Application of Melting Curve Method in Genotyping and Qualitative Analysis of Human Papillomavirus

Abstract

The present invention discloses an application of the melting curve method in the genotyping and qualitative analysis of human papillomavirus. In the present invention, specific primers and corresponding specific fluorescent probes are designed for the E6 / E7 DNA of 18 high-risk and intermediate-risk types of HPV and the internal reference β-globin, and multiple targets in a reaction can be simultaneously detected by using asymmetric PCR amplification combined with the melting curve technology. The present invention can perform qualitative and genotyping detection of 18 types of human papillomavirus. The 18 types of HPV in the present invention refer to HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The present invention uses the carcinogenic gene E6 / E7 DNA of HPV as the detection target, which can avoid the missed detection caused by using HPV L1 DNA as the detection target. At the same time, the melting curve technology used in the present invention has the advantages of high detection sensitivity, good specificity, and high throughput.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and particularly relates to the application of a melting curve method in the typing and qualitative analysis of human papillomavirus. Background Art

[0002] Human papillomavirus (HPV) is divided into high-risk types and low-risk types according to its carcinogenicity. Cervical cancer is mainly caused by persistent infection with high-risk HPV, and high-risk HPV can be detected in 99.7% of cervical cancers. In addition, high-risk HPV can also cause cancers in the anus, vagina, vulva, penis, head and neck and other parts. Low-risk HPV can cause diseases such as anogenital warts and recurrent respiratory papillomatosis. In 2017, a position paper of the World Health Organization (WHO) showed that HPV infection was associated with approximately 4.5% of newly diagnosed cancer cases globally. Results published by the International Agency for Research on Cancer (IARC) showed that in 2018, it was estimated that nearly 570,000 women newly developed cervical cancer globally, and more than 310,000 women died from cervical cancer; among them, there were nearly 110,000 newly diagnosed cases of cervical cancer and nearly 50,000 death cases in China. Cervical cancer has become a public health problem seriously threatening women's health.

[0003] Currently, about 200 HPV types have been identified from the human body. The differentiation of HPV types is mainly based on a 291bp fragment located on the L1 ORF gene, and the difference in the L1 gene between different HPV types exceeds 10%. According to the main infection site, it is divided into cutaneous types and mucosal types; according to the carcinogenic potential, it is divided into high-risk types and low-risk types. In 2012, IARC divided HPV into three groups: Group 1 carcinogens (human carcinogens), including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59; Group 2A carcinogens (substances that are probably carcinogenic to humans), including HPV68; Group 2B carcinogens (substances that may be carcinogenic to humans), including HPV26, 53, 66, 73 and 82, etc.; Group 3, the carcinogenicity to humans is not yet determined, including HPV6 and 11, etc.

[0004] The nucleic acid detection of HPV is another cervical cancer screening technology widely used in clinics after cytological examination, and it makes up for the deficiencies of cytology with its high sensitivity and negative predictive value, being fast, convenient, and capable of high-throughput and automated operation. The typing detection of HPV is meaningful for clinically tracking the persistent infection status of HPV, and for the post-treatment follow-up evaluation of CIN and cervical cancer.

[0005] The present invention provides a method for rapid genotyping and qualitative detection of 18 high- and medium-risk HPV types based on melting curve technology, which can simultaneously genotype 18 HPV types in three reaction tubes, with high sensitivity, good specificity, and clear clinical significance and potential application value. Summary of the Invention

[0006] The object of the present invention is to provide an application of melting curve method in the genotyping and qualitative analysis of human papillomavirus.

[0007] By using the technical principle of PCR amplification combined with melting curve analysis, specific primer-probe pairs for the E6 / E7 region of HPV are designed to make accurate clinical auxiliary diagnosis for patients infected with human papillomavirus, thereby enriching the technical means for clinical detection of HPV.

[0008] The technical solution of the present invention is as follows:

[0009] A primer-probe combination for simultaneously detecting multiple human papillomaviruses, including any two or more of the following primer-probe groups;

[0010] The primer-probe group for detecting HPV16: Among them, the primer sequences for detecting HPV16 are shown as SEQ ID No.1 and SEQ ID No.2; the probe sequence for detecting HPV16 is shown as SEQ ID No.3;

[0011] The primer-probe group for detecting HPV35: Among them, the primer sequences for detecting HPV35 are shown as SEQ ID No.4 and SEQ ID No.5; the probe sequence for detecting HPV35 is shown as SEQ ID No.6;

[0012] The primer-probe group for detecting HPV18: Among them, the primer sequences for detecting HPV18 are shown as SEQ ID No.7 and SEQ ID No.8; the probe sequence for detecting HPV18 is shown as SEQ ID No.9;

[0013] The primer-probe group for detecting HPV45: Among them, the primer sequences for detecting HPV45 are shown as SEQ ID No.10 and SEQ ID No.11; the probe sequence for detecting HPV45 is shown as SEQ ID No.12;

[0014] The primer-probe group for detecting HPV31, among which, the primer sequences for detecting HPV31 are shown as SEQ ID No.13 and SEQ ID No.14; the probe sequence for detecting HPV31 is shown as SEQ ID No.15;

[0015] Primer-probe sets for detecting HPV52, wherein the primer sequences for detecting HPV52 are shown as SEQ ID No.16 and SEQ ID No.17; the probe sequence for detecting HPV52 is shown as SEQ ID No.18;

[0016] Primer-probe sets for detecting HPV33, wherein the primer sequences for detecting HPV33 are shown as SEQ ID No.19 and SEQ ID No.20; the probe sequence for detecting HPV33 is shown as SEQ ID No.21;

[0017] Primer-probe sets for detecting HPV58, wherein the primer sequences for detecting HPV58 are shown as SEQ ID No.22 and SEQ ID No.23; the probe sequence for detecting HPV58 is shown as SEQ ID No.24;

[0018] Primer-probe sets for detecting HPV56, wherein the primer sequences for detecting HPV56 are shown as SEQ ID No.25 and SEQ ID No.26; the probe sequence for detecting HPV56 is shown as SEQ ID No.27;

