High-risk human papilloma virus nucleic acid typing detection kit

Abstract

The present application relates to the field of molecular biology, in particular to a high-risk human papilloma virus nucleic acid typing detection kit. The kit uses internal standard positive quality control, detects beta-globin in human genome to determine whether the clinical sample is suitable for amplification detection, the internal standard quality control can be used to monitor whether there is PCR inhibitor in the reaction system, and ensures that there is no false negative result of human papilloma virus. The kit is used for high-efficiency typing detection of high-risk human papilloma virus, specific primers and probes are designed, and the system is optimized in multiple ways, so that the detection sensitivity and specificity are ensured.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a nucleic acid typing detection kit for high-risk human papillomavirus. Background Technology

[0002] Cervical cancer is one of the most common gynecological malignancies, ranking second in incidence among female malignant tumors in my country, after breast cancer. In 2018, there were over 569,000 new cases of cervical cancer globally, with over 311,000 deaths. In 2015, my country had approximately 111,000 new cases and 34,000 deaths. It is now clearly established that persistent infection with high-risk human papillomavirus (HPV) is a necessary factor for the development of cervical cancer and precancerous lesions; that is, HPV infection is the most critical link in the process of cervical cancer development.

[0003] Currently, more than 200 HPV subtypes have been discovered and identified, with approximately 54 types capable of infecting the genital tract mucosa. According to relevant national guidelines, HPV is classified into two main categories—high-risk HPV and low-risk HPV—based on the likelihood of HPV causing cervical cancer and the varying carcinogenicity of HPV after infecting the human genital tract mucosa. The 14 high-risk HPV types confirmed by the WHO and IRAC to cause cervical cancer include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

[0004] HPV genotyping can predict an individual's risk of developing the disease and determine the screening interval; it can also distinguish between persistent and transient infections, enabling early treatment to reduce morbidity and mortality. Furthermore, it can be used for post-operative follow-up to determine whether lesions have been completely removed, facilitating post-treatment monitoring and guiding the research and use of HPV vaccines.

[0005] Over the years, numerous methods have been used for HPV detection, such as cytological examination, immunohistochemical detection of HPV antigens, and HPV antibody detection. However, these methods have been gradually phased out due to their inability to perform HPV genotyping and their drawbacks, including low sensitivity, poor specificity, and long processing times. Currently, HPV detection primarily employs molecular biology methods, such as nucleic acid hybridization, gene chips, and polymerase chain reaction (PCR). Among these, quantitative real-time PCR (qPCR) is the most widely used method. This method is time-efficient, highly sensitive, and can be automated, eliminating the need for manual operation, thus becoming the mainstream HPV detection method.

[0006] Currently, there are various kits in this field that use quantitative real-time PCR to detect HPV viruses. The mainstream kits are divided into two categories: one type covers high-risk HPV virus types and can perform genotyping of all HPV types within the detection range; the other type covers high-risk HPV virus types and can perform genotyping of some HPV types within the detection range.

[0007] The first type is a kit that can detect 15 types of HPV. This kit requires four tubes for testing, meaning each sample needs four wells for type identification. Therefore, in one experiment, a 96-well PCR plate, excluding positive and negative controls, can only process a maximum of 22 samples. Although this can achieve type detection, the detection efficiency is low. For some hospitals with a daily sample volume of more than 100 cases, at least five rounds of sample processing and PCR amplification are required, which seriously wastes the time of laboratory doctors. Especially in application scenarios such as large-scale cervical cancer screening, the low detection efficiency of this kit is even more prominent and obvious.

[0008] The second type includes kits that can detect 14 high-risk HPV types and perform genotyping of types 16 and 18. The advantage of this kit is that it uses single-well testing; excluding positive and negative controls, up to 94 samples can be processed in a single experiment on a commonly used 96-well microplate, resulting in high detection efficiency. However, its disadvantage is that it can only distinguish between types 16 and 18 and cannot genotype other types. Because different HPV types require different treatment regimens, this often causes confusion for clinicians. Furthermore, when patients repeatedly test positive over many years, this type of kit cannot distinguish whether the infection is persistent with the same type across the 12 types. As is well known, persistent infection with the same HPV type for many years significantly increases the probability of cervical cancer. Therefore, there is an urgent clinical need for kits that are both highly efficient and capable of genotyping.

