Primer probe group, kit and detection method for multiple PCR (Polymerase Chain Reaction) detection of papilloma virus DNA (Deoxyribonucleic Acid)

Abstract

The invention relates to the technical field of PCR (Polymerase Chain Reaction) detection, and discloses a high-risk human papilloma virus DNA (Deoxyribose Nucleic Acid) multiplex PCR detection kit, which adopts a primer probe group and comprises a PCR mixed solution, a negative reference substance and a positive reference substance, wherein the PCR mixed solution comprises a primer probe group. Female first-stage urine is used as a detection sample, and compared with a cervix uteri sample, sampling is more convenient, the experience feeling is better, and the willingness of female cervical cancer screening can be provided; meanwhile, the content of cervical exfoliated cells in the urine is also increased by using the first-stage urine, and the clinical sensitivity of clinical HPV detection can be remarkably improved by matching with a provided cell enrichment method.

Description

Technical Field

[0001] The present invention relates to the technical field of PCR detection, and in particular to a primer probe set, a kit and a detection method for multiplex PCR detection of papillomavirus DNA. Background Art

[0002] It has been confirmed that persistent infection with high-risk human papillomavirus is a necessary factor for the occurrence of cervical cancer and precancerous lesions, especially HPV types 16 and 18, which are most closely related to cervical cancer. High-risk human papillomavirus detection has also been included in the cervical cancer diagnosis and treatment guidelines as one of the important means of early screening for cervical cancer. Common methods for high-risk HPV detection include fluorescent PCR, reverse dot hybridization, gene sequencing, PCR-melting curve method, gene chip method, etc.

[0003] Fluorescence PCR technology is currently the most widely used detection technology in the field of molecular diagnosis. Due to its high detection sensitivity and strong specificity, it is widely used in many fields such as pathogen detection, early tumor screening, and food testing. Its core principle is PCR amplification and fluorescence signal detection. The PCR amplification process simulates the semi-conservative replication process of DNA in the body. In the presence of biological enzymes, dNTPs, metal ions and other substances, the primers that are complementary to the target gene nucleic acid sequence are used as guides to synthesize new nucleic acid fragments. The reaction conditions include denaturation, annealing and extension. The fluorescent signal is detected by a probe that is complementary to the target sequence and labeled with a reporter group and a quencher group at the 5' and 3' ends respectively. Common probes include the most commonly used Taqman probes, molecular beacon probes, scorpion probes, MGB probes, etc. Different types of probes are used in different specific detection fields.

[0004] Most of the high-risk HPV detection products currently on the market are based on the detection principle of the fluorescent PCR method, and perform full or partial typing (generally distinguishing between types 16 and 18, and not distinguishing other types) of 14 high-risk types of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The detection process mainly includes: a) collection of clinical cervical samples (cervical exfoliated cells); b) nucleic acid extraction of cervical samples to obtain HPV virus nucleic acid; c) fluorescent PCR detection.

[0005] However, there are several problems in clinical practice: a) Cervical samples are used as the test sample type, and the collection method is "invasive", which results in a poor user experience and is the main reason why many women are unwilling to undergo HPV screening for cervical cancer; b) The detection time is too long, especially the PCR detection process, which generally takes about 1.5 to 2 hours and has low screening efficiency; c) The reagent operation is complicated, and multiple reagent components need to be manually mixed and prepared before PCR testing. Summary of the invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art and to propose a primer probe set, a kit and a detection method for multiple PCR detection of papillomavirus DNA.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A primer probe set for multiplex PCR detection of high-risk human papillomavirus DNA, comprising:

[0009] Targets HPV16, including:

[0010] Upstream primer 5′-CCACCTTCCAGCAGATGTTGACTCTACGCTTCGGTTGT-3′;

[0011] Downstream primer 5′-CCACCTTCCAGCAGATGTTGCCCATTAACAGGTCTTCC-3′;

[0012] Probe 5′-CGTACAAAGCACACACGTAGACATTCG-3′;

[0013] Targets HPV18, including:

[0014] Upstream primer 5′-CCACCTTCCAGCAGATGTTCACAACATAGCTGGGCACT-3′;

[0015] Downstream primer 5′-CCACCTTCCAGCAGATGTGTTGGAGTCGTTCCTGTCGT-3′;

[0016] Probe 5′-AGGCCAGTGCCATTCGTGCTGC-3′;

[0017] Targets HPV31, including:

[0018] Upstream primer 5′-CCACCTTCCAGCAGATGTCCTCGTACTGAAACCCAAGTG-3′;

[0019] Downstream primer 5′-CCACCTTCCAGCAGATGTGCTGTCGGGTAATTGCTCAT-3′;

[0020] Probe 5′-TGCGTGGAGAAACACCTACGTTGC-3′;

[0021] Targets HPV33, including:

