequence is shown in SEQ ID No.27;

[0115] Primers and probes for detecting HPV56 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.28 and SEQ ID No.29 respectively; the probe sequence is shown in SEQ ID No.30;

[0116] Primers and probes for detecting HPV58 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.31 and SEQ ID No.32 respectively; the probe sequence is shown in SEQ ID No.33;

[0117] Primers and probes for detecting HPV59 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.34 and SEQ ID No.35 respectively; the probe sequence is shown in SEQ ID No.36;

[0118] Primers and probes for detecting HPV66 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.37 and SEQ ID No.38 respectively; the probe sequence is shown in SEQ ID No.39;

[0119] Primers and probes for detecting HPV68 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.40 and SEQ ID No.41 respectively; the probe sequence is shown in SEQ ID No.42;

[0120] Primers and probes for detecting internal reference HBB: wherein the upstream and downstream primer sequences are shown as SEQ ID No.43 and SEQ ID No.44 respectively; the probe sequence is shown as SEQ ID No.45.

[0121] The detection method of this embodiment realizes one-step detection without multiple amplifications, saving time and reagents. Single-tube reaction, 14 high-risk HPVs are detected together to avoid missed detection. Internal reference calibration can assess whether there are inhibitory factors affecting the results. Melting curve typing has high specificity and intuitive results. In non-clinical diagnostic applications, it can meet a variety of non-medical needs such as scientific research.

[0122] Another embodiment of the present invention provides a method for simultaneously detecting 14 types of human papillomavirus using asymmetric multicolor multiplex PCR melting curves, wherein the nucleic acid extraction method used adopts a release agent method, and specifically comprises the following steps:

[0123] 1) Nucleic acid release: The extraction reagent used was the nucleic acid release reagent kit produced by Shenzhen Dexia Biomedical Engineering Co., Ltd. The sample used the human papillomavirus (HPV cell) single typing quality control product - human papillomavirus (HPV16) cell quality control product (product number GW-IPF030, concentration 1x105 copies / mL) produced by Jingliang Technology (Shenzhen) Co., Ltd. to dilute the cell sample to an appropriate concentration.

[0124] The extraction process is as follows: add the cell quality control product to the release agent (100 μL) at a ratio of 1:1, manually shake the release agent left and right 15 times, and let it stand at room temperature for 3 minutes; add it to the PCR reaction reagent for amplification.

[0125] 2) Configuration of the reaction system: 5.64 μL amplification reaction solution, 14.36 μL primer probe reaction solution, 5 μL nucleic acid sample.

[0126] 3) Amplification: The sample with release was placed in a fluorescent quantitative PCR instrument for detection. The experimental detection procedure was as follows: decontamination stage: 50°C for 2 min; pre-denaturation stage: 95°C for 3 min; denaturation, annealing and extension stage: 95°C for 10 s, 60°C for 25 s, fluorescence detection; melting stage: 95°C for 2 min, 45°C for 2 min; continuous heating mode was used from 45°C to 90°C, 0.3°C / s, and fluorescence was collected.

[0127] 4) Result analysis: After the test, the results shall be interpreted according to the following standards.

[0128] Samples with the same four peaks as the control in the FAM channel are positive for HPV31 (TM51.9±1.5), HPV52 (TM60.87±1.5), HPV68 (TM66.69±1.5) and HPV45 (TM71.18±1.5). Samples with only one or two Tm value melting peaks can refer to the TM value corresponding to the specific typing.

[0129] Samples with four peaks in the HEX channel that are the same as the control are positive for HPV33 (TM55.87±1.5), HPV56 (TM 62.87±1.5), HPV16 (TM69.37±1.5), and HPV74 (TM74.34±1.5). Samples with only one or two Tm value melting peaks can refer to the TM value corresponding to the specific typing;

[0130] Samples with three peaks in the ROX channel that are the same as the control are positive for HPV35 (TM57.20±1.5), HPV58 (TM 64.40±1.5), and HPV18 (TM71.73±1.5). Samples with only one or two Tm value melting peaks can refer to the TM value corresponding to the specific typing;

[0131] Samples with the same four peaks as the control in the CY5 channel are positive for HPV39 (TM57.52±1.5), HPV59 (TM 66.67±1.5), HPV51 (TM72.05±1.5) and internal reference (TM77.54±1.5). Samples with only one or two Tm value melting peaks can refer to the TM value corresponding to the specific typing.