[0009] In conclusion, further development of a kit that is both highly efficient in detection and capable of genotyping all high-risk HPV types is of significant practical importance. Summary of the Invention

[0010] In view of this, the present invention provides a high-risk human papillomavirus nucleic acid typing detection kit and detection method.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides a primer-probe combination having:

[0013] I. Nucleotide sequences as shown in SEQ ID No. 1–60; or

[0014] II. Nucleotide sequences obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequences shown in SEQ ID No. 1–60, and having the same or similar function as the nucleotide sequences shown in SEQ ID No. 1–60; or

[0015] III. A nucleotide sequence having at least 80% homology with the nucleotide sequences shown in SEQ ID No. 1 to 60, or a nucleotide sequence having the same or similar function as the nucleotide sequences shown in SEQ ID No. 1 to 60.

[0016] In some specific embodiments of the present invention, the primer-probe combination includes one or more of the following combinations:

[0017] Combination X:

[0018] I. The upstream primer has the nucleotide sequence shown in SEQ ID No. (3X-2); and

[0019] II. The downstream primer has the nucleotide sequence shown in SEQ ID No. (3X-1); and

[0020] Probe III has the nucleotide sequence shown in SEQ ID No. 3X; or

[0021] IV. Nucleotide sequences obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequences shown in I, II, or III, and which have the same or similar function as the nucleotide sequences shown in I, II, or III; or

[0022] V. A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in I, II or III;

[0023] X is selected from any integer from 1 to 20;

[0024] When X=1, human papillomavirus types 16, 31, and 35 are detected.

[0025] When X=2, human papillomavirus types 33, 52, and 58 are detected.

[0026] When X=3, human papillomavirus types 51, 56, and 66 are detected.

[0027] When X=4, human papillomavirus types 18, 45, and 68 are detected.

[0028] When X=5, human papillomavirus types 39 and 59 are detected.

[0029] When X=6, an internal reference is detected; the internal reference is human genome β-Globin.

[0030] When X=7, human papillomavirus type 16 is detected;

[0031] When X=8, human papillomavirus type 31 is detected;

[0032] When X=9, human papillomavirus type 35 is detected;

[0033] When X=10, human papillomavirus type 33 is detected;

[0034] When X=11, human papillomavirus type 52 is detected;

[0035] When X=12, human papillomavirus type 58 is detected;

[0036] When X=13, human papillomavirus type 51 is detected;

[0037] When X=14, human papillomavirus type 56 is detected;

[0038] When X=15, human papillomavirus type 66 is detected;

[0039] When X=16, human papillomavirus type 18 is detected;

[0040] When X=17, human papillomavirus type 45 is detected;

[0041] When X=18, human papillomavirus type 68 is detected;

[0042] When X=19, human papillomavirus type 39 is detected;

[0043] When X=20, human papillomavirus type 59 is detected.

[0044] The present invention also provides the application of the primer-probe combination in the preparation of reagents or kits for detecting human papillomavirus.

[0045] In some specific embodiments of the present invention, the human papillomavirus includes high-risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

[0046] The present invention also provides detection reagents, including the aforementioned primer-probe combination and acceptable auxiliaries.

[0047] In some specific embodiments of the present invention, the detection reagent includes human papillomavirus primer-probe mixture 1, human papillomavirus primer-probe mixture 2, human papillomavirus primer-probe mixture 3, and human papillomavirus primer-probe mixture 4, wherein:

[0048] The human papillomavirus primer-probe mixture 1 includes combinations 1, 2, 3, 4, 5, and 6 of the primer-probe combinations. In combination 1, the probes are labeled with a fluorescent reporter group FAM and a fluorescent quencher group BHQ1 at both ends. In combination 2, the probes are labeled with a fluorescent reporter group HEX and a quencher group MGB at both ends. In combination 3, the probes are labeled with a fluorescent reporter group ROX and a quencher group MGB at both ends. In combination 4, the probes are labeled with a fluorescent reporter group CY5 and a fluorescent quencher group BHQ2 at both ends. In combination 5, the probes are labeled with a fluorescent reporter group Quasar705 and a fluorescent quencher group BHQ3 at both ends. In combination 6, the probes are labeled with a fluorescent reporter group Atto425 and a fluorescent quencher group BHQ1 at both ends.