[0022] Upstream primer 5′-CCACCTTCCAGCAGATGTTCAAGACACTGAGGAAAAACCA-3′;

[0023] Downstream primer 5′-CCACCTTCCAGCAGATGTTTGCATTCCACGCACTGTAG-3′; probe 5′-CGAACATTGCATGATTTGTGCCA-3′;

[0024] Targets HPV35, including:

[0025] Upstream primer 5′-CCACCTTCCAGCAGATGTAAAAACCGCTGTGTCCAGTT-3′; downstream primer 5′-CCACCTTCCAGCAGATGTGACATACACCGACCTGTCCA-3′; probe 5′-CGATTCCATAACATCGGTGGACGG-3′;

[0026] Targets HPV39, including:

[0027] Upstream primer 5′-CCACCTTCCAGCAGATGTGCCGGTTGACCTTGTATGTC-3′; downstream primer 5′-CCACCTTCCAGCAGATGTGTTCATCCCGTCTGGCTAGT-3′; probe 5′-CCCGACCATGCAGTTAATCACCA-3′;

[0028] Targets HPV45, including:

[0029] Upstream primer 5′-CCACCTTCCAGCAGATGTAGGAGGAAAACGATGAAGCA-3′; downstream primer 5′-CCACCTTCCAGCAGATGTAAGCTCAATTCTGCCGTCAC-3′; probe 5′-CGAGCCGAACCACAGCGTCA-3′;

[0030] Targets HPV51, including:

[0031] Upstream primer 5′-CCACCTTCCAGCAGATGTTATGCGTGACCAGCTACCAG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTACTTGAACACCTGCAACACG-3′; probe 5′-CGGGCTGGACAGGCTACGTG-3′;

[0032] Targets HPV52, including:

[0033] Upstream primer 5′-CCACCTTCCAGCAGATGTATTATGGGTCGTTGGACAGG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTCGTTACACTTGGGTCACAGG-3′; probe 5′-TGTTCAGAGTGTTGGAGACCCCGA-3′;

[0034] Targets HPV56, including:

[0035] Upstream primer 5′-CCACCTTCCAGCAGATGTTGAGTGTAAGTTTGTGGTGCAG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTCAGAGTGGGCACGTTACTGT-3′; probe 5′-TGGACATTCACAGTACCAAAGAGGACC-3′;

[0036] Targets HPV58, including:

[0037] Upstream primer 5′-CCACCTTCCAGCAGATGTATATTTCGGGTCGTTGGACA-3′;

[0038] Downstream primer 5′-CCACCTTCCAGCAGATGTTAGCGTTGGGTTGTTTCCTC-3′;

[0039] Probe 5′-TCCAACACACTGCACAGCGCC-3′;

[0040] Targets HPV59, including:

[0041] Upstream primer 5′-CCACCTTCCAGCAGATGTATGGCACGCTTTGAGGAT-3′;

[0042] Downstream primer 5′-CCACCTTCCAGCAGATGTTGCGAATATCATGCAGAGGA-3′;

[0043] Probe 5′-CACAACGACCATACAAACTGCCTGA-3′;

[0044] Targets HPV66, including:

[0045] Upstream primer 5′-CCACCTTCCAGCAGATGTTCAGTGTATGGGGCAACATT-3′;

[0046] Downstream primer 5′-CCACCTTCCAGCAGATGTATTGTTTTTCCTCCGGTGTT-3′;

[0047] Probe 5′-AGGTGCTACCGATGTCAATGTCCG-3′;

[0048] Targets HPV68, including:

[0049] Upstream primer 5′-CCACCTTCCAGCAGATGTCCGTGCAGGAAATTGTGTTA-3′;

[0050] Downstream primer 5′-CCACCTTCCAGCAGATGTGCATGGTCGGGTTCATCTAT-3′;

[0051] Probe 5′-TCACGAGCAATTAGGAGATTCAGACGA-3′;

[0052] Universal primer, CCACCTTCCAGCAGATGT-3′;

[0053] Internal standards, including:

[0054] Upstream primer 5′-CTCTTCCAGCCTTCCTTCCT-3′;

[0055] Downstream primer 5′-TGTTGGCGTACAGGTCTTTG-3′;

[0056] Probe 5′-CATGAAGTGTGACGTGGACATCCG-3′.

[0057] A kit for multiplex PCR detection of high-risk human papillomavirus DNA, using a primer probe set, including a PCR mixture, a negative control substance, and a positive control substance;

[0058] The PCR mixture includes a primer probe set.

[0059] Preferably, the final concentration of the HPV primer is 0.05-0.2 μM, the final concentration of the HPV probe is 0.1-0.4 μM, the final concentrations of the internal standard primer and the probe are 0.2-0.8 μM and 0.1-0.4 μM respectively, and the final concentration of the universal primer is 0.2-0.8 μM.

[0060] Preferably, the PCR mixture also includes PCR Mix, the main components of which include fast Taq enzyme, UDG enzyme, dNTPs, and Buffer.