[0132] The primer-probe combination, kit and detection method for detecting human papillomavirus of the present invention realize detection of 14 target genes in one reaction tube by designing specific primers and corresponding specific fluorescent probes for E6 / E7 DNA and internal reference HBB of 14 high-risk HPVs (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68), and furthermore, by utilizing asymmetric PCR combined with a multi-color multiple melting curve method, the method has the advantages of high detection sensitivity, good specificity, high throughput, simple operation and low cost.

[0133] The present invention will be further described below through some specific embodiments.

[0134] Example 1 A human papillomavirus (HPV) 14-type typing detection kit

[0135] 1) Design and synthesis of specific primers and probes

[0136] In the NCBI database, search for E6 / E7 sequences of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68, and download 30-40 E6 / E7 sequences of each type of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68, respectively. Use sequence alignment software clustalx1.83 to find the most conservative fragments of each type that do not cross with other types to design primers. After determining the conservative sequence, oligo7.0 was used to design primers and probes. Reference products were purchased from Jingliang Technology (Shenzhen) Co., Ltd. Primers and probes were synthesized by Hunan Aikerui Biotechnology Co., Ltd. The primer and probe sequences used for detection are shown in Table 1 below:

[0137] Table 1. Primer and probe sequences of 14 high-risk HPV types

[0138]

[0139]

[0140] 2) Sample processing

[0141] The samples were released, transported and stored using the exfoliated cell quality control product of Shenzhen Jingliang Technology Co., Ltd. and the release agent of Shenzhen Dexia Biomedical Engineering Co., Ltd. Sample release process: Add the cell quality control product to the release agent (100μL) at a ratio of 1:1, shake the release agent 15 times manually, and let it stand at room temperature for 3 minutes; add it to the PCR reaction reagent for amplification. After the sample is released, conduct downstream inspection in time. If it cannot be detected in time, store it at room temperature for no more than 24 hours. The amount of primers and probes added.

[0142] Table 2. Reagent configuration and primer probe concentrations

[0143]

[0144]

[0145] 3) Amplification and programming

[0146] Table 3. Amplification program settings table

[0147]

[0148] 4) Result analysis

[0149] If all targets of FAM / HEX / ROX / CY5 have no melting peak and amplification, the internal reference must have a melting peak, and the TM value corresponds to 77.54±1.5. If the internal reference has no melting peak, it means that the results need to be redone, and the reagents need to be replaced or the samples need to be resampled. If all targets of FAM / HEX / ROX / CY5 have melting peaks, the internal reference can be a negative result; samples with the same four peaks as the reference substance in the FAM channel are positive for HPV31 (TM51.9±1.5), HPV52 (TM60.87±1.5), HPV68 (TM66.69±1.5) and HPV45 (TM71.18±1.5), and samples with only one or two Tm value melting peaks can refer to the specific typing corresponding to the TM value; samples with the same four peaks as the reference substance in the HEX channel are HPV33 (TM55.87±1.5), HPV56 (TM 62.87±1.5), HPV16 (TM69.37±1.5) and HPV74 (TM 74.34±1.5) was positive, and samples with only one or two Tm value melting peaks could refer to the TM value for specific typing; samples with three peaks in the ROX channel that were the same as the reference substance were positive for HPV35 (TM57.20±1.5), HPV58 (TM 64.40±1.5), and HPV18 (TM71.73±1.5), and samples with only one or two Tm value melting peaks could refer to the TM value for specific typing; samples with four peaks in the CY5 channel that were the same as the reference substance were positive for HPV39 (TM57.52±1.5), HPV59 (TM 66.67±1.5), HPV51 (TM72.05±1.5), and internal reference (TM77.54±1.5), and samples with only one or two Tm value melting peaks could refer to the TM value for specific typing;