[0049] The human papillomavirus primer-probe mixture 2 includes combinations 7, 8, 9, 10, 11, and 6 of the primer-probe combinations. In combination 7, the probes are labeled with a fluorescent reporter group FAM and a fluorescent quencher group BHQ1 at both ends. In combination 8, the probes are labeled with a fluorescent reporter group HEX and a fluorescent quencher group BHQ1 at both ends. In combination 9, the probes are labeled with a fluorescent reporter group ROX and a fluorescent quencher group BHQ2 at both ends. In combination 10, the probes are labeled with a fluorescent reporter group CY5 and a fluorescent quencher group BHQ2 at both ends. In combination 11, the probes are labeled with a fluorescent reporter group Quasar705 and a fluorescent quencher group BHQ3 at both ends. In combination 6, the probes are labeled with a fluorescent reporter group Atto425 and a fluorescent quencher group BHQ1 at both ends.

[0050] The human papillomavirus primer-probe mixture 3 includes combinations 13, 14, 15, 16, 17, and 6 of the primer-probe combinations. In combination 13, the probes are labeled with a fluorescent reporter group FAM and a fluorescent quencher group BHQ1 at both ends. In combination 14, the probes are labeled with a fluorescent reporter group HEX and a fluorescent quencher group BHQ1 at both ends. In combination 15, the probes are labeled with a fluorescent reporter group ROX and a fluorescent quencher group BHQ2 at both ends. In combination 16, the probes are labeled with a fluorescent reporter group CY5 and a fluorescent quencher group BHQ2 at both ends. In combination 17, the probes are labeled with a fluorescent reporter group Quasar705 and a fluorescent quencher group BHQ3 at both ends. In combination 6, the probes are labeled with a fluorescent reporter group Atto425 and a fluorescent quencher group BHQ1 at both ends.

[0051] The human papillomavirus primer-probe mixture 4 includes combinations 12, 18, 19, 20, and 6 of the primer-probe combinations. In combination 12, the probes are labeled with a fluorescent reporter group FAM and a fluorescent quencher group BHQ1 at both ends. In combination 18, the probes are labeled with a fluorescent reporter group HEX and a fluorescent quencher group BHQ1 at both ends. In combination 19, the probes are labeled with a fluorescent reporter group ROX and a fluorescent quencher group BHQ2 at both ends. In combination 20, the probes are labeled with a fluorescent reporter group CY5 and a fluorescent quencher group BHQ2 at both ends. In combination 6, the probes are labeled with a fluorescent reporter group Quasar705 and a fluorescent quencher group BHQ3 at both ends.

[0052] The probes include a Taqman probe and an MGB probe. The two ends of the Taqman probe are labeled with a fluorescent reporter group and a fluorescent quencher group, respectively, and the two ends of the MGB probe are labeled with a fluorescent reporter group and a quencher group MGB, respectively.

[0053] The present invention also provides the application of the aforementioned detection reagent in the preparation of a kit for detecting human papillomavirus.

[0054] In some specific embodiments of the present invention, the human papillomavirus includes high-risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

[0055] The present invention also provides a kit comprising the primer-probe combination or the detection reagents described herein.