[0061] Preferably, the negative control substance is physiological saline containing human genomic DNA, and the positive control substance is physiological saline containing human genomic DNA, HPV16 plasmid, HPV18 plasmid and HPV31 plasmid.

[0062] Preferably, the HPV16 probe is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quencher at the 3' end; the HPV18 probe is labeled with a CY5 fluorescent group at the 5' end and a BHQ3 quencher at the 3' end; the HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 probes are all labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher at the 3' end; the internal standard probes are all labeled with a VIC fluorescent group at the 5' end and a BHQ1 quencher at the 3' end.

[0063] A method for detecting high-risk human papillomavirus DNA multiplex PCR, using a kit for detecting high-risk human papillomavirus DNA multiplex PCR, comprises the following steps:

[0064] a) Centrifuge the collected first-pass urine sample to be tested, discard part of the supernatant, retain 200-500 μl of liquid and resuspend;

[0065] b) Using the nucleic acid extraction reagent to extract the urine sample and the DNA of the negative control and positive control in the kit as nucleic acid templates for later use;

[0066] c) Take the PCR mixture in the kit and dispense it into PCR reaction tubes;

[0067] d) Pipette the extracted urine sample, negative control, and positive control nucleic acid templates and add them to the PCR reaction tubes filled with PCR premix solution, mix and centrifuge;

[0068] e) performing fluorescence PCR detection on a fluorescence quantitative PCR instrument;

[0069] f) After PCR amplification, use the analysis software of the fluorescent PCR instrument to analyze the results of the test samples, negative controls, and positive controls, and use the fluorescent signal values ​​of the FAM / VIC / ROX / CY5 channels to determine the effectiveness of the experiment and the high-risk HPV infection status of the samples.

[0070] Preferably, the collected first-pass urine sample to be tested is centrifuged at 3000 rpm for 20 minutes, part of the supernatant is discarded, and 200-500 μl of liquid is retained and then resuspended.

[0071] Preferably, the reaction procedure of the fluorescent PCR detection is: 1) 98°C for 1 min; 2) 98°C for 1-3 s, 60-62°C for 5-15 s, for 5 cycles; 3) 98°C for 1-3 s, 58°C for 5-15 s, for 35 cycles.

[0072] Preferably, in c), 10 μl of the PCR mixture in the kit is dispensed into a PCR reaction tube; in d), 10 μl of the extracted nucleic acid templates of the urine sample, negative control, and positive control are respectively added into the PCR reaction tubes dispensed with the PCR premix, mixed, and centrifuged.

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] 1) The present invention uses female first-pass urine as the detection sample. Compared with cervical samples, sampling is more convenient and the experience is better, which can provide women with a willingness for cervical cancer screening; at the same time, the use of first-pass urine also increases the content of cervical exfoliated cells in urine, and combined with the provided cell enrichment method, it can significantly improve the clinical sensitivity of clinical HPV detection.

[0075] 2) The present invention uses a PCR Mix (containing a fast Taq enzyme) with better performance and higher amplification efficiency as the core raw material, which greatly shortens the detection time of PCR. At the same time, the special components in the PCR Mix can reduce the inhibitory effect of residual impurities in urine samples on PCR;

[0076] 3) Different from the primer probes used in conventional kits, the primers in the primer probe set of the present invention are specially designed primers, and a common label sequence is introduced at the 5' end of the HPV-specific primer, which can reduce the concentration of each HPV typing-specific primer so that it only needs to be used and consumed in the 5 PCR cycles of the first stage. Since the label sequence is introduced into the PCR product of the first stage, the amplification of the second stage only requires a set of universal primers with the same label sequence to complete the remaining 35 cycles of amplification detection. The advantage of this method is that it reduces the risk of primer dimers easily generated due to too many primer probes or the mutual interference effect between primers in the amplification process, thereby improving the sensitivity and specificity of the detection.

[0077] 4) Different from conventional test kits, the present invention combines essential PCR components such as primer probes, Taq enzyme, and PCR Buffer into one PCR mixed solution component, thereby simplifying the clinical operation process while ensuring detection performance and reagent stability. DETAILED DESCRIPTION

[0078] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0079] Example 1

[0080] A primer probe set for multiplex PCR detection of high-risk human papillomavirus DNA, comprising:

[0081] Targets HPV16, including:

[0082] Upstream primer 5′-CCACCTTCCAGCAGATGTTGACTCTACGCTTCGGTTGT-3′;

[0083] Downstream primer 5′-CCACCTTCCAGCAGATGTTGCCCATTAACAGGTCTTCC-3′;

[0084] Probe 5′-CGTACAAAGCACACACGTAGACATTCG-3′;

[0085] Targets HPV18, including:

[0086] Upstream primer 5′-CCACCTTCCAGCAGATGTTCACAACATAGCTGGGCACT-3′;