[0019] Primer-probe sets for detecting HPV66, wherein the primer sequences for detecting HPV66 are shown as SEQ ID No.28 and SEQ ID No.29; the probe sequence for detecting HPV66 is shown as SEQ ID No.30;

[0020] Primer-probe sets for detecting HPV39, wherein the primer sequences for detecting HPV39 are shown as SEQ ID No.31 and SEQ ID No.32; the probe sequence for detecting HPV39 is shown as SEQ ID No.33;

[0021] Primer-probe sets for detecting HPV68, wherein the primer sequences for detecting HPV68 are shown as SEQ ID No.34 and SEQ ID No.35; the probe sequence for detecting HPV68 is shown as SEQ ID No.36;

[0022] Primer-probe sets for detecting HPV51, wherein the primer sequences for detecting HPV51 are shown as SEQ ID No.37 and SEQ ID No.38; the probe sequence for detecting HPV51 is shown as SEQ ID No.39;

[0023] Primer-probe sets for detecting HPV82, wherein the primer sequences for detecting HPV82 are shown as SEQ ID No.40 and SEQ ID No.41; the probe sequence for detecting HPV82 is shown as SEQ ID No.42;

[0024] Primer-probe sets for detecting HPV26, wherein the primer sequences for detecting HPV26 are shown as SEQ ID No.43 and SEQ ID No.44; the probe sequence for detecting HPV26 is shown as SEQ ID No.45;

[0025] Primer-probe sets for detecting HPV53, wherein the primer sequences for detecting HPV53 are shown as SEQ ID No.46 and SEQ ID No.47; the probe sequence for detecting HPV53 is shown as SEQ ID No.48;

[0026] Primer-probe sets for detecting HPV59, wherein the primer sequences for detecting HPV59 are shown as SEQ ID No.49 and SEQ ID No.50; the probe sequence for detecting HPV59 is shown as SEQ ID No.51;

[0027] Primer-probe sets for detecting HPV73, wherein the primer sequences for detecting HPV73 are shown as SEQ ID No.52 and SEQ ID No.53; the probe sequence for detecting HPV73 is shown as SEQ ID No.54.

[0028] The primer-probe sets further include a primer-probe combination for detecting the human β-globin internal reference gene;

[0029] The primer sequences for detecting the human β-globin internal reference gene are shown as SEQ ID No.55 and SEQ ID No.56

[0030] The probe sequence for detecting the human β-globin internal reference gene is shown as SEQ ID No.57.

[0031] The present invention also provides a kit comprising the primer-probe sets as described above.

[0032] The present invention also provides an application of the melting curve method in the genotyping and qualitative analysis of different human papillomaviruses. The application is the genotyping and qualitative analysis of HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82, and includes the following steps:

[0033] S1. Design specific primer probes according to the E6 / E7 DNA of HPV types;

[0034] S2. Design a pair of internal reference gene primer probes according to the human β-globin gene;

[0035] S3. Combine the primer probes of HPV16, HPV35, HPV18, HPV45, HPV31, HPV52 and the internal reference gene with the materials required for PCR to form the reaction solution of HPV-A group; Combine the primer probes of HPV33, HPV58, HPV56, HPV66, HPV39, HPV68 and the internal reference gene with the materials required for PCR to form the reaction solution of HPV-B group; Combine the primer probes of HPV51, HPV82, HPV53, HPV26, HPV59, HPV73 and the internal reference gene with the materials required for PCR to form the reaction solution of HPV-C group;

[0036] S4. After adding the sample nucleic acid to the reaction solution, use the asymmetric PCR amplification technology combined with melting curve analysis to perform amplification on a fluorescence PCR instrument;

[0037] S5. Determine whether the sample is positive according to the fluorescence amplification signals of different channels, and perform typing on the positive nucleic acid using the melting curve analysis results in the corresponding fluorescence channels.

[0038] In the reaction solution of HPV-A group:

[0039] The HPV types corresponding to the melting curve analysis in the FAM channel are HPV16 and HPV35 in sequence;

[0040] The HPV types corresponding to the melting curve analysis in the CY5 channel are HPV18 and HPV45 in sequence;

[0041] The HPV types corresponding to the melting curve analysis in the ROX channel are HPV31 and HPV52 in sequence;

[0042] The internal reference gene β-globin corresponds to the melting curve analysis in the VIC channel;

[0043] In the reaction solution of HPV-B group:

[0044] The HPV types corresponding to the melting curve analysis in the FAM channel are HPV33 and HPV58 in sequence;

[0045] The HPV types corresponding to the melting curve analysis in the CY5 channel are HPV56 and HPV66 in sequence;

[0046] The HPV types corresponding to the melting curve analysis in the ROX channel are HPV39 and HPV68 in sequence;

[0047] The melting curve analysis of the VIC channel corresponds to the internal reference gene β-globin;

[0048] In the reaction solution of the HPV-C group:

[0049] The HPV types corresponding to the melting curve analysis of the FAM channel are HPV51 and HPV82 in sequence;

[0050] The HPV types corresponding to the melting curve analysis of the CY5 channel are HPV26 and HPV53 in sequence;

[0051] The HPV types corresponding to the melting curve analysis of the ROX channel are HPV59 and HPV73 in sequence;

[0052] The melting curve analysis of the VIC channel corresponds to the internal reference gene β-globin.

