Reagent and kit for detecting human papilloma virus genotyping by using four-channel fluorescently-labeled melting curve method and application

Abstract

The invention relates to the technical field of gene detection, in particular to a reagent and a kit for detecting human papilloma virus genotyping by using a four-channel fluorescently-labeled melting curve method and application of the reagent and the kit. A 4X Lyo-PCR buffer freeze-drying amplification reagent is matched with a designed specific primer and probe combination; the kit can be directly prepared into freeze-dried powder by using a unique all-component freeze-drying technology, and can be directly redissolved during use, so that the sensitivity of a freeze-dried reagent is improved to 0.2 copy / uL, and meanwhile, the kit can amplify and classify 28 human papilloma viruses in a PCR reaction, so that the 28 human papilloma viruses can be subjected to typing detection at the same time, and the kit is suitable for large-scale popularization and application. The sensitivity of each type reaches 5 copies / reaction, meanwhile, an internal reference system of endogenous ribonuclease P is introduced and used for monitoring sample sampling and sample extraction quality so as to avoid false negative of a detection result, and higher detection flux, sensitivity and specificity are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, and in particular to a reagent, a freeze-dried kit and an application thereof for detecting human papillomavirus gene typing by a melting curve method using four-channel fluorescent labels. Background Art

[0002] Human papillomavirus (HPV) is a group of DNA viruses that can infect human skin and mucous membranes. Currently, there are more than 200 different types of HPV, which are divided into high-risk, intermediate-risk and low-risk types according to their carcinogenicity. Human papillomavirus nucleic acid typing detection is widely used in clinical practice and is meaningful in early screening of cervical cancer and follow-up evaluation after treatment of cervical cancer.

[0003] The distinction of HPV types is mainly based on the fragments on the L1 gene, and the difference in L1 genes between different HPV types exceeds 10%. High-risk types include HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, medium-risk types include HPV26, 53, 66, 73 and 82, and low-risk types include HPV6 and 11. There are many methods for human papillomavirus typing detection, including fluorescent PCR, flow-through hybridization, and multi-channel fluorescent labeled melting curve method. Among them, the flow-through hybridization method cannot detect high-risk and low-risk types at the same time, has poor accuracy, and the sample size for one test is limited, time-consuming, and costly, which limits its wide application in clinical practice. The fluorescent PCR method is limited by the types of fluorescent dyes, and the single-tube detection throughput is low, usually within 5-fold detection throughput. At present, the melting curve method of multi-channel fluorescence labeling is based on the probe method and the melting curve analysis method, combined with multi-color fluorescence detection. It can simultaneously detect multiple targets in a reaction, thereby realizing single-tube multi-typing. The amplified product does not need to be opened, which is time-saving. It has been widely used in HPV typing detection reagents; however, this method requires specific amplification of multiple sites in the same reaction system, has high requirements for primer specificity, is prone to non-specificity, and thus causes false positives. Therefore, primer pairing and competitive amplification will affect the amplification efficiency.

[0004] Existing reagents for detecting HPV typing using the melting curve method have problems such as low multiplex system amplification efficiency, fewer detection types, low detection sensitivity, and low single-tube typing detection throughput, which makes clinical sample processing time-consuming and labor-intensive. In addition, the main component of molecular diagnostic reagents, enzymes, need to be frozen for storage, and low-temperature conditions such as dry ice are also required during transportation, resulting in high transportation costs.

[0005] In summary, based on the multiplex PCR melting curve method for HPV typing, it is urgent to further develop 28 human papillomavirus typing detection methods and detection kits with more ideal detection effects. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the present invention provides a reagent, a lyophilized kit and an application for detecting human papillomavirus genotyping using a four-channel fluorescently labeled melting curve method, aiming to provide a detection reagent with high specificity, low cost, convenient transportation, high sensitivity and easy use.

[0007] On the one hand, the present invention provides a reagent for a four-channel fluorescently labeled melting curve method for detecting human papillomavirus genotyping kit, wherein the reagent is a 4X Yo-PCR buffer lyophilizable amplification reagent, composed of: TrisHcL 276mM, (NH4)2SO48mM, KCL 660mM, MgSO440mM, Tween-206.5% (v / v), trehalose 10% (m / v), Dextran-406% (m / v), PEG200002.5% (m / v), dATP 4mM, dCTP 4mM, dGTP 4mM, dTTP 1.6mM, dUTP 6.4mM, Taq enzyme 1.6U / μL, and UDG enzyme 0.16U / μL.