[0056] This invention provides a high-risk human papillomavirus (HPV) nucleic acid typing detection kit, which contains at least seven components: HPV PCR reaction solution A, HPV primer-probe mixture 1, HPV primer-probe mixture 2, HPV primer-probe mixture 3, HPV primer-probe mixture 4, HPV positive control, and HPV negative control. Wherein:

[0057] The human papillomavirus PCR reaction solution A is a commercially available multiplex PCR amplification MIX;

[0058] The human papillomavirus (HPV) positive control includes HPV type 16 clone plasmid, HPV type 31 clone plasmid, HPV type 35 clone plasmid, HPV type 33 clone plasmid, HPV type 52 clone plasmid, HPV type 58 clone plasmid, HPV type 51 clone plasmid, HPV type 56 clone plasmid, HPV type 66 clone plasmid, HPV type 18 clone plasmid, HPV type 45 clone plasmid, HPV type 68 clone plasmid, HPV type 39 clone plasmid, HPV type 59 clone plasmid, and human genome β-Globin clone plasmid. The HPV positive control is composed of a mixture of these 15 plasmids and can be specifically amplified using the primer and probe combination.

[0059] In some specific embodiments of the present invention, the concentration of each plasmid is 1.00-5.00E+05 copies / mL.

[0060] This invention also provides a method for detecting human papillomavirus, wherein the sample to be tested is subjected to any of the following tests:

[0061] (I) The primer-probe combination described above; or

[0062] (II) The aforementioned detection reagent; or

[0063] (III) The kit described above.

[0064] In some specific embodiments of the present invention, the detection method includes:

[0065] When dealing with testing institutions with large clinical sample sizes or large-scale cervical cancer screening, the following testing methods can be used:

[0066] Step 1: Take out the detection reagent from the kit and mix the human papillomavirus PCR reaction solution A and human papillomavirus primer probe mixture 1 as follows: the amount of human papillomavirus PCR reaction solution A added is 12.5 μL per person, and the amount of human papillomavirus primer probe mixture 1 added is 7.5 μL per person. The final concentration of each primer is 0.1-0.5 μmol / L, and the final concentration of each probe is 0.05-0.25 μmol / L. Shake to mix well, and dispense 20 μL into each PCR reaction tube.

[0067] The second step is to extract nucleic acids from clinical samples using the magnetic bead method;

[0068] The third step involves adding 5 μL each of the negative control, positive control, and extracted clinical sample nucleic acid to separate PCR reaction tubes containing the reaction solution, mixing, and centrifuging.

[0069] The fourth step is to perform amplification and detection on a 6-channel real-time PCR instrument. The reaction program is as follows: (1) 25℃, 5-10 min; (2) 95℃, 1-5 min; (3) 95℃, 5-15 sec; 60℃, 20-30 sec, for 40-45 cycles.

[0070] The fifth step is to determine the positive or negative result of the target based on the amplification curve and Ct value after the PCR amplification test. When the amplification curve is a standard S-shape and the target Ct value is less than or equal to 39, and the β-Globin amplification curve is a standard S-shape and the Ct value is less than or equal to 40, the target is considered positive. The next step is determined based on the test results. If the sample test result is negative, no further steps are required, and the result is reported as negative. If the test result is positive for a certain channel, a second test is performed. Specifically, if the test result is positive for the FAM channel, it indicates that the sample is positive for one or more of human papillomavirus (HPV) types 16, 31, and 35. In this case, repeat the steps one through four above with HPV PCR reaction solution A and HPV primer-probe mixture 2 to confirm the positive type of the sample. If the test result is positive for the HEX channel, it indicates that the sample is positive for one or more of HPV types 33, 52, and 58. In this case, mix HPV PCR reaction solution A with HPV primer-probe mixture 2 and HPV primer-probe mixture 4 respectively for a second test. If the test result is positive for the ROX channel, it indicates that the sample is positive for HPV types 51, 52, and 35. If the sample is positive for one or more of the following types: 6, 66, then the human papillomavirus (HPV) PCR reaction solution A and HPV primer-probe mixture 3 should be mixed for a second test. If the test result is positive for the CY5 channel, it indicates that the sample is positive for one or more of the following types: HPV 18, 45, 68. In this case, the HPV PCR reaction solution A should be mixed with HPV primer-probe mixture 3 and HPV primer-probe mixture 4 respectively for a second test. If the test result is positive for the Quasar 705 channel, it indicates that the sample is positive for one or more of the following types: HPV 39, 59. In this case, the HPV PCR reaction solution A should be mixed with HPV primer-probe mixture 4 for a second test.