[0087] Downstream primer 5′-CCACCTTCCAGCAGATGTGTTGGAGTCGTTCCTGTCGT-3′;

[0088] Probe 5′-AGGCCAGTGCCATTCGTGCTGC-3′;

[0089] Targets HPV31, including:

[0090] Upstream primer 5′-CCACCTTCCAGCAGATGTCCTCGTACTGAAACCCAAGTG-3′;

[0091] Downstream primer 5′-CCACCTTCCAGCAGATGTGCTGTCGGGTAATTGCTCAT-3′;

[0092] Probe 5′-TGCGTGGAGAAACACCTACGTTGC-3′;

[0093] Targets HPV33, including:

[0094] Upstream primer 5′-CCACCTTCCAGCAGATGTTCAAGACACTGAGGAAAAACCA-3′;

[0095] Downstream primer 5′-CCACCTTCCAGCAGATGTTTGCATTCCACGCACTGTAG-3′;

[0096] Probe 5′-CGAACATTGCATGATTTGTGCCA-3′;

[0097] Targets HPV35, including:

[0098] Upstream primer 5′-CCACCTTCCAGCAGATGTAAAAACCGCTGTGTCCAGTT-3′;

[0099] Downstream primer 5′-CCACCTTCCAGCAGATGTGACATACACCGACCTGTCCA-3′;

[0100] Probe 5′-CGATTCCATAACATCGGTGGACGG-3′;

[0101] Targets HPV39, including:

[0102] Upstream primer 5′-CCACCTTCCAGCAGATGTGCCGGTTGACCTTGTATGTC-3′; downstream primer 5′-CCACCTTCCAGCAGATGTGTTCATCCCGTCTGGCTAGT-3′; probe 5′-CCCGACCATGCAGTTAATCACCA-3′;

[0103] Targets HPV45, including:

[0104] Upstream primer 5′-CCACCTTCCAGCAGATGTAGGAGGAAAACGATGAAGCA-3′; downstream primer 5′-CCACCTTCCAGCAGATGTAAGCTCAATTCTGCCGTCAC-3′; probe 5′-CGAGCCGAACCACAGCGTCA-3′;

[0105] Targets HPV51, including:

[0106] Upstream primer 5′-CCACCTTCCAGCAGATGTTATGCGTGACCAGCTACCAG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTACTTGAACACCTGCAACACG-3′; probe 5′-CGGGCTGGACAGGCTACGTG-3′;

[0107] Targets HPV52, including:

[0108] Upstream primer 5′-CCACCTTCCAGCAGATGTATTATGGGTCGTTGGACAGG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTCGTTACACTTGGGTCACAGG-3′; probe 5′-TGTTCAGAGTGTTGGAGACCCCGA-3′;

[0109] Targets HPV56, including:

[0110] Upstream primer 5′-CCACCTTCCAGCAGATGTTGAGTGTAAGTTTGTGGTGCAG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTCAGAGTGGGCACGTTACTGT-3′; probe 5′-TGGACATTCACAGTACCAAAGAGGACC-3′;

[0111] Targets HPV58, including:

[0112] Upstream primer 5′-CCACCTTCCAGCAGATGTATATTTCGGGTCGTTGGACA-3′; downstream primer 5′-CCACCTTCCAGCAGATGTTAGCGTTGGGTTGTTTCCTC-3′; probe 5′-TCCAACACACTGCACAGCGCC-3′;

[0113] Targets HPV59, including:

[0114] Upstream primer 5′-CCACCTTCCAGCAGATGTATGGCACGCTTTGAGGAT-3′;

[0115] Downstream primer 5′-CCACCTTCCAGCAGATGTTGCGAATATCATGCAGAGGA-3′;

[0116] Probe 5′-CACAACGACCATACAAACTGCCTGA-3′;

[0117] Targets HPV66, including:

[0118] Upstream primer 5′-CCACCTTCCAGCAGATGTTCAGTGTATGGGGCAACATT-3′;

[0119] Downstream primer 5′-CCACCTTCCAGCAGATGTATTGTTTTTCCTCCGGTGTT-3′;

[0120] Probe 5′-AGGTGCTACCGATGTCAATGTCCG-3′;

[0121] Targets HPV68, including:

[0122] Upstream primer 5′-CCACCTTCCAGCAGATGTCCGTGCAGGAAATTGTGTTA-3′;

[0123] Downstream primer 5′-CCACCTTCCAGCAGATGTGCATGGTCGGGTTCATCTAT-3′;

[0124] Probe 5′-TCACGAGCAATTAGGAGATTCAGACGA-3′;

[0125] Universal primer, CCACCTTCCAGCAGATGT-3′;

[0126] Internal standards, including:

[0127] Upstream primer 5′-CTCTTCCAGCCTTCCTTCCT-3′;

[0128] Downstream primer 5′-TGTTGGCGTACAGGTCTTTG-3′;

[0129] Probe 5′-CATGAAGTGTGACGTGGACATCCG-3′.