[0008] Another aspect of the present invention is to provide a full-component freeze-drying technology, including: a refrigeration stage and a drying stage; the refrigeration stage includes -45°C pre-feed shelf refrigeration and a first refrigeration stage; the drying stage includes a first drying stage at -40°C, 0.06 mbar for 120 minutes, a second drying stage at -35°C, 0.06 mbar for 120 minutes, a third drying stage at -30°C, 0.06 mbar for 120 minutes, a fourth drying stage at -25°C, 0.06 mbar for 120 minutes and a first analytical drying stage at 25°C, 0 mbar for 120 minutes.

[0009] Based on the above-mentioned reagent or full-component freeze-drying technology, the present invention also provides a freeze-dried kit, including freeze-dried powder, a primer and probe combination for human papillomavirus genotyping, a negative control and a positive control, wherein the positive control is a mixture of 28 typing plasmids and an internal reference plasmid; the negative control is an internal reference plasmid.

[0010] Furthermore, the primer and probe combination for human papillomavirus genotyping includes: a first combination for 18 medium- and high-risk human papillomavirus genotyping detections, a second combination for 10 low-risk human papillomavirus genotyping detections, and an internal reference combination;

[0011] The nucleotide sequences of the specific primers and probes of the first combination are shown in SEQ ID NOs. 1-54;

[0012] The nucleotide sequences of the specific primers and probes of the second combination are shown in SEQ ID NOs.55-84;

[0013] The nucleotide sequences of the primers and probes of the internal reference combination are shown in SEQ ID NOs.85-87;

[0014] Wherein, the probe includes an MGB probe and a locked nucleic acid probe, and the probe nucleotide sequences modified by the locked nucleic acid probe are shown in SEQ ID NO.66 and SEQ ID NO.75; the internal reference gene is an internal reference gene of endogenous ribonuclease RNase P.

[0015] Preferably, the first combination includes the following primers and probes:

[0016] A primer and probe combination for HPV-16, the nucleotide sequence of which includes the specific amplification primer sequence shown in SEQ ID NO.1-2 and the probe sequence shown in SEQ ID NO.3;

[0017] A primer and probe combination for HPV-18, the nucleotide sequence of which includes the specific amplification primer sequence shown in SEQ ID NO.4-5 and the probe sequence shown in SEQ ID NO.6;

[0018] A primer and probe combination for HPV-26, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.7-8 and the probe sequence shown in SEQ ID NO.9;

[0019] A primer and probe combination for HPV-31, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.10-11 and the probe sequence shown in SEQ ID NO.12;

[0020] A primer and probe combination for HPV-33, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.13-14 and the probe sequence shown in SEQ ID NO.15;

[0021] A primer and probe combination for HPV-35, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.16-17 and the probe sequence shown in SEQ ID NO.18;

[0022] A primer and probe combination for HPV-39, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.19-20 and the probe sequence shown in SEQ ID NO.21;

[0023] A primer and probe combination for HPV-45, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.22-23 and the probe sequence shown in SEQ ID NO.24;

[0024] A primer and probe combination for HPV-51, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.25-26 and the probe sequence shown in SEQ ID NO.27;

[0025] A primer and probe combination for HPV-52, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.28-29 and the probe sequence shown in SEQ ID NO.30;

[0026] A primer and probe combination for HPV-53, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.31-32 and the probe sequence shown in SEQ ID NO.33;

[0027] A primer and probe combination for HPV-56, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.34-35 and the probe sequence shown in SEQ ID NO.36;

[0028] A primer and probe combination for HPV-58, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.37-38 and the probe sequence shown in SEQ ID NO.39;

[0029] A primer and probe combination for HPV-59, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.40-41 and the probe sequence shown in SEQ ID NO.42;

[0030] A primer and probe combination for HPV-66, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.43-44 and the probe sequence shown in SEQ ID NO.45;

[0031] A primer and probe combination for HPV-68, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.46-47 and the probe sequence shown in SEQ ID NO.48;