[0053] The primer and probe sequences in the reaction solution of the HPV-A group are as follows:

[0054] The primer sequences for detecting HPV16 are shown as SEQ ID No.1 and SEQ ID No.2; for detecting HPV16

[0055] The probe sequence is shown as SEQ ID No.3;

[0056] The primer sequences for detecting HPV35 are shown as SEQ ID No.4 and SEQ ID No.5; the probe sequence for detecting HPV35 is shown as SEQ ID No.6;

[0057] The primer sequences for detecting HPV18 are shown as SEQ ID No.7 and SEQ ID No.8; the probe sequence for detecting HPV18 is shown as SEQ ID No.9;

[0058] The primer sequences for detecting HPV45 are shown as SEQ ID No.10 and SEQ ID No.11; the probe sequence for detecting HPV45 is shown as SEQ ID No.12;

[0059] The primer sequences for detecting HPV31 are shown as SEQ ID No.13 and SEQ ID No.14; the probe sequence for detecting HPV31 is shown as SEQ ID No.15;

[0060] The primer sequences for detecting HPV52 are shown as SEQ ID No.16 and SEQ ID No.17; the probe sequence for detecting HPV52 is shown as SEQ ID No.18;

[0061] The primer sequences for detecting human β-globin internal reference gene are as shown in SEQ ID No.55 and SEQ ID No.56;

[0062] The probe sequence for detecting human β-globin internal reference gene is as shown in SEQ ID No.57;

[0063] The primer and probe sequences in the HPV-B group reaction solution are as follows:

[0064] The primer sequences for detecting HPV33 are as shown in SEQ ID No.19 and SEQ ID No.20; the probe sequence for detecting HPV33 is as shown in SEQ ID No.21;

[0065] The primer sequences for detecting HPV58 are as shown in SEQ ID No.22 and SEQ ID No.23; the probe sequence for detecting HPV58 is as shown in SEQ ID No.24;

[0066] The primer sequences for detecting HPV56 are as shown in SEQ ID No.25 and SEQ ID No.26; the probe sequence for detecting HPV56 is as shown in SEQ ID No.27;

[0067] The primer sequences for detecting HPV66 are as shown in SEQ ID No.28 and SEQ ID No.29; the probe sequence for detecting HPV66 is as shown in SEQ ID No.30;

[0068] The primer sequences for detecting HPV39 are as shown in SEQ ID No.31 and SEQ ID No.32; the probe sequence for detecting HPV39 is as shown in SEQ ID No.33;

[0069] The primer sequences for detecting HPV68 are as shown in SEQ ID No.34 and SEQ ID No.35; the probe sequence for detecting HPV68 is as shown in SEQ ID No.36;

[0070] The primer sequences for detecting human β-globin internal reference gene are as shown in SEQ ID No.55 and SEQ ID No.56;

[0071] The probe sequence for detecting human β-globin internal reference gene is as shown in SEQ ID No.57;

[0072] The primer and probe sequences in the HPV-C group reaction solution are as follows:

[0073] The primer sequences for detecting HPV51 are shown in SEQ ID No.37 and SEQ ID No.38; the probe sequence for detecting HPV51 is shown in SEQ ID No.39;

[0074] The primer sequences for detecting HPV82 are shown in SEQ ID No.40 and SEQ ID No.41; the probe sequence for detecting HPV82 is shown in SEQ ID No.42;

[0075] The primer sequences for detecting HPV26 are shown in SEQ ID No.43 and SEQ ID No.44; the probe sequence for detecting HPV26 is shown in SEQ ID No.45;

[0076] The primer sequences for detecting HPV53 are shown in SEQ ID No.46 and SEQ ID No.47; the probe sequence for detecting HPV53 is shown in SEQ ID No.48;

[0077] The primer sequences for detecting HPV59 are shown in SEQ ID No.49 and SEQ ID No.50; the probe sequence for detecting HPV59 is shown in SEQ ID No.51;

[0078] The primer sequences for detecting HPV73 are shown in SEQ ID No.52 and SEQ ID No.53; the probe sequence for detecting HPV73 is shown in SEQ ID No.54;

[0079] The primer sequences for detecting the human β-globin internal reference gene are shown in SEQ ID No.55 and SEQ ID No.56;

[0080] The probe sequence for detecting the human β-globin internal reference gene is shown in SEQ ID No.57.

[0081] The present invention provides a kit for simultaneously detecting multiple human papillomaviruses by using melting curves, including an HPV-A group reaction solution, an HPV-B group reaction solution, and an HPV-C group reaction solution;

[0082] The primer and probe sequences in the HPV-A group reaction solution are as follows:

[0083] The primer sequences for detecting HPV16 are shown in SEQ ID No.1 and SEQ ID No.2; for detecting HPV16

[0084] the probe sequence is shown in SEQ ID No.3;

[0085] The primer sequences for detecting HPV35 are shown in SEQ ID No.4 and SEQ ID No.5; the probe sequence for detecting HPV35 is shown in SEQ ID No.6;

[0086] The primer sequences for detecting HPV18 are shown in SEQ ID No.7 and SEQ ID No.8; the probe sequence for detecting HPV18 is shown in SEQ ID No.9;

[0087] The primer sequences for detecting HPV45 are shown in SEQ ID No.10 and SEQ ID No.11; the probe sequence for detecting HPV45 is shown in SEQ ID No.12;

[0088] The primer sequences for detecting HPV31 are shown in SEQ ID No.13 and SEQ ID No.14; the probe sequence for detecting HPV31 is shown in SEQ ID No.15;

[0089] The primer sequences for detecting HPV52 are shown in SEQ ID No.16 and SEQ ID No.17; the probe sequence for detecting HPV52 is shown in SEQ ID No.18;

[0090] The primer sequences for detecting the human β-globin internal reference gene are shown in SEQ ID No.55 and SEQ ID No.56;

[0091] The probe sequence for detecting the human β-globin internal reference gene is shown in SEQ ID No.57;

[0092] The primer and probe sequences in the HPV-B group reaction solution are as follows:

[0093] The primer sequences for detecting HPV33 are shown in SEQ ID No.19 and SEQ ID No.20; the probe sequence for detecting HPV33 is shown in SEQ ID No.21;

[0094] The primer sequences for detecting HPV58 are shown in SEQ ID No.22 and SEQ ID No.23; the probe sequence for detecting HPV58 is shown in SEQ ID No.24;

[0095] The primer sequences for detecting HPV56 are shown in SEQ ID No.25 and SEQ ID No.26; the probe sequence for detecting HPV56 is shown in SEQ ID No.27;