[0130] A kit for multiplex PCR detection of high-risk human papillomavirus DNA, using a primer probe set, including a PCR mixture, a negative control substance, and a positive control substance;

[0131] The PCR mixture includes a primer probe set.

[0132] In this example, the final concentration of the HPV primer is 0.05 μM, the final concentration of the HPV probe is 0.1 μM, the final concentrations of the internal standard primer and the probe are 0.2 μM and 0.1 μM, respectively, and the final concentration of the universal primer is 0.2 μM.

[0133] In this embodiment, the PCR mixture also includes PCR Mix, the main components of which include fast Taq enzyme, UDG enzyme, dNTPs, and Buffer.

[0134] In this embodiment, the negative control substance is physiological saline containing human genomic DNA, and the positive control substance is physiological saline containing human genomic DNA, HPV16 plasmid, HPV18 plasmid and HPV31 plasmid.

[0135] In this embodiment, the HPV16 probe is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quencher at the 3' end; the HPV18 probe is labeled with a CY5 fluorescent group at the 5' end and a BHQ3 quencher at the 3' end; the HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 probes are all labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher at the 3' end; the internal standard probes are all labeled with a VIC fluorescent group at the 5' end and a BHQ1 quencher at the 3' end.

[0136] Compared with the prior art, the present invention:

[0137] 1) Compared with cervical samples, first-pass urine of women is used as the test sample. Sampling is more convenient and the experience is better, which can provide women with the willingness for cervical cancer screening. At the same time, the use of first-pass urine also increases the content of cervical exfoliated cells in urine. Combined with the provided cell enrichment method, it can significantly improve the clinical sensitivity of clinical HPV detection.

[0138] 2) Using PCR Mix (including fast Taq enzyme) with better performance and higher amplification efficiency as the core raw material greatly shortens the detection time of PCR. At the same time, the special components in PCR Mix can reduce the inhibitory effect of residual impurities in urine samples on PCR;

[0139] 3) Different from the primer probes used in conventional kits, the primers in the primer probe set of the present invention are specially designed primers, and a common label sequence is introduced at the 5' end of the HPV-specific primer, which can reduce the concentration of each HPV typing-specific primer so that it only needs to be used and consumed in the 5 PCR cycles of the first stage. Since the label sequence is introduced into the PCR product of the first stage, the amplification of the second stage only requires a set of universal primers with the same label sequence to complete the remaining 35 cycles of amplification detection. The advantage of this method is that it reduces the risk of primer dimers easily generated due to too many primer probes or the mutual interference effect between primers in the amplification process, thereby improving the sensitivity and specificity of the detection.

[0140] 4) Different from conventional test kits, the present invention combines essential PCR components such as primer probes, Taq enzyme, and PCR Buffer into one PCR mixed solution component, thereby simplifying the clinical operation process while ensuring detection performance and reagent stability.

[0141] A method for detecting high-risk human papillomavirus DNA multiplex PCR, using a kit for detecting high-risk human papillomavirus DNA multiplex PCR, comprises the following steps:

[0142] a) Centrifuge the collected first-pass urine sample to be tested, discard part of the supernatant, retain 200 μl of liquid and resuspend;

[0143] b) Using the nucleic acid extraction reagent to extract the urine sample and the DNA of the negative control and positive control in the kit as nucleic acid templates for later use;

[0144] c) Take the PCR mixture in the kit and dispense it into PCR reaction tubes;

[0145] d) Pipette the extracted urine sample, negative control, and positive control nucleic acid templates and add them to the PCR reaction tubes filled with PCR premix solution, mix and centrifuge;

[0146] e) performing fluorescence PCR detection on a fluorescence quantitative PCR instrument;

[0147] f) After PCR amplification, use the analysis software of the fluorescent PCR instrument to analyze the results of the test samples, negative controls, and positive controls, and use the fluorescent signal values ​​of the FAM / VIC / ROX / CY5 channels to determine the effectiveness of the experiment and the high-risk HPV infection status of the samples.

[0148] In this embodiment, the collected first-pass urine sample to be tested was centrifuged at 3000 rpm for 20 minutes, part of the supernatant was discarded, and 200 μl of liquid was retained and then resuspended.

[0149] In this embodiment, the reaction procedure of the fluorescent PCR detection is: 1) 98°C for 1 min; 2) 98°C for 1-3 s, 60-62°C for 5-15 s, 5 cycles; 3) 98°C for 1-3 s, 58°C for 5-15 s, 35 cycles.

[0150] In this embodiment, in c), 10 μl of the PCR mixture in the kit is dispensed into a PCR reaction tube; in d), 10 μl of the extracted nucleic acid templates of the urine sample, negative control, and positive control are respectively added to the PCR reaction tubes dispensed with the PCR premix, mixed, and centrifuged.