[0032] A primer and probe combination for HPV-73, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.49-50 and the probe sequence shown in SEQ ID NO.51;

[0033] A primer and probe combination for HPV-82, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.52-53 and the probe sequence shown in SEQ ID NO.54;

[0034] Preferably, the second combination comprises the following primers and probes:

[0035] A primer and probe combination for HPV-6, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.55-56 and the probe sequence shown in SEQ ID NO.57;

[0036] A primer and probe combination for HPV-11, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.58-59 and the probe sequence shown in SEQ ID NO.60;

[0037] A primer and probe combination for HPV-40, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.61-62 and the probe sequence shown in SEQ ID NO.63;

[0038] A primer and probe combination for HPV-42, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.64-65 and the probe sequence shown in SEQ ID NO.66;

[0039] A primer and probe combination for HPV-43, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.67-68 and the probe sequence shown in SEQ ID NO.69;

[0040] A primer and probe combination for HPV-44, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.70-71 and the probe sequence shown in SEQ ID NO.72;

[0041] A primer and probe combination for HPV-54, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.73-74 and the probe sequence shown in SEQ ID NO.75;

[0042] A primer and probe combination for HPV-61, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.76-77 and the probe sequence shown in SEQ ID NO.78;

[0043] A primer and probe combination for HPV-81, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.79-80 and the probe sequence shown in SEQ ID NO.81;

[0044] A primer and probe combination for HPV-83, the nucleotide sequence of which includes the specific amplification primer sequences shown in SEQ ID NO.82-83 and the probe sequence shown in SEQ ID NO.84;

[0045] Furthermore, the 5' end of the MGB probe is modified with a fluorescent group, and the 3' end is modified with a quenching group. Preferably, the fluorescent group includes any one of FAM, HEX, ROX or CY5; and the quenching group includes any one of BHQ1, BHQ2 or MGB.

[0046] Further, the lyophilized powder is composed of a primer and probe combination for human papillomavirus genotyping and a 4XLyo-PCRbuffer lyophilizable amplification reagent;

[0047] Preferably, the lyophilized powder comprises a first lyophilized powder and a second lyophilized powder;

[0048] The first lyophilized powder is composed of a first combination of primers and probes for human papillomavirus genotyping and an internal reference combination;

[0049] The second lyophilized powder consists of a second combination of primers and probes for human papillomavirus genotyping and an internal reference combination.

[0050] Furthermore, the preparation process of the freeze-dried powder is:

[0051] Combine 6.25 μL of 4X Lyo-PCR buffer freeze-dried amplification reagent, 0.01-0.02 μL of upstream primer (100 mM) for human papillomavirus genotyping, 0.1-0.2 μL of downstream primer (100 mM) and 0.01-0.02 μL of probe (100 mM) to prepare a 20 μL / reaction freeze-dried reaction solution, divide the reaction solution into 0.2 ml PCR eight-tube strips, put them into a freeze dryer and freeze-dry for 16 hours to obtain a formed amplification reagent freeze-dried powder.

[0052] Furthermore, the freeze-drying kit also includes a freeze-drying reaction tube A and a freeze-drying reaction tube B, wherein the freeze-drying reaction tube A contains the first freeze-dried powder, and the freeze-drying reaction tube B contains the second freeze-dried powder.

[0053] The present invention also provides an application of the freeze-dried kit in detecting human papillomavirus genotyping using a melting curve method with four-channel fluorescence labeling.

[0054] Furthermore, a method for detecting human papillomavirus genotyping using a four-channel fluorescently labeled melting curve method comprises the following steps:

[0055] (1) Use a fully automatic nucleic acid extraction instrument to extract the prepared sample, with a sample addition volume of 200 μL and an elution volume of 100 μL;

[0056] (2) Add 25 μL of purified nucleic acid into the freeze-dried tube to reconstitute the freeze-dried powder. After reconstitution, directly place the tube into a fluorescent PCR instrument with a four-channel melting curve analysis system to run the amplification program.

[0057] (3) Interpret the amplification results based on the TM value range of each probe.