[0071] For testing institutions or hospitals with a small clinical sample size, where the daily sample size does not exceed 31 cases, the following testing methods can be used:

[0072] Step 1: Take out the detection reagents from the kit and mix the human papillomavirus PCR reaction solution A with human papillomavirus primer and probe mixture 2, human papillomavirus primer and probe mixture 3, and human papillomavirus primer and probe mixture 4 respectively to prepare three reagents ①, ②, and ③. In reagent ①, the amount of human papillomavirus PCR reaction solution A added for each reaction is 12.5 μL per person, and the amount of human papillomavirus primer and probe mixture 2 added is 7.5 μL per person. In reagent ②, the amount of human papillomavirus PCR reaction solution A added for each reaction is... The dosage is 12.5 μL per person, the dosage of human papillomavirus primer and probe mixture 3 is 7.5 μL per person, the dosage of human papillomavirus PCR reaction solution A for each reaction in reagent ③ is 12.5 μL per person, and the dosage of human papillomavirus primer and probe mixture 4 is 7.5 μL per person. The final concentration of each primer is 0.1-0.5 μmol / L, and the final concentration of each probe is 0.05-0.25 μmol / L. After the three reagents are prepared, they are shaken to mix well, and 20 μL of each reaction is dispensed into PCR reaction tubes.

[0073] The second step is to extract nucleic acids from clinical samples using the magnetic bead method;

[0074] The third step involves adding 5 μL each of the negative control, positive control, and extracted clinical sample nucleic acid to separate PCR reaction tubes containing the reaction solution, mixing, and centrifuging.

[0075] The fourth step is to perform amplification and detection on a 6-channel real-time PCR instrument. The reaction program is as follows: (1) 25℃, 5-10 min; (2) 95℃, 1-5 min; (3) 95℃, 5-15 sec; 60℃, 20-30 sec, for 40-45 cycles.

[0076] The fifth step is to determine the positive or negative status of the target based on the amplification curve and Ct value after the PCR amplification test. When the amplification curve is a standard S-shape and the Ct value of the target is less than or equal to 39, and the β-Globin amplification curve is a standard S-shape and the Ct value is less than or equal to 40, the target is considered positive. The type of human papillomavirus is then determined based on the test reagents used and the positive channel.

[0077] This kit uses an internal standard positive control, which determines whether a clinical sample is suitable for amplification detection by detecting β-Globin in the human genome. This internal standard control can be used to monitor the presence of PCR inhibitors in the reaction system to ensure that there are no false negative results for human papillomavirus.

[0078] This kit is designed for efficient typing and detection of high-risk human papillomavirus types. By designing specific primers and probes and optimizing the system in multiple ways, the kit ensures high detection sensitivity and specificity.

[0079] This kit is used for high-efficiency typing and detection of high-risk human papillomavirus (HPV) types. It has two main applications: First, for testing institutions with large clinical sample volumes or large-scale cervical cancer screening, a single-well amplification test is performed for initial screening. On a standard PCR instrument, up to 94 clinical samples can be tested in one experiment. The other two wells are used for positive and negative controls. Since most people are HPV negative, these samples do not require secondary testing and are reported as negative. However, for positive samples, secondary testing is required to determine the specific type.

[0080] The second application scenario is when the hospital's daily sample volume does not exceed 31 cases. In order to save medical staff's time, a two-stage testing strategy can be used directly. Human papillomavirus PCR reaction solution A is mixed with human papillomavirus primer and probe mixture 2, human papillomavirus primer and probe mixture 3, and human papillomavirus primer and probe mixture 4 in proportion, and then the samples are added for amplification. High-risk human papillomavirus typing can be performed directly, and high-risk typing can be completed in one experiment.