[0150] Example 2 Evaluation of HPV lyophilized reagent template amount

[0151] 1) Lyophilized reagents

[0152] The freeze-drying evaluation of 14 HPV test kits was performed, and the buffer used was low-salt ion and glycerol-free enzyme from Shenzhen Dexia Biomedical Engineering Co., Ltd. The freeze-drying procedure used the procedure in Table 4 (Sihuan Furui freeze dryer)

[0153] Table 4. HPV lyophilization procedure

[0154]

[0155] 2) Evaluation of template quantity

[0156] The samples used the exfoliated cell quality control product of Jingliang Technology (Shenzhen) Co., Ltd., and after mixing four types of HPV 16 / 18 / internal reference / 31, the cells were diluted 2 times, 5 times, 10 times and 20 times with ddH2O, and then released with the release agent of Shenzhen Dexia Biomedical Engineering Co., Ltd. Sample release process: add the cell quality control product to the release agent (100μL) at a ratio of 1:1, manually shake the release agent left and right 15 times after adding the sample, and let it stand at room temperature for 3 minutes; add it to the freeze-dried reagent for amplification according to Table 5, and test it according to Table 6.

[0157] Table 5. Reaction system configuration table

[0158]

[0159] Table 6. Evaluation test table of different template amounts

[0160]

[0161] 3) Amplification and programming

[0162] Table 7. Amplification program settings table

[0163]

[0164] 4) Result analysis

[0165] The amplification results are guided by the melting peak. The specific melting TM values ​​are shown in Table 8.

[0166] Table 8. Amplification and melting results

[0167]

[0168] Judging from the results, when the release agent was fully added to the template into the freeze-dried reagent, the detection limit was improved and no inhibitory effect occurred. Therefore, the freeze-dried reagent can be combined with the release agent of Shenzhen Dexia Biomedical Engineering Co., Ltd. to achieve full sample addition.

[0169] Example 3 Specificity Assessment

[0170] The method of the present invention is used to verify the specificity of the present invention for pathogens with similar HPV infection sites and symptoms and HPVs outside the detection range of the present invention (HPV6 / 11 / 26 / 53 / 73 / 82, etc.). According to the HPV registration review guidelines, the concentration of bacteria is 106CFU / mL and the concentration of viruses is 105PFU / mL or higher; the results of the pathogens in Table 9 show that no corresponding melting peaks appear, so the method of the present invention has no cross-specificity for pathogens outside the invention.

[0171] Table 9. Specific cross-pathogens

[0172]

[0173] Example 4 Testing of clinical samples

[0174] 14 high-risk HPV typing samples were obtained from the hospital, and the samples were confirmed by typing. According to the method of Example 1, the clinical samples were extracted with a release agent and the freeze-dried reagent was used for testing. The experimental results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, each type can be accurately detected.

[0175] In summary, the primer-probe combination, kit and detection method for detecting human papillomavirus provided by the present invention use asymmetric multicolor multiplex PCR melting curves to simultaneously detect 14 types of human papillomavirus, and have the following effects:

[0176] (1) One tube can detect 14 high-risk HPV types specified by the WHO. One sample addition can detect 14 high-risk positive and negative types, reducing the number of operating steps, the use of consumables, and the cost.

[0177] (2) The use of a release agent to extract and preserve samples greatly shortens the extraction time. At the same time, the release agent can be used as a sample preservation liquid to facilitate sample transportation and preservation. The nucleic acid extraction is completed during the sample transportation process.

[0178] (3) Freeze-dried reagents: The reagents are freeze-dried to facilitate later transportation and storage. At the same time, freeze-dried reagents can be added to the entire sample (25 μl) to increase the sample volume and improve detection performance.

[0179] (4) The detection throughput is high, one sample per tube is tested, and it can be combined with automation to perform large-scale auxiliary diagnosis or early screening.