[0151] Example 2

[0152] The difference between this embodiment and embodiment 1 is that:

[0153] In this embodiment, a kit for multiplex PCR detection of high-risk human papillomavirus DNA uses a primer probe set, including a PCR mixture, a negative control substance, and a positive control substance;

[0154] The PCR mixture includes a primer probe set.

[0155] In this example, the final concentration of the HPV primer is 0.2 μM, the final concentration of the HPV probe is 0.4 μM, the final concentrations of the internal standard primer and the probe are 0.8 μM and 0.4 μM, respectively, and the final concentration of the universal primer is 0.8 μM.

[0156] In this embodiment, the PCR mixture also includes PCR Mix, the main components of which include fast Taq enzyme, UDG enzyme, dNTPs, and Buffer.

[0157] In this embodiment, the negative control substance is physiological saline containing human genomic DNA, and the positive control substance is physiological saline containing human genomic DNA, HPV16 plasmid, HPV18 plasmid and HPV31 plasmid.

[0158] In this embodiment, the HPV16 probe is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quencher at the 3' end; the HPV18 probe is labeled with a CY5 fluorescent group at the 5' end and a BHQ3 quencher at the 3' end; the HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 probes are all labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher at the 3' end; the internal standard probes are all labeled with a VIC fluorescent group at the 5' end and a BHQ1 quencher at the 3' end.

[0159] A method for detecting high-risk human papillomavirus DNA multiplex PCR, using a kit for detecting high-risk human papillomavirus DNA multiplex PCR, comprises the following steps:

[0160] a) Centrifuge the collected first-pass urine sample to be tested, discard part of the supernatant, retain 500 μl of liquid and resuspend;

[0161] b) Using the nucleic acid extraction reagent to extract the urine sample and the DNA of the negative control and positive control in the kit as nucleic acid templates for later use;

[0162] c) Take the PCR mixture in the kit and dispense it into PCR reaction tubes;

[0163] d) Pipette the extracted urine sample, negative control, and positive control nucleic acid templates and add them to the PCR reaction tubes filled with PCR premix solution, mix and centrifuge;

[0164] e) performing fluorescence PCR detection on a fluorescence quantitative PCR instrument;

[0165] f) After PCR amplification, use the analysis software of the fluorescent PCR instrument to analyze the results of the test samples, negative controls, and positive controls, and use the fluorescent signal values ​​of the FAM / VIC / ROX / CY5 channels to determine the effectiveness of the experiment and the high-risk HPV infection status of the samples.

[0166] In this embodiment, the collected first-pass urine sample to be tested is centrifuged at 3000 rpm for 20 minutes, part of the supernatant is discarded, and 200-500 μl of liquid is retained and then resuspended.

[0167] In this embodiment, the reaction procedure of the fluorescent PCR detection is: 1) 98°C for 1 min; 2) 98°C for 3 s, 62°C for 15 s, for 5 cycles; 3) 98°C for 3 s, 58°C for 15 s, for 35 cycles.

[0168] In this embodiment, in c), 10 μl of the PCR mixture in the kit is dispensed into a PCR reaction tube; in d), 10 μl of the extracted nucleic acid templates of the urine sample, negative control, and positive control are respectively added to the PCR reaction tubes dispensed with the PCR premix, mixed, and centrifuged.

[0169] Example 2

[0170] The difference between this embodiment and embodiment 1 is that:

[0171] In this embodiment, a kit for multiplex PCR detection of high-risk human papillomavirus DNA uses a primer probe set, including a PCR mixture, a negative control substance, and a positive control substance;

[0172] The PCR mixture includes a primer probe set.

[0173] In this example, the final concentration of the HPV primer is 0.2 μM, the final concentration of the HPV probe is 0.4 μM, the final concentrations of the internal standard primer and the probe are 0.8 μM and 0.4 μM, respectively, and the final concentration of the universal primer is 0.8 μM.

[0174] In this embodiment, the PCR mixture also includes PCR Mix, the main components of which include fast Taq enzyme, UDG enzyme, dNTPs, and Buffer.

[0175] In this embodiment, the negative control substance is physiological saline containing human genomic DNA, and the positive control substance is physiological saline containing human genomic DNA, HPV16 plasmid, HPV18 plasmid and HPV31 plasmid.

[0176] In this embodiment, the HPV16 probe is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quencher at the 3' end; the HPV18 probe is labeled with a CY5 fluorescent group at the 5' end and a BHQ3 quencher at the 3' end; the HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 probes are all labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher at the 3' end; the internal standard probes are all labeled with a VI C fluorescent group at the 5' end and a BHQ1 quencher at the 3' end.