[0081] The beneficial effects of this invention are:

[0082] Firstly, for testing institutions with large sample volumes or large-scale cervical cancer screening, this kit can perform initial screening of clinical samples with a single well. Since most people in the normal population are HPV negative, samples with negative results are directly reported as negative. When a positive result is found, a second experiment is performed for genotyping. This method can filter out most negative samples in the first initial screening, allowing a typical 96-well PCR experiment to detect up to 94 positive samples. Other manufacturers typically require 4 or 8 reaction wells per sample, and after deducting positive and negative controls, a single experiment can only detect a maximum of 24 or 10 samples. Therefore, this kit significantly increases throughput and saves testing personnel time. This kit not only meets the clinical need for genotyping of high-risk HPV types but also greatly improves testing efficiency and saves medical staff time.

[0083] Secondly, the human papillomavirus primer-probe mixture 1 in this kit contains 6 pairs of primers and probes, each labeled with a different fluorescent emitting group, corresponding to different channels of the real-time PCR instrument. Therefore, different channels correspond to different detection types. Thus, the primers and probes in the human papillomavirus primer-probe mixture 1 of this patent application cover 2 or 3 types. In human papillomavirus primer-probe mixture 2, human papillomavirus primer-probe mixture 3, and human papillomavirus primer-probe mixture 4, each primer probe specifically detects only that type. All the above primer probes have been carefully compared and designed, and have been screened from multiple pairs of primer probes through a large number of clinical samples, ensuring both detection sensitivity and high specificity.

[0084] Third, this kit can screen out most negative samples during the initial screening, and the few positive samples undergo a second test. However, this second test is not a simple re-screening from all 14 high-level types. Instead, after the first test, it records the channel that shows a positive signal. This channel contains a maximum of 3 types, meaning the positive type is one of these 3 types. Therefore, in the second test, a targeted primer-probe mixture is used for detection. One or two wells are sufficient to determine the infection type. Thus, if a clinical sample is negative, one well is enough to complete the test; if it is positive, two or three wells are sufficient for positive genotyping. Other manufacturers generally require four or more wells for genotyping. Therefore, this kit achieves the purpose of HPV genotyping while saving reagents, making it highly economical.

[0085] Fourth, this kit is suitable not only for testing institutions with large sample sizes or during large-scale cervical cancer screening, but also for testing institutions with smaller sample sizes. If the sample size in a single test does not exceed 31 cases, this kit can use a 3-well testing scheme to directly perform genotyping on all samples in one experiment. A maximum of 31 samples can be tested in one experiment, with the remaining two wells used for positive and negative controls. Therefore, this kit meets the needs of multiple application scenarios. When the sample size is large or during large-scale cervical cancer screening, it provides a rapid and efficient genotyping solution; when the sample size is small, a 3-well rapid testing scheme is used, truly achieving the goal of balancing genotyping and efficiency. Whether in large or small hospitals or large-scale screening, this kit can demonstrate its unique characteristics. Attached Figure Description

[0086] To more clearly illustrate the technical solutions of the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0087] Figure 1In Example 1, reaction solution A and mixture 1 were mixed in a certain proportion to prepare amplification reagent, and the original curve obtained by amplifying and detecting the FAM channel of LCBD002 sample was obtained.

[0088] Figure 2 In Example 1, reaction solution A and mixture 1 were mixed in a certain proportion to prepare amplification reagent, and the original curve obtained by amplifying and detecting the CY5 channel of LCBD023 sample was obtained.

[0089] Figure 3 In Example 1, reaction solution A and mixture 1 were mixed in a certain proportion to prepare amplification reagent, and the original curves obtained by amplifying and detecting the HEX channel of LC007-LC012 samples were obtained.

[0090] Figure 4 The results of linear regression analysis on the Ct values ​​of positive samples in the test results of the reagent kit of this application and the Jiangsu Tongke test kit in Example 1 are shown.