[0180] (5) Good specificity. In addition to the 14 designed HPV types, there is no non-specificity for other types and other pathogens with the same infection site and the same symptoms.

[0181] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0182] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0183] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A primer-probe combination for detecting human papillomavirus, characterized in that: Includes the following 14 primer probe sets: Primers and probes for detecting HPV16 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; the probe sequence is shown in SEQ ID No. 3; Primers and probes for detecting HPV18 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively; the probe sequence is shown in SEQ ID No. 6; Primers and probes for detecting HPV31 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the probe sequence is shown in SEQ ID No.9; Primers and probes for detecting HPV33 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No. 10 and SEQ ID No. 11 respectively; the probe sequence is shown as SEQ ID No. 12; Primers and probes for detecting HPV35 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 13 and SEQ ID No. 14 respectively; the probe sequence is shown in SEQ ID No. 15; Primers and probes for detecting HPV39 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 16 and SEQ ID No. 17 respectively; the probe sequence is shown in SEQ ID No. 18; Primers and probes for detecting HPV45 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No. 19 and SEQ ID No. 20 respectively; the probe sequence is shown as SEQ ID No. 21; Primers and probes for detecting HPV51 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 22 and SEQ ID No. 23 respectively; the probe sequence is shown in SEQ ID No. 24; Primers and probes for detecting HPV52 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No. 25 and SEQ ID No. 26 respectively; the probe sequence is shown as SEQ ID No. 27; Primers and probes for detecting HPV56 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 28 and SEQ ID No. 29 respectively; the probe sequence is shown in SEQ ID No. 30; Primers and probes for detecting HPV58 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No.31 and SEQ ID No.32 respectively; the probe sequence is shown as SEQ ID No.33; Primers and probes for detecting HPV59 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.34 and SEQ ID No.35 respectively; the probe sequence is shown in SEQ ID No.36; Primers and probes for detecting HPV66 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.37 and SEQ ID No.38 respectively; the probe sequence is shown in SEQ ID No.39; Primers and probes for detecting HPV68 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No.40 and SEQ ID No.41, respectively; the probe sequence is shown as SEQ ID No.42; Primers and probes for detecting internal reference HBB: wherein the upstream and downstream primer sequences are shown as SEQ ID No.43 and SEQ ID No.44 respectively; the probe sequence is shown as SEQ ID No.

45.

2. The primer-probe combination according to claim 1, characterized in that: The probes in the primer probe set are respectively labeled with different fluorescent light-emitting groups.

3. Use of the primer-probe combination of claim 1 or 2 in the preparation of a kit, the function of the kit is to use the melting curve method to type and qualitatively analyze 14 different human papillomaviruses, and the 14 human papillomaviruses are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68.

4. A kit, characterized in that: Comprising the primer-probe combination of claim 1 or 2.

5. The kit according to claim 4, characterized in that It also includes nucleic acid reaction solution, positive control and negative control.

6. The kit according to claim 5, characterized in that The nucleic acid reaction solution includes PCR buffer, MgCl2, dNTPs, DNA polymerase, UNG enzyme and DNase-free water.

7. The kit according to claim 6, characterized in that The PCR buffer is 10×PCR buffer, which includes Tris-HCL, KCl, Tween 20, Brij35, PEG-6000, BSA and water; Among them, the pH value of Tris-HCL is 8.5-9.0, and the final concentration is 50-200 mM; the final concentration of KCl is 200-500 mM, the final concentration of Briji35 is 0.01%-0.2% (mass volume ratio); the final concentration of Tween 20 is 0.01%-0.1% (mass volume ratio), the final concentration of BSA is 0.2 mg / ml-1 mg / ml, and the final concentration of PEG-6000 is 1%-8% (mass volume ratio).