[0058] Furthermore, the amplification program includes UNG enzyme treatment, pre-denaturation treatment, PCR cycle program and melting analysis program in sequence;

[0059] The UNG enzyme treatment is: 37°C, 5 min; the pre-denaturation treatment is 95°C, 5 min; the PCR cycle program is: 95°C, 15 s, 57°C, 30 s, 78°C, 20 s, for 55 cycles; the melting analysis program is: 95°C, 2 min; 40°C, 2 min; heating to 40-80°C at a rate of 0.04°C / s.

[0060] The advantages of the present invention are:

[0061] 1. The present invention provides a four-channel fluorescently labeled melting curve method for detecting human papillomavirus genotyping kit special reagent 4X Lyo-PCR buffer, which is suitable for multiple system amplification and can complete the amplification and typing of 28 types of human papillomavirus in one PCR reaction, thereby improving the detection throughput and detection sensitivity and reducing the detection cost;

[0062] 2. The present invention uses a multiplex system amplification reagent 4X Lyo-PCR buffer with a designed specific primer and probe combination, and uses a full-component freeze-drying technology to directly prepare a freeze-dried powder. When using, the purified nucleic acid sample only needs to be directly added to the dry powder for re-dissolution, which reduces the difficulty of experimental operation.

[0063] 3. Compared with the prior art, the kit provided by the present invention can use sample nucleic acid to directly reconstitute, so that the sensitivity of the freeze-dried reagent is increased to 0.2copy / uL. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The kit is created by the present invention;

[0065] Figure 2This is a graph showing the test results of 18 types of HPV positive samples in the freeze-dried reaction tube A created by the present invention;

[0066] Figure 3 This is a graph showing the test results of 10 types of HPV-positive samples using the freeze-dried reaction tube B created by the present invention. DETAILED DESCRIPTION

[0067] The following will be combined with the drawings in the embodiments of the invention to clearly and completely describe the technical solutions in the embodiments of the invention. Obviously, the described embodiments are only part of the embodiments of the invention, not all of them. Based on the embodiments in the invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the invention.

[0068] It should be noted that the various installation methods and technical terms mentioned in the invention are technical terms that have long been clearly known in the relevant technical field, so no further explanation is given. In addition, the same reference numerals are used for the same components, but this does not affect and should not constitute an accurate understanding of the technical solution by those skilled in the art.

[0069] Example 1 Design of primers and probes

[0070] The whole genome sequences of 28 types of HPV were searched in the NCBI database for comparison analysis. Through sequence comparison, the L1 region was confirmed as the target gene, and primers and probes for amplification of each type were designed. At the same time, the full-length sequence of the internal reference gene endogenous ribonuclease RNase P was searched to design amplification primers and probes.

[0071] Finally, unique amplification primer probes for 28 types of human papillomavirus were obtained, and asymmetric PCR and multiplex PCR melting curve technology were used to achieve simultaneous detection of 28 types of HPV typing, including a first combination and a second combination; the nucleotide sequences of the specific primers and probes of the first combination are shown in SEQ ID NO.1-54, see Table 1 for details; the nucleotide sequences of the specific primers and probes of the second combination are shown in SEQ ID NO.55-84, see Table 2 for details; the probes include MGB probes and locked nucleic acid probes, and the nucleotide sequences of the probes modified by the locked nucleic acid probes are shown in SEQ ID NO.66 and SEQ ID NO.75;

[0072] It also includes an internal reference combination, wherein the internal reference gene is an internal reference gene of endogenous ribonuclease RNase P; the nucleotide sequences of the primers and probes of the internal reference combination are shown in SEQ ID NOs.85-87.

[0073] The first combination and the internal reference combination are used for genotyping detection of 18 medium- and high-risk human papillomaviruses; the second combination and the internal reference combination are used for genotyping detection of 10 low-risk human papillomaviruses;

[0074] The 5' end of the MGB probe is modified with a fluorescent group, and the 3' end is modified with a quenching group;

[0075] The fluorescent group includes any one of FAM, HEX, ROX or CY5;

[0076] The quenching group includes any one of BHQ1, BHQ2 or MGB.

[0077] Table 1 First combination nucleotide sequence information table

[0078]

[0079]

[0080]

[0081] Table 2 Nucleotide sequence information of the second combination and the internal reference combination

[0082]

[0083]

[0084] Example 2 Preparation of lyophilized kit

[0085] Unless otherwise specified, all reagents and materials used in the present invention are purchased from the market.