[0096] The primer sequences for detecting HPV66 are shown in SEQ ID No.28 and SEQ ID No.29; the probe sequence for detecting HPV66 is shown in SEQ ID No.30;

[0097] The primer sequences for detecting HPV39 are shown in SEQ ID No.31 and SEQ ID No.32; the probe sequence for detecting HPV39 is shown in SEQ ID No.33;

[0098] The primer sequences for detecting HPV68 are shown in SEQ ID No.34 and SEQ ID No.35; the probe sequence for detecting HPV68 is shown in SEQ ID No.36;

[0099] The primer sequences for detecting the human β-globin internal reference gene are shown in SEQ ID No.55 and SEQ ID No.56;

[0100] The probe sequence for detecting the human β-globin internal reference gene is shown in SEQ ID No.57;

[0101] The primer and probe sequences in the HPV-C group reaction solution are as follows:

[0102] The primer sequences for detecting HPV51 are shown in SEQ ID No.37 and SEQ ID No.38; the probe sequence for detecting HPV51 is shown in SEQ ID No.39;

[0103] The primer sequences for detecting HPV82 are shown in SEQ ID No.40 and SEQ ID No.41; the probe sequence for detecting HPV82 is shown in SEQ ID No.42;

[0104] The primer sequences for detecting HPV26 are shown in SEQ ID No.43 and SEQ ID No.44; the probe sequence for detecting HPV26 is shown in SEQ ID No.45;

[0105] The primer sequences for detecting HPV53 are shown in SEQ ID No.46 and SEQ ID No.47; the probe sequence for detecting HPV53 is shown in SEQ ID No.48;

[0106] The primer sequences for detecting HPV59 are shown in SEQ ID No.49 and SEQ ID No.50; the probe sequence for detecting HPV59 is shown in SEQ ID No.51;

[0107] The primer sequences for detecting HPV73 are shown as SEQ ID No.52 and SEQ ID No.53; the probe sequence for detecting HPV73 is SEQ ID No.54;

[0108] The primer sequences for detecting the human β-globin internal reference gene are shown as SEQ ID No.55 and SEQ ID No.56;

[0109] The probe sequence for detecting the human β-globin internal reference gene is SEQ ID No.57.

[0110] The described kit further includes a nucleic acid amplification reaction solution, a positive control, and a negative control.

[0111] The described nucleic acid amplification reaction solution is composed of 2×PCR buffer, MgCl2 with a final concentration of 2 - 4 mM, 0.05 - 0.5 mM dNTPs (dATP:dTTP:dCTP:dGTP:dUTP = 1:1:1:1:1), 2 - 4 U DNA polymerase, 0.05 - 0.1 U UNG enzyme, and RNase-free water.

[0112] The present invention also provides a method for detecting human papillomavirus using the above-mentioned primer-probe combination, and the method includes the steps of sample collection and viral nucleic acid extraction, loading and sealing of the reaction solution, and amplification and detection of nucleic acids.

[0113] Preferably, in the above technical solution, the main components of the positive control are synthetic genes with different concentrations and DNA of the C33a human cervical cancer cell line;

[0114] Preferably, in the above technical solution, the main component of the negative control is DNA of the C33a human cervical cancer cell line.

[0115] The application of the kit as described above in the genotyping and qualitative analysis of HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82 also belongs to the protection scope of the present invention.

[0116] As another embodiment of the present invention, a method for simultaneously detecting multiple human papillomaviruses using a melting curve specifically includes the following steps:

[0117] 1. Configuration of the reaction system: 10 - 20 μL of nucleic acid amplification reaction solution, 10 - 20 μL of primer-probe reaction solution, 5 - 20 μL of nucleic acid of the sample to be tested;

[0118] 2. Amplification of real-time fluorescence PCR: After adding the prepared PCR reaction solution with the detection template, place it in a fluorescence quantitative PCR instrument for detection. The specific experimental detection procedure is as follows: UNG enzyme treatment at 50°C for 5 min; pre-denaturation at 95°C for 10 min; the conditions for denaturation, annealing, extension, and fluorescence detection are: 95°C for 10 s, 60°C for 30 - 40 s, 45 cycles, fluorescence detection at 60°C; melting segment at 95°C for 30 s, 46°C for 20 s, collect fluorescence from 46°C to 75°C, 1 cycle.

[0119] 3. Result analysis: After the experiment, make a judgment according to the following criteria:

[0120] In the positive control tube, there should be only one melting peak in the VIC channel after melting curve analysis, and there should be two melting peaks in other channels. In the negative control, there should be only one melting peak in the VIC channel, and there should be no obvious melting peaks in other channels. There should be one melting peak in the internal reference channel (VIC) of reaction tubes A, B, and C. If the above requirements are met, it indicates that the experiment is successful. Then analyze the sample tubes:

[0121] Tube A: Taking the control reaction tube as a reference, samples with two peaks (Tm value deviation less than 1.5°C) in the FAM channel that are the same as those in the control are positive for HPV16 and HPV35. Samples with only one low-Tm melting peak are positive for HPV16. Samples with only one high-Tm melting peak are positive for HPV35. Taking the control reaction tube as a reference, samples with two peaks (Tm value deviation less than 1.5°C) in the CY5 channel that are the same as those in the control are positive for HPV18 and HPV45. Samples with only one low-Tm melting peak are positive for HPV18. Samples with only one high-Tm melting peak are positive for HPV45. Taking the control reaction tube as a reference, samples with two peaks (Tm value deviation less than 1.5°C) in the ROX channel that are the same as those in the control are positive for HPV31 and HPV52. Samples with only one low-Tm melting peak are positive for HPV31. Samples with only one high-Tm melting peak are positive for HPV52.