8. The kit according to claim 6, characterized in that The final concentration of the MgCl2 is 1 to 5 mM, the dNTPs include dATP, dTTP, dCTP, dGTP and dUTP, and the ratios are 1:0.5:1:1:0.5, respectively, and the final concentration is 0.1 mM to 0.7 mM; the DNA polymerase is a high-tolerance fast Tag enzyme, and the final concentration is 2 to 5 U; the final concentration of the UNG enzyme is 0.05 U to 0.3 U at a temperature of 50°C.

9. The kit according to claim 6, characterized in that The positive control includes artificially synthesized HPV gene plasmids of different concentrations and at least one HPV typing in each fluorescent channel.

10. A method for detecting human papillomavirus for non-disease diagnosis purposes, characterized in that: include: 1) Designing HPV primer probes based on the conserved sequences of E6 / E7 of 14 human papillomaviruses; the 14 human papillomaviruses are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68; 2) Designing primers and probes for the internal reference gene HBB based on the conserved sequence of the internal reference gene HBB; 3) 14 human papillomavirus and internal reference gene primer probes are mixed according to the proportion to form primer probe MIX; 4) Mix the primer probe MIX with the reaction solution MIX, and add the template to perform PCR amplification; 5) Determine whether the sample is positive based on the fluorescence amplification signals of different channels, and perform typing of the positive nucleic acid using the melting curve analysis results in the corresponding fluorescence channel; The HPV types corresponding to the melting curve analysis of the FAM channel were HPV31, HPV52, HPV68, and HPV45; The HPV types corresponding to the melting curve analysis of the HEX channel were HPV33, HPV56, HPV16, and HPV66; The HPV types corresponding to the melting curve analysis of the ROX channel were HPV35, HPV58, and HPV18; The HPV types corresponding to the melting curve analysis of the Cy5 channel were HPV39, HPV59, HPV51, and the internal reference; Wherein, the HPV primer probe and the internal reference gene HBB primer probe are as follows: Primers and probes for detecting HPV16 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; the probe sequence is shown in SEQ ID No. 3; Primers and probes for detecting HPV18 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively; the probe sequence is shown in SEQ ID No. 6; Primers and probes for detecting HPV31 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively; the probe sequence is shown in SEQ ID No.9; Primers and probes for detecting HPV33 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No. 10 and SEQ ID No. 11 respectively; the probe sequence is shown as SEQ ID No. 12; Primers and probes for detecting HPV35 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 13 and SEQ ID No. 14 respectively; the probe sequence is shown in SEQ ID No. 15; Primers and probes for detecting HPV39 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 16 and SEQ ID No. 17 respectively; the probe sequence is shown in SEQ ID No. 18; Primers and probes for detecting HPV45 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No. 19 and SEQ ID No. 20 respectively; the probe sequence is shown as SEQ ID No. 21; Primers and probes for detecting HPV51 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 22 and SEQ ID No. 23 respectively; the probe sequence is shown in SEQ ID No. 24; Primers and probes for detecting HPV52 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No. 25 and SEQ ID No. 26 respectively; the probe sequence is shown as SEQ ID No. 27; Primers and probes for detecting HPV56 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No. 28 and SEQ ID No. 29 respectively; the probe sequence is shown in SEQ ID No. 30; Primers and probes for detecting HPV58 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No.31 and SEQ ID No.32 respectively; the probe sequence is shown as SEQ ID No.33; Primers and probes for detecting HPV59 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.34 and SEQ ID No.35 respectively; the probe sequence is shown in SEQ ID No.36; Primers and probes for detecting HPV66 typing: wherein the upstream and downstream primer sequences are shown in SEQ ID No.37 and SEQ ID No.38 respectively; the probe sequence is shown in SEQ ID No.39; Primers and probes for detecting HPV68 typing: wherein the upstream and downstream primer sequences are shown as SEQ ID No.40 and SEQ ID No.41, respectively; the probe sequence is shown as SEQ ID No.42; Primers and probes for detecting internal reference HBB: wherein the upstream and downstream primer sequences are shown as SEQ ID No.43 and SEQ ID No.44 respectively; the probe sequence is shown as SEQ ID No.45.

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