[0177] Example 3

[0178] The difference between this embodiment and embodiment 1 is that:

[0179] In this embodiment, a method for detecting high-risk human papillomavirus DNA by multiplex PCR is provided, using a kit for detecting high-risk human papillomavirus DNA by multiplex PCR, and comprises the following steps:

[0180] a) Centrifuge the collected first-pass urine sample to be tested, discard part of the supernatant, retain 300 μl of liquid and resuspend;

[0181] b) Using the nucleic acid extraction reagent to extract the urine sample and the DNA of the negative control and positive control in the kit as nucleic acid templates for later use;

[0182] c) Take the PCR mixture in the kit and dispense it into PCR reaction tubes;

[0183] d) Pipette the extracted urine sample, negative control, and positive control nucleic acid templates and add them to the PCR reaction tubes filled with PCR premix solution, mix and centrifuge;

[0184] e) performing fluorescence PCR detection on a fluorescence quantitative PCR instrument;

[0185] f) After PCR amplification, use the analysis software of the fluorescent PCR instrument to analyze the results of the test samples, negative controls, and positive controls, and use the fluorescent signal values ​​of the FAM / VIC / ROX / CY5 channels to determine the effectiveness of the experiment and the high-risk HPV infection status of the samples.

[0186] In this embodiment, the collected first-pass urine sample to be tested is centrifuged at 3000 rpm for 20 minutes, part of the supernatant is discarded, and 350 μl of liquid is retained and then resuspended.

[0187] In this embodiment, the reaction procedure of the fluorescent PCR detection is: 1) 98°C for 1 min; 2) 98°C for 1 s, 62°C for 5 s, 5 cycles; 3) 98°C for 2 s, 58°C for 10 s, 35 cycles.

[0188] In this embodiment, in c), 10 μl of the PCR mixture in the kit is dispensed into a PCR reaction tube; in d), 10 μl of the extracted nucleic acid templates of the urine sample, negative control, and positive control are respectively added to the PCR reaction tubes dispensed with the PCR premix, mixed, and centrifuged.

[0189] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A primer probe set for multiplex PCR detection of high-risk human papillomavirus DNA, characterized in that: include: Targets HPV16, including: Upstream primer 5′-CCACCTTCCAGCAGATGTTGACTCTACGCTTCGGTTGT-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTTGCCCATTAACAGGTCTTCC-3′; Probe 5′-CGTACAAAGCACACACGTAGACATTCG-3′; Targets HPV18, including: Upstream primer 5′-CCACCTTCCAGCAGATGTTCACAACATAGCTGGGCACT-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTGTTGGAGTCGTTCCTGTCGT-3′; Probe 5′-AGGCCAGTGCCATTCGTGCTGC-3′; Targets HPV31, including: Upstream primer 5′-CCACCTTCCAGCAGATGTCCTCGTACTGAAACCCAAGTG-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTGCTGTCGGGTAATTGCTCAT-3′; Probe 5′-TGCGTGGAGAAACACCTACGTTGC-3′; Targets HPV33, including: Upstream primer 5′-CCACCTTCCAGCAGATGTTCAAGACACTGAGGAAAAACCA-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTTTGCATTCCACGCACTGTAG-3′; Probe 5′-CGAACATTGCATGATTTGTGCCA-3′; Targets HPV35, including: Upstream primer 5′-CCACCTTCCAGCAGATGTAAAAACCGCTGTGTCCAGTT-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTGACATACACCGACCTGTCCA-3′; Probe 5′-CGATTCCATAACATCGGTGGACGG-3′; Targets HPV39, including: Upstream primer 5′-CCACCTTCCAGCAGATGTGCCGGTTGACCTTGTATGTC-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTGTTCATCCCGTCTGGCTAGT-3′; probe 5′-CCCGACCATGCAGTTAATCACCA-3′; Targets HPV45, including: Upstream primer 5′-CCACCTTCCAGCAGATGTAGGAGGAAAACGATGAAGCA-3′; downstream primer 5′-CCACCTTCCAGCAGATGTAAGCTCAATTCTGCCGTCAC-3′; probe 5′-CGAGCCGAACCACAGCGTCA-3′; Targets HPV51, including: Upstream primer 5′-CCACCTTCCAGCAGATGTTATGCGTGACCAGCTACCAG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTACTTGAACACCTGCAACACG-3′; probe 5′-CGGGCTGGACAGGCTACGTG-3′; Targets HPV52, including: Upstream primer 5′-CCACCTTCCAGCAGATGTATTATGGGTCGTTGGACAGG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTCGTTACACTTGGGTCACAGG-3′; probe 5′-TGTTCAGAGTGTTGGAGACCCCGA-3′; Targets HPV56, including: Upstream primer 5′-CCACCTTCCAGCAGATGTTGAGTGTAAGTTTGTGGTGCAG-3′; downstream primer 5′-CCACCTTCCAGCAGATGTCAGAGTGGGCACGTTACTGT-3′; probe 5′-TGGACATTCACAGTACCAAAGAGGACC-3′; Targets HPV58, including: Upstream primer 5′-CCACCTTCCAGCAGATGTATATTTCGGGTCGTTGGACA-3′; downstream primer 5′-CCACCTTCCAGCAGATGTTAGCGTTGGGTTGTTTCCTC-3′; probe 5′-TCCAACACACTGCACAGCGCC-3′; Targets HPV59, including: Upstream primer 5′-CCACCTTCCAGCAGATGTATGGCACGCTTTGAGGAT-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTTGCGAATATCATGCAGAGGA-3′; probe 5′-CACAACGACCATACAAACTGCCTGA-3′; Targets HPV66, including: Upstream primer 5′-CCACCTTCCAGCAGATGTTCAGTGTATGGGGCAACATT-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTATTGTTTTTCCTCCGGTGTT-3′; Probe 5′-AGGTGCTACCGATGTCAATGTCCG-3′; Targets HPV68, including: Upstream primer 5′-CCACCTTCCAGCAGATGTCCGTGCAGGAAATTGTGTTA-3′; Downstream primer 5′-CCACCTTCCAGCAGATGTGCATGGTCGGGTTCATCTAT-3′; Probe 5′-TCACGAGCAATTAGGAGATTCAGACGA-3′; Universal primer, CCACCTTCCAGCAGATGT-3′; Internal standards, including: Upstream primer 5′-CTCTTCCAGCCTTCCTTCCT-3′; Downstream primer 5′-TGTTGGCGTACAGGTCTTTG-3′; Probe 5′-CATGAAGTGTGACGTGGACATCCG-3′.