[0122] Tube B: Using the control reaction tube as a reference, samples with two peaks identical to the control in the FAM channel (Tm value deviation less than 1.5 °C) are positive for HPV33 and HPV58. Samples with only one low-Tm melting peak are positive for HPV33, and samples with only one high-Tm melting peak are positive for HPV58. Using the control reaction tube as a reference, samples with two peaks identical to the control in the CY5 channel (Tm value deviation less than 1.5 °C) are positive for HPV56 and HPV66. Samples with only one low-Tm melting peak are positive for HPV56, and samples with only one high-Tm melting peak are positive for HPV66. Using the control reaction tube as a reference, samples with two peaks identical to the control in the ROX channel (Tm value deviation less than 1.5 °C) are positive for HPV39 and HPV68. Samples with only one low-Tm melting peak are positive for HPV39, and samples with only one high-Tm melting peak are positive for HPV68.

[0123] Tube C: Using the control reaction tube as a reference, samples with two peaks identical to the control in the FAM channel (Tm value deviation less than 1.5 °C) are positive for HPV51 and HPV82. Samples with only one low-Tm melting peak are positive for HPV51, and samples with only one high-Tm melting peak are positive for HPV82. Using the control reaction tube as a reference, samples with two peaks identical to the control in the CY5 channel (Tm value deviation less than 1.5 °C) are positive for HPV26 and HPV53. Samples with only one low-Tm melting peak are positive for HPV26, and samples with only one high-Tm melting peak are positive for HPV53. Using the control reaction tube as a reference, samples with two peaks identical to the control in the ROX channel (Tm value deviation less than 1.5 °C) are positive for HPV59 and HPV73. Samples with only one low-Tm melting peak are positive for HPV59, and samples with only one high-Tm melting peak are positive for HPV73.

[0124] Compared with the prior art, the present invention has the following beneficial effects:

[0125] (1) The present invention uses the oncogene E6 / E7 DNA of each HPV type as the detection target, which can avoid the missed detection caused by using HPVL1 DNA as the detection target in the traditional method.

[0126] (2) High detection throughput: It can achieve multi-target gene detection in one fluorescence channel, greatly improving the throughput of nucleic acid detection and being suitable for cervical cancer screening.

[0127] (3) High accuracy: Typing positive samples according to the melting curve results, the result interpretation is simple and convenient, and the interpretation result is more accurate.

[0128] (4) Good specificity: It has high specificity and no cross-reactivity with other HPV types not within the detection scope of the present invention, such as HPV6, 11, 40, 42, 43, 44, 54, 61, 67, 69, 70, 71, 72, 81 and 83. It also has no cross-reactivity with other pathogens that have the same infection site or similar infection symptoms and are common, such as cytomegalovirus, Epstein-Barr virus, herpes simplex virus type II, Neisseria gonorrhoeae, Treponema pallidum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia trachomatis, Trichomonas vaginalis, Streptococcus viridans, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Bacteroides fragilis, Mycobacterium smegmatis, Acinetobacter baumannii, Corynebacterium parvum, Corynebacterium vaginale and Candida albicans.

[0129] (5) High sensitivity: The minimum detection limit of each HPV type can reach 500 copies / mL. Brief Description of the Drawings

[0130] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objectives and advantages of the present invention will become more obvious:

[0131] Figure 1 It is the detection result of the samples of HPV16, 35, 18, 45, 31 and 52 types in tube A;

[0132] Figure 2 It is the detection result of the samples of HPV33, 58, 56, 66, 39 and 68 types in tube B;

[0133] Figure 3 It is the detection result of the samples of HPV51, 82, 26, 53, 59 and 73 types in tube C. Detailed Embodiment

[0134] The present invention will be described in detail below in conjunction with the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several adjustments and improvements can still be made. These all belong to the protection scope of the present invention.

[0135] Embodiment 1 Design of Specific Primers and Probes

[0136] The whole genome or gene sequences of regions such as E6 and E7 of different HPV types were searched and downloaded through NCBI. After sequence alignment, specific amplification primers and Taqman probes for the above-mentioned pathogens and internal reference β-globin were designed using Beacon Designer 8 and synthesized by Shanghai Shuoying Biotechnology Co., Ltd. The primer and probe sequences for detection are shown in Table 1 below:

[0137] Table 1 Primer and Probe Sequence Information

[0138]

[0139]

[0140] Example 2 Sample Processing

[0141] 1. Sample Type: Cervical exfoliated cells

[0142] 2. Sample Collection:

[0143] Use a cervical sampling swab to collect cervical exfoliated cells at the cervical os. Insert the brush parallel into the uterus, gently insert the middle bristles of the brush deep into the endocervical canal so that the shorter bristles can fully contact the ectocervix. Fix it gently by hand, gently press forward against the sampler, and then turn the sampler 5 - 10 circles in the same direction. Slowly remove the sampler and place it in a special cell preservation solution. Repeatedly push the broom-shaped sampler to the bottom of the bottle and rinse it up and down about 10 times. Finally, quickly rotate the broom-shaped sampler in the solution to further rinse down the cell sample. Discard the sampler, tighten the bottle cap, and send it for inspection in a sealed manner.

[0144] 3. Sample Preservation and Transportation

[0145] The sample should be detected in a timely manner after collection. It should be detected within 6 months when stored at 2 - 30°C, stored at -20 ± 5°C for no more than 12 months, and is valid for a long time at -70°C or below. Avoid repeated freezing and thawing of the sample, and the number of freeze-thaw cycles should not exceed 5 times. The transportation time at 2 - 30°C should not exceed 7 days.

[0146] 4. Nucleic Acid Extraction

[0147] The nucleic acid extraction of the sample uses the DNAMini Kit of QIAGEN Company or the nucleic acid extraction or purification reagent of Jiangsu Biosino Bio-Technology & Science Co., Ltd., and the nucleic acid extraction is carried out according to the instructions.