[0086] 1. Preparation of 4X Lyo-PCR buffer freeze-dried amplification reagent

[0087] TrisHcL, (NH4)2SO4, KCL, MgSO4, Tween 20, trehalose, Dextran-40, PEG20000, dATP, dCTP, dGTP, dTTP, dUTP, Taq enzyme, and UDG enzyme were designed according to fixed concentrations and amounts to obtain a 4X Lyo-PCR buffer lyophilizable amplification reagent, as shown in Table 3 for details.

[0088] 2. Primer and probe combinations

[0089] By combining the first combination for detecting 18 medium- and high-risk human papillomavirus genotyping and the internal reference combination, a primer and probe combination corresponding to the first human papillomavirus genotyping is obtained;

[0090] By combining the second combination for detecting 10 low-risk human papillomavirus genotypings with the internal reference combination, a primer and probe combination corresponding to the second human papillomavirus genotyping is obtained;

[0091] 3. Preparation of freeze-dried powder

[0092] The first human papillomavirus genotyping primer and probe combination and the second human papillomavirus genotyping primer and probe combination of step 2 are mixed with the designed 4X Lyo-PCR buffer lyophilizable amplification reagent, TaqDNA polymerase, UDG enzyme, dUTP and dNTP to prepare 20 μl / reaction of the first human papillomavirus nucleic acid reaction solution and the second human papillomavirus nucleic acid reaction solution, respectively.

[0093] The reaction solution was divided into freeze-dried reaction tube A and freeze-dried reaction tube B respectively. A vacuum freeze-drying device was used to freeze-dry the amplification reagent for 16 hours according to the freeze-drying program shown in Table 4. The amplification reagent was freeze-dried into a dry powder state to obtain a first freeze-dried powder and a second freeze-dried powder of the formed amplification reagent. The reagents in the freeze-dried powder state can be stored for a long time at 4-8°C or even at room temperature.

[0094] As shown in Table 4, the freeze-drying procedure includes: a refrigeration stage and a drying stage; the refrigeration stage includes -45°C pre-feed shelf refrigeration and a refrigeration first stage; the drying stage includes a first drying stage at -40°C, 0.06 mbar for 120 min, a second drying stage at -35°C, 0.06 mbar for 120 min, a third drying stage at -30°C, 0.06 mbar for 120 min, a fourth drying stage at -25°C, 0.06 mbar for 120 min, and a first analytical drying stage at 25°C, 0 mbar for 120 min.

[0095] 4. Preparation of lyophilized kit

[0096] After the freeze-dried powder and the second freeze-dried powder are taken out of the box, the room needs to be dehumidified before being taken out of the box to control the humidity below 30%, the tube cover of the reaction tube is covered, and the freeze-dried reaction tube A and the freeze-dried reaction tube B are sealed together in blister packaging to prevent moisture, and the corresponding freeze-dried test kit is obtained.

[0097] Table 3 4X Lyo-PCR buffer freeze-dried amplification reagent components and concentration information

[0098]

[0099]

[0100] Table 4 Lyophilization procedure for 4X Lyo-PCR buffer lyophilizable amplification reagent

[0101]

[0102] Example 3 Detection

[0103] The fluorescent PCR instrument with four-channel melting curve analysis in this example is Hongshi 96S

[0104] The test contains two freeze-dried reaction tubes. Freeze-dried reaction tube A can quickly type and qualitatively detect 18 medium- and high-risk human papillomaviruses in a single reaction tube, and freeze-dried reaction tube B can quickly type and qualitatively detect 10 low-risk human papillomaviruses in a single reaction tube.

[0105] A method for detecting human papillomavirus nucleic acid typing using a freeze-dried kit for human papillomavirus genotyping comprises the following steps:

[0106] (1) Sampling: Use a sterile swab to collect a sample from the cervix or vagina (the sample is known to be positive).

[0107] (2) Nucleic acid extraction: 200 μL of the collected sample was added to the lysis buffer and extracted using a fully automatic nucleic acid extraction instrument with an elution volume of 100 μL.

[0108] (3) Sample addition and amplification: Add 25 μL of purified nucleic acid into the freeze-dried tube, reconstitute the freeze-dried powder, mix thoroughly, and directly place the reconstituted sample into a fluorescent PCR instrument with four-channel melting curve analysis to run the amplification program.