2. A kit for multiplex PCR detection of high-risk human papillomavirus DNA, using the primer probe set of claim 1, characterized in that: Includes PCR mixture, negative control, and positive control; The PCR mixture includes a primer probe set.

3. A kit for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 2, characterized in that: The final concentration of the HPV primer is 0.05-0.2 μM, the final concentration of the HPV probe is 0.1-0.4 μM, the final concentrations of the internal standard primer and the probe are 0.2-0.8 μM and 0.1-0.4 μM respectively, and the final concentration of the universal primer is 0.2-0.8 μM.

4. A kit for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 3, characterized in that: The PCR mixture also includes PCR Mix, the main components of which include fast Taq enzyme, UDG enzyme, dNTPs, and Buffer.

5. A kit for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 4, characterized in that: The negative control substance is physiological saline containing human genomic DNA, and the positive control substance is physiological saline containing human genomic DNA, HPV16 plasmid, HPV18 plasmid and HPV31 plasmid.

6. A kit for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 1, characterized in that: The HPV16 probe is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quenching group at the 3' end; the HPV18 probe is labeled with a CY5 fluorescent group at the 5' end and a BHQ3 quenching group at the 3' end; the HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 probes are all labeled with a FAM fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end; the internal standard probes are all labeled with a VIC fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end.

7. A method for multiplex PCR detection of high-risk human papillomavirus DNA, using a kit for multiplex PCR detection of high-risk human papillomavirus DNA according to any one of claims 2 to 6, characterized in that: The following steps are involved: a) Centrifuge the collected first-pass urine sample to be tested, discard part of the supernatant, retain 200-500 μl of liquid and resuspend; b) Using the nucleic acid extraction reagent to extract the urine sample and the DNA of the negative control and positive control in the kit as nucleic acid templates for later use; c) Take the PCR mixture in the kit and dispense it into PCR reaction tubes; d) Pipette the extracted urine sample, negative control, and positive control nucleic acid templates and add them to the PCR reaction tubes filled with PCR premix solution, mix and centrifuge; e) performing fluorescence PCR detection on a fluorescence quantitative PCR instrument; f) After PCR amplification, use the analysis software of the fluorescent PCR instrument to analyze the results of the test samples, negative controls, and positive controls, and use the fluorescent signal values ​​of the FAM / VIC / ROX / CY5 channels to determine the effectiveness of the experiment and the high-risk HPV infection status of the samples.

8. The method for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 7, characterized in that: The collected first-pass urine sample to be tested was centrifuged at 3000 rpm for 20 minutes, part of the supernatant was discarded, and 200-500 μl of liquid was retained and resuspended.

9. The method for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 8, characterized in that: The reaction procedure of the fluorescent PCR detection is: 1) 98°C for 1 min; 2) 98°C for 1-3 s, 60-62°C for 5-15 s, 5 cycles; 3) 98°C for 1-3 s, 58°C for 5-15 s, 35 cycles.

10. The method for multiplex PCR detection of high-risk human papillomavirus DNA according to claim 9, characterized in that: In c), 10 μl of the PCR mixture in the kit is dispensed into a PCR reaction tube; in d), 10 μl of the extracted nucleic acid templates of the urine sample, negative control, and positive control are respectively added into the PCR reaction tubes dispensed with the PCR premix, mixed and centrifuged.