[0148] Example 3 Preparation of Reaction System

[0149] Prepare reaction system tubes A, B, and C according to the following operations: The total volume is 40 μL. 15 μL of nucleic acid amplification reaction solution, 15 μL each of HPV-A group reaction solution, HPV-B group reaction solution, and HPV-C group reaction solution, and 10 μL of nucleic acid;

[0150] Among them:

[0151] The primers and probes of HPV16, 35, 18, 45, 31, 52 and the internal reference gene are combined with the materials required for PCR to form the HPV-A group reaction solution;

[0152] The primers and probes of HPV33, 58, 56, 66, 39, 68 and the internal reference gene are combined with the materials required for PCR to form the reaction solution of HPV-B group;

[0153] The primers and probes of HPV51, 82, 53, 26, 59, 73 and the internal reference gene are combined with the materials required for PCR to form the reaction solution of HPV-C group.

[0154] The dosages of specific primers and probes are shown in Table 2 below.

[0155] Table 2 Dosages of primers and probes

[0156]

[0157]

[0158] Detect the nucleic acid extracted in Example 4

[0159] Prepare the amplification reaction system according to the above concentrations, and set the PCR cycle parameters according to the procedure in Table 3 (set according to the operation instructions of the instrument). Table 3 PCR cycle parameters

[0160]

[0161] Among them:

[0162] In the reaction solution of HPV-A group:

[0163] The HPV types corresponding to the melting curve analysis in the FAM channel are HPV16 and HPV35 in sequence;

[0164] The HPV types corresponding to the melting curve analysis in the CY5 channel are HPV18 and HPV45 in sequence;

[0165] The HPV types corresponding to the melting curve analysis in the ROX channel are HPV31 and HPV52 in sequence;

[0166] The melting curve analysis in the VIC channel corresponds to the internal reference gene β-globin;

[0167] In the reaction solution of HPV-B group:

[0168] The HPV types corresponding to the melting curve analysis in the FAM channel are HPV33 and HPV58 in sequence;

[0169] The HPV types corresponding to the melting curve analysis in the CY5 channel are HPV56 and HPV66 in sequence;

[0170] The HPV types corresponding to the melting curve analysis in the ROX channel are HPV39 and HPV68 in sequence;

[0171] The melting curve analysis of the VIC channel corresponds to the internal reference gene β-globin;

[0172] In the reaction solution of the HPV-C group:

[0173] The HPV types corresponding to the melting curve analysis of the FAM channel are HPV51 and HPV82 in sequence;

[0174] The HPV types corresponding to the melting curve analysis of the CY5 channel are HPV26 and HPV53 in sequence;

[0175] The HPV types corresponding to the melting curve analysis of the ROX channel are HPV59 and HPV73 in sequence;

[0176] The melting curve analysis of the VIC channel corresponds to the internal reference gene β-globin.

[0177] Result interpretation of Example 5

[0178] In the positive control tube, there should be only one melting peak in the VIC channel after melting curve analysis, and there should be two melting peaks in other channels. In the negative control, there should be only one melting peak in the VIC channel, and there should be no obvious melting peaks in other channels. There should be one melting peak in the internal reference channel (VIC) of reaction tubes A, B, and C. If the above requirements are met, it indicates that this experiment is successful, and the sample tubes are analyzed:

[0179] Tube A: With the reference of the control reaction tube, samples with two peaks (Tm value deviation less than 1.5 °C) in the FAM channel that are the same as the control are positive for HPV16 and HPV35. Samples with only one low-Tm melting peak are positive for HPV16. Samples with only one high-Tm melting peak are positive for HPV35. With the reference of the control reaction tube, samples with two peaks (Tm value deviation less than 1.5 °C) in the CY5 channel that are the same as the control are positive for HPV18 and HPV45. Samples with only one low-Tm melting peak are positive for HPV18. Samples with only one high-Tm melting peak are positive for HPV45. With the reference of the control reaction tube, samples with two peaks (Tm value deviation less than 1.5 °C) in the ROX channel that are the same as the control are positive for HPV31 and HPV52. Samples with only one low-Tm melting peak are positive for HPV31. Samples with only one high-Tm melting peak are positive for HPV52.

[0180] Tube B: Using the control reaction tube as a reference, samples with two peaks identical to the control in the FAM channel (Tm value deviation less than 1.5 °C) are positive for HPV33 and HPV58. Samples with only one low-Tm melting peak are positive for HPV33, and samples with only one high-Tm melting peak are positive for HPV58. Using the control reaction tube as a reference, samples with two peaks identical to the control in the CY5 channel (Tm value deviation less than 1.5 °C) are positive for HPV56 and HPV66. Samples with only one low-Tm melting peak are positive for HPV56, and samples with only one high-Tm melting peak are positive for HPV66. Using the control reaction tube as a reference, samples with two peaks identical to the control in the ROX channel (Tm value deviation less than 1.5 °C) are positive for HPV39 and HPV68. Samples with only one low-Tm melting peak are positive for HPV39, and samples with only one high-Tm melting peak are positive for HPV68.

[0181] Tube C: Using the control reaction tube as a reference, samples with two peaks identical to the control in the FAM channel (Tm value deviation less than 1.5 °C) are positive for HPV51 and HPV82. Samples with only one low-Tm melting peak are positive for HPV51, and samples with only one high-Tm melting peak are positive for HPV82. Using the control reaction tube as a reference, samples with two peaks identical to the control in the CY5 channel (Tm value deviation less than 1.5 °C) are positive for HPV26 and HPV53. Samples with only one low-Tm melting peak are positive for HPV26, and samples with only one high-Tm melting peak are positive for HPV53. Using the control reaction tube as a reference, samples with two peaks identical to the control in the ROX channel (Tm value deviation less than 1.5 °C) are positive for HPV59 and HPV73. Samples with only one low-Tm melting peak are positive for HPV59, and samples with only one high-Tm melting peak are positive for HPV73.

[0182] Example 6 Performance Evaluation

[0183] 1. Specificity Analysis

[0184] The method of the present invention was used to verify the specificity of the present invention for pathogens with the same infection site and similar symptoms as human papillomavirus and human papillomaviruses outside the detection range of the present invention. The concentration of bacteria was 10 6 CFU / mL, the concentration of virus was 10 5 PFU / mL; the concentration of Chlamydia trachomatis was 10 5 IFU / mL; the concentration of Trichomonas vaginalis was 10 5 cells / mL; the concentration of other HPV types was 10 6copies / mL. The results of detecting the pathogens in Table 4 showed that no obvious melting peaks were observed and the results were negative. Therefore, the above pathogens will not interfere with the detection of the present invention within the concentration range studied.