[0109] (4) Result analysis: According to the designed amplification program and the probe TM value range, the amplification results are interpreted. If the sample internal reference is amplified normally, the result is valid; otherwise, the result is invalid.

[0110] As shown in Table 5, the designed amplification program includes UNG enzyme treatment, pre-denaturation treatment, PCR cycle program and melting analysis program in sequence. The UNG enzyme treatment is: 37°C, 5 min; the pre-denaturation treatment is 95°C, 5 min; the PCR cycle program is: 95°C, 15 s, 57°C, 30 s, 78°C, 20 s, for 55 cycles; the melting analysis program is: 95°C, 2 min; 40°C, 2 min; heating to 40-80°C at a rate of 0.04°C / s.

[0111] The probe TM value ranges of freeze-dried reaction tube A and freeze-dried reaction tube B in each fluorescence channel are shown in Table 6.

[0112] According to the TM value range of each probe designed in Table 6, the amplification results are interpreted. If there is a melting peak within the Tm value range, it means that the sample internal reference is amplified normally and the result is valid, otherwise the result is invalid.

[0113] like Figure 2 Lyophilize reaction tube A and Figure 3 The positive sample test results of freeze-dried reaction tube B show that each subtype of human papillomavirus positive will produce a melting peak in the corresponding temperature range in the corresponding fluorescence channel. In each fluorescence channel of freeze-dried reaction tube A and freeze-dried reaction tube B, 3 to 5 different human papillomavirus subtypes are evenly distributed in the temperature range of 40-80°C:

[0114] The human papillomavirus subtypes distributed in the FAM fluorescence channel of freeze-dried reaction tube A are HPV-52, HPV-35, HPV-68, HPV-18, and HPV-31; the human papillomavirus subtypes distributed in the HEX fluorescence channel are HPV-66, HPV-82, and HPV-73; the human papillomavirus subtypes distributed in the ROX fluorescence channel are HPV-39, HPV-56, HPV-53, HPV-16, and HPV-26; and the human papillomavirus subtypes distributed in the CY5 fluorescence channel are HPV-58, HPV-59, HPV-33, HPV-45, and HPV-51.

[0115] The human papillomavirus subtypes in the FAM fluorescence channel of freeze-dried reaction tube B are HPV-11, HPV-40, and HPV-81, the human papillomavirus subtypes distributed in the HEX fluorescence channel are internal reference genes, the human papillomavirus subtypes distributed in the ROX fluorescence channel are HPV-83, HPV-06, HPV-44, and HPV-43, and the human papillomavirus subtypes distributed in the CY5 fluorescence channel are HPV-42, HPV-52, and HPV-61.

[0116] The above test results indicate that the 28 types of human papillomavirus in the kit of the present invention are amplified and typed in one PCR reaction, and the 28 types of human papillomavirus can be typed and detected simultaneously, with a sensitivity of 5 copies / reaction for each type. At the same time, an internal reference system of endogenous ribonuclease P is introduced to monitor the quality of sample sampling and sample extraction to avoid false negative test results, thereby having higher sensitivity and specificity.

[0117] Table 5 Amplification procedures

[0118]

[0119] Table 6 Types and Tm ranges of 28 HPV genotypes in four channels

[0120]

[0121] For those skilled in the art, the invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the invention is defined by the appended claims rather than the above description, so it is intended to include all changes within the meaning and scope of the equivalent elements of the claims. Any reference numerals in the claims should not be regarded as limiting the claims involved.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any minor modifications, equivalent substitutions and improvements made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the technical solution of the present invention.

Claims

1. A reagent for a four-channel fluorescently labeled melting curve method for detecting human papillomavirus genotyping kit, characterized in that: The reagent is a 4XLyo-PCR buffer lyophilizable amplification reagent, which is composed of: TrisHCl 276mM, (NH4)2SO48mM, KCl 660mM, MgSO440mM, Tween-206.5% (v / v), trehalose 10% (m / v), Dextran-406% (m / v), PEG200002.5% (m / v), dATP 4mM, dCTP 4mM, dGTP 4mM, dTTP 1.6mM, dUTP 6.4mM, Taq enzyme 1.6U / μL, and UDG enzyme 0.16U / μL.