[0185] Table 4 Pathogen names

[0186]

[0187] 2. Sensitivity analysis

[0188] The sensitivity of the present invention was determined by gradient dilution of the positive reference product calibrated by digital PCR. After gradient dilution of the positive reference products of each HPV type as required, 5×10 6 copies, 5×10 5 copies / mL, 5×10 4 copies / mL, 5×10 3 copies / mL, 5×10 2 copies / mL, 5×10 1 copies / mL of six concentration gradients were used to determine the sensitivity of the present invention, and each concentration gradient was detected 20 times repeatedly.

[0189] Probit regression analysis was performed on the data using SSPS software. The concentration when the positive detection rate of the virus sample ≥ 95% was the lowest detection limit of the present invention. In addition, the HPV standard product was gradient diluted to the lowest detection limit concentration (500 copies / mL), and each was detected 20 times repeatedly to verify the sensitivity of the present invention. Statistical analysis was performed on the experimental results, and the detection rates of 18 types of the HPV standard product at a concentration of 500 copies / mL were all greater than 95%. Therefore, the sensitivity of the present invention was determined to be 500 copies / mL.

[0190] Example 7 Detection of clinical samples

[0191] Clinical positive samples of 18 types of HPV: including HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV82. The positive samples were all provided by the hospital and had been typed and confirmed with typing reagents.

[0192] Sample processing, reaction system preparation, nucleic acid detection and result interpretation were carried out according to Example 2, Example 3, Example 4 and Example 5.

[0193] The experimental results are as Figure 1 ,Figure 2 and Figure 3 As shown, each type can be accurately detected. Figure 1 They are the test results of samples of HPV types 16, 35, 18, 45, 31, and 52 in tube A. Figure 2 They are the test results of samples of HPV types 33, 58, 56, 66, 39, and 68 in tube B. Figure 3 They are the test results of samples of HPV types 51, 82, 26, 53, 59, and 73 in tube C.

[0194] The present invention discloses a method for simultaneously detecting multiple human papillomaviruses (HPV) by using a melting curve technology. In the present invention, specific primers and corresponding specific fluorescent probes are designed for the E6 / E7 DNA of 18 high-risk and intermediate-risk types of HPV and the internal reference β-globin, and multiple targets in one reaction can be simultaneously detected by using asymmetric PCR amplification combined with the melting curve technology. The present invention can perform qualitative and typing detection of 18 human papillomaviruses. The 18 HPV types in the present invention refer to HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The present invention uses the carcinogenic gene E6 / E7 DNA of HPV as the detection target, which can avoid missed detection caused by using HPV L1 DNA as the detection target. At the same time, the melting curve technology used in the present invention has the advantages of high detection sensitivity, good specificity, and high throughput.

[0195] It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A primer-probe combination for simultaneously detecting multiple human papillomaviruses, characterized in that, Comprising the following primer-probe sets; Primer-probe set for detecting HPV16: Among them, the primer sequences for detecting HPV16 are shown as SEQ ID No.1 and SEQ ID No.2; the probe sequence for detecting HPV16 is shown as SEQ ID No.3; Primer-probe set for detecting HPV35: Among them, the primer sequences for detecting HPV35 are shown as SEQ ID No.4 and SEQ ID No.5; the probe sequence for detecting HPV35 is shown as SEQ ID No.6; Primer-probe set for detecting HPV18: Among them, the primer sequences for detecting HPV18 are shown as SEQ ID No.7 and SEQ ID No.8; the probe sequence for detecting HPV18 is shown as SEQ ID No.9; Primer-probe set for detecting HPV45: Among them, the primer sequences for detecting HPV45 are shown as SEQ ID No.10 and SEQ ID No.11; the probe sequence for detecting HPV45 is shown as SEQ ID No.12; Primer-probe set for detecting HPV31, among which, the primer sequences for detecting HPV31 are shown as SEQ ID No.13 and SEQ ID No.14; the probe sequence for detecting HPV31 is shown as SEQ ID No.15; Primer-probe set for detecting HPV52, among which, the primer sequences for detecting HPV52 are shown as SEQ ID No.16 and SEQ ID No.17; the probe sequence for detecting HPV52 is shown as SEQ ID No.18; Primer-probe set for detecting HPV33, among which, the primer sequences for detecting HPV33 are shown as SEQ ID No.19 and SEQ ID No.20; the probe sequence for detecting HPV33 is shown as SEQ ID No.21; Primer-probe set for detecting HPV58, among which, the primer sequences for detecting HPV58 are shown as SEQ ID No.22 and SEQ ID No.23; the probe sequence for detecting HPV58 is shown as SEQ ID No.24; Primer-probe set for detecting HPV56, among which, the primer sequences for detecting HPV56 are shown as SEQ ID No.25 and SEQ ID No.26; the probe sequence for detecting HPV56 is shown as SEQ ID No.27; Primer-probe set for detecting HPV66, among which, the primer sequences for detecting HPV66 are shown as SEQ ID No.28 and SEQ ID No.29; the probe sequence for detecting HPV66 is shown as SEQ ID No.30; Primer-probe set for detecting HPV39, among which, the primer sequences for detecting HPV39 are shown as SEQ ID No.31 and SEQ ID No.32; the probe sequence for detecting HPV39 is shown as SEQ ID No.33; Primer-probe sets for detecting HPV68, wherein the primer sequences for detecting HPV68 are shown as SEQ ID No.34 and SEQ ID No.35; the probe sequence for detecting HPV68 is shown as SEQ ID No.36; Primer-probe sets for detecting HPV51, wherein the primer sequences for detecting HPV51 are shown as SEQ ID No.37 and SEQ ID No.38; the probe sequence for detecting HPV51 is shown as SEQ ID No.39; Primer-probe sets for detecting HPV82, wherein the primer sequences for detecting HPV82 are shown as SEQ ID No.40 and SEQ ID No.41; the probe sequence for detecting HPV82 is shown as SEQ ID No.42; Primer-probe sets for detecting HPV26, wherein the primer sequences for detecting HPV26 are shown as SEQ ID No.43 and SEQ ID No.44; the probe sequence for detecting HPV26 is shown as SEQ ID No.45; Primer-probe sets for detecting HPV53, wherein the primer sequences for detecting HPV53 are shown as SEQ ID No.46 and SEQ ID No.47; the probe sequence for detecting HPV53 is shown as SEQ ID No.48; Primer-probe sets for detecting HPV59, wherein the primer sequences for detecting HPV59 are shown as SEQ ID No.49 and SEQ ID No.50; the probe sequence for detecting HPV59 is shown as SEQ ID No.51; Primer-probe sets for detecting HPV73, wherein the primer sequences for detecting HPV73 are shown as SEQ ID No.52 and SEQ ID No.53; the probe sequence for detecting HPV73 is shown as SEQ ID No.

54.

2. The primer-probe set according to claim 1, wherein The primer-probe sets further include a primer-probe combination for detecting the human β-globin internal reference gene; The primer sequences for detecting the human β-globin internal reference gene are shown as SEQ ID No.55 and SEQ ID No.56; The probe sequence for detecting the human β-globin internal reference gene is shown as SEQ ID No.

57.

3. A kit comprising the primer-probe sets as claimed in claim 1 or 2.

4. A kit for simultaneously detecting multiple human papillomaviruses using melting curves, characterized in that: It includes HPV-A group reaction solution, HPV-B group reaction solution and HPV-C group reaction solution; The primer-probe sequences in the HPV-A group reaction solution are as follows: The primer sequences for detecting HPV16 are shown as SEQ ID No.1 and SEQ ID No.2; the probe sequence for detecting HPV16 is shown as SEQ ID No.3; The primer sequences for detecting HPV35 are shown as SEQ ID No.4 and SEQ ID No.5; the probe sequence for detecting HPV35 is shown as SEQ ID No.6; The primer sequences for detecting HPV18 are shown in SEQ ID No.7 and SEQ ID No.8; the probe sequence for detecting HPV18 is shown in SEQ ID No.9; The primer sequences for detecting HPV45 are shown in SEQ ID No.10 and SEQ ID No.11; the probe sequence for detecting HPV45 is shown in SEQ ID No.12; The primer sequences for detecting HPV31 are shown in SEQ ID No.13 and SEQ ID No.14; The probe sequence for detecting HPV31 is shown in SEQ ID No.15; The primer sequences for detecting HPV52 are shown in SEQ ID No.16 and SEQ ID No.17; The probe sequence for detecting HPV52 is shown in SEQ ID No.18; The primer sequences for detecting the human β-globin internal reference gene are shown in SEQ ID No.55 and SEQ ID No.56; The probe sequence for detecting the human β-globin internal reference gene is shown in SEQ ID No.57; The primer and probe sequences in the HPV-B group reaction solution are as follows: The primer sequences for detecting HPV33 are shown in SEQ ID No.19 and SEQ ID No.20; the probe sequence for detecting HPV33 is shown in SEQ ID No.21; The primer sequences for detecting HPV58 are shown in SEQ ID No.22 and SEQ ID No.23; the probe sequence for detecting HPV58 is shown in SEQ ID No.24; The primer sequences for detecting HPV56 are shown in SEQ ID No.25 and SEQ ID No.26; the probe sequence for detecting HPV56 is shown in SEQ ID No.27; The primer sequences for detecting HPV66 are shown in SEQ ID No.28 and SEQ ID No.29; The probe sequence for detecting HPV66 is shown in SEQ ID No.30; The primer sequences for detecting HPV39 are shown in SEQ ID No.31 and SEQ ID No.32; The probe sequence for detecting HPV39 is shown in SEQ ID No.33; The primer sequences for detecting HPV68 are shown in SEQ ID No.34 and SEQ ID No.35; The probe sequence for detecting HPV68 is shown in SEQ ID No.36; The primer sequences for detecting the human β-globin internal reference gene are shown in SEQ ID No.55 and SEQ ID No.56; The probe sequence for detecting the human β-globin internal reference gene is shown in SEQ ID No.57; The primer and probe sequences in the HPV-C group reaction solution are as follows: The primer sequences for detecting HPV51 are shown in SEQ ID No.37 and SEQ ID No.38; the probe sequence for detecting HPV51 is shown in SEQ ID No.39; The primer sequences for detecting HPV82 are shown as SEQ ID No.40 and SEQ ID No.41; the probe sequence for detecting HPV82 is SEQ ID No.42; The primer sequences for detecting HPV26 are shown as SEQ ID No.43 and SEQ ID No.44; the probe sequence for detecting HPV26 is SEQ ID No.45; The primer sequences for detecting HPV53 are shown as SEQ ID No.46 and SEQ ID No.47; the probe sequence for detecting HPV53 is SEQ ID No.48; The primer sequences for detecting HPV59 are shown as SEQ ID No.49 and SEQ ID No.50; the probe sequence for detecting HPV59 is SEQ ID No.51; The primer sequences for detecting HPV73 are shown as SEQ ID No.52 and SEQ ID No.53; The probe sequence for detecting HPV73 is SEQ ID No.54; The primer sequences for detecting the human β-globin internal reference gene are SEQ ID No.55 and SEQ ID No.56; The probe sequence for detecting the human β-globin internal reference gene is SEQ ID No.

57.

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

6. The kit according to claim 5, wherein: The nucleic acid amplification reaction solution consists of 2×PCR buffer, MgCl2 with a final concentration of 2 - 4 mM, dNTPs at 0.05 - 0.5 mM, 2 - 4 U of DNA polymerase, 0.05 - 0.1 U of UNG enzyme, and RNase-free water.

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