2. A four-channel fluorescently labeled melting curve method for detecting human papillomavirus genotyping kit, characterized in that: The kit uses a full-component freeze-drying technology to process the amplification reagent and primer probe composition of claim 1, wherein the full-component freeze-drying technology includes: a refrigeration stage and a drying stage; the refrigeration stage includes a pre-feed shelf refrigeration at -45°C and a refrigeration first stage; the drying stage includes a first drying stage at -40°C, 0.06mbar for 120min, a second drying stage at -35°C, 0.06mbar for 120min, a third drying stage at -30°C, 0.06mbar for 120min, a fourth drying stage at -25°C, 0.06mbar for 120min, and a first analytical drying stage at 25°C, 0mbar for 120min.

3. The kit according to claim 2, characterized in that The kit comprises lyophilized powder, a primer and probe combination for human papillomavirus genotyping, a negative control and a positive control; wherein the positive control is a mixed solution of 28 typing plasmids and an internal reference plasmid; and the negative control is an internal reference plasmid.

4. The kit according to claim 3, characterized in that The primer and probe combination for human papillomavirus genotyping includes: a first combination for 18 medium- and high-risk human papillomavirus genotyping detections, a second combination for 10 low-risk human papillomavirus genotyping detections, and an internal reference combination; The nucleotide sequences of the specific primers and probes of the first combination are shown in SEQ ID NOs. 1-54; The nucleotide sequences of the specific primers and probes of the second combination are shown in SEQ ID NOs.55-84; The nucleotide sequences of the primers and probes of the internal reference combination are shown in SEQ ID NOs.85-87; Wherein, the probe includes an MGB probe and a locked nucleic acid probe, and the probe nucleotide sequence modified by the locked nucleic acid probe is shown in SEQ ID NO.63 and SEQ ID NO.75; the internal reference gene is an internal reference gene of endogenous ribonuclease RNase P.

5. The kit according to claim 4, characterized in that The 5' end of the MGB probe is modified with a fluorescent group, and the 3' end is modified with a quenching group; Wherein, the fluorescent group includes any one of FAM, HEX, ROX or CY5; the quenching group includes any one of BHQ1, BHQ2 or MGB.

6. The kit according to claim 3, wherein the lyophilized powder consists of a primer and probe combination for human papillomavirus genotyping and a 4X Lyo-PCR buffer lyophilizable amplification reagent; in, The lyophilized powder includes a first lyophilized powder and a second lyophilized powder; the first lyophilized powder includes a first combination of primers and probes for human papillomavirus genotyping and an internal reference combination; the second lyophilized powder includes a second combination of primers and probes for human papillomavirus genotyping and an internal reference combination.

7. The kit according to claim 6, characterized in that The kit further comprises a freeze-drying reaction tube A and a freeze-drying reaction tube B, wherein the freeze-drying reaction tube A contains a first freeze-dried powder, and the freeze-drying reaction tube B contains a second freeze-dried powder.

8. Use of the kit according to any one of claims 2 to 7 in detecting human papillomavirus genotyping using a four-channel fluorescently labeled melting curve method.

9. The method for detecting human papillomavirus genotyping using a four-channel fluorescently labeled melting curve method according to claim 8, characterized in that: The following steps are involved: (1) Use a fully automatic nucleic acid extraction instrument to extract the prepared sample, with a sample addition volume of 200 μL and an elution volume of 100 μL; (2) Add 25 μL of purified nucleic acid into the freeze-dried tube to reconstitute the freeze-dried powder. After reconstitution, directly place the tube into a fluorescent PCR instrument with a four-channel melting curve analysis system to run the amplification program. (3) Interpret the amplification results based on the TM value range of each probe.

10. The detection method using a melting curve method with four-channel fluorescent labeling according to claim 9, characterized in that: The amplification program includes UNG enzyme treatment, pre-denaturation treatment, PCR cycle program and melting analysis program in sequence; The UNG enzyme treatment is: 37°C, 5 min; the pre-denaturation treatment is 95°C, 5 min; the PCR cycle program is: 95°C, 15 s, 57°C, 30 s, 78°C, 20 s, for 55 cycles; the melting analysis program is: 95°C, 2 min; 40°C, 2 min; heating to 40-80°C at a rate of 0.04°C / s.

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