[0091] Figure 5 The results of linear regression analysis of the Ct values ​​of positive samples in the test results of the reagent kit of this application and the Jiangsu Tongke test kit in Example 3 are shown. Detailed Implementation

[0092] This invention discloses a high-risk human papillomavirus (HPV) nucleic acid typing detection kit. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0093] The nucleotide sequences of the primers and probes for specifically amplifying multiple HPV subtypes and the internal control β-Globin provided by this invention are shown in the table below:

[0094] Table 1

[0095]

[0096] The high-risk human papillomavirus nucleic acid typing detection kit provided by this invention includes the following components: ① PCR reaction solution A, which is a commercially available multiplex PCR amplification MIX, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product name: ① Universal TaqMan mμLtiplex qPCR master mix (UDG plus), product number 11212ES03; ② Human papillomavirus primer and probe mixture 1, including primer and probe combinations as shown in Combinations 1, 2, 3, 4, 5, and 6; ③ Human papillomavirus primer and probe mixture 2, including primer and probe combinations as shown in Combinations 7, 8, 9, 10, 11, and 6; ④ Human papillomavirus primer and probe mixture 3, including primer and probe combinations as shown in Combinations 13, 14, 15, 16, 17, and 6; ⑤ Human papillomavirus primer and probe mixture 2. 4. Needle mixture, including primer and probe combinations as shown in combinations 12, 18, 19, 20, and 6; ⑥ Human papillomavirus positive control, including human papillomavirus cloning plasmids of types 16, 31, 35, 33, 52, 58, 51, 56, 66, 18, 45, 68, 39, and 59, and human genome β-Globin cloning plasmid. Each plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the concentration was 1.00-5.00E+05 copies / mL; ⑦ Human papillomavirus negative control, which is ultrapure water.

[0097] Each primer and probe in the human papillomavirus primer-probe mixture 1-4 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The final concentration of each primer in the reaction system was 0.1-0.5 μmol / L, and the final concentration of each probe in the reaction system was 0.05-0.25 μmol / L.

[0098] This invention provides a method for using a high-risk human papillomavirus nucleic acid typing detection kit:

[0099] The first step involves mixing human papillomavirus (HPV) PCR reaction solution A and HPV primer-probe mixture 1 as follows: According to the instructions for use of PCR amplification MIX from Yisheng Company, the amount of HPV PCR reaction solution A added is 12.5 μL per person, and the amount of HPV primer-probe mixture 1 added is 7.5 μL per person. The final concentration of each primer is 0.1-0.5 μmol / L, and the final concentration of each probe is 0.05-0.25 μmol / L. The mixture is then vortexed and aliquoted into PCR reaction tubes at a rate of 20 μL per reaction. The second step involves extracting nucleic acid from clinical samples using the magnetic bead method.

[0100] The third step involves adding 5 μL each of the negative control, positive control, and extracted clinical sample nucleic acid to separate PCR reaction tubes containing the reaction solution, mixing, and centrifuging.

[0101] The fourth step is to perform amplification and detection on a 6-channel fluorescence quantitative PCR instrument purchased from Shanghai Hongshi Medical Technology Co., Ltd. The reaction program is as follows: (1) 25℃, 5-10min; (2) 95℃, 1-5min; (3) 95℃, 5-15sec; 60℃, 20-30sec, for 40-45 cycles.

[0102] The fifth step is to determine the positive or negative result of the target based on the amplification curve and Ct value after the PCR amplification test. When the amplification curve is a standard S-shape and the target Ct value is less than or equal to 39, and the β-Globin amplification curve is a standard S-shape and the Ct value is less than or equal to 40, the target is considered positive. The next step is determined based on the test results. If the sample test result is negative, no further steps are required, and the result is reported as negative. If the test result is positive for a certain channel, a second test is performed. Specifically, if the test result is positive for the FAM channel, it indicates that the sample is positive for one or more of human papillomavirus (HPV) types 16, 31, and 35. In this case, repeat the steps one through four above with HPV PCR reaction solution A and HPV primer-probe mixture 2 to confirm the positive type of the sample. If the test result is positive for the HEX channel, it indicates that the sample is positive for one or more of HPV types 33, 52, and 58. In this case, mix HPV PCR reaction solution A with HPV primer-probe mixture 2 and HPV primer-probe mixture 4 respectively for a second test. If the test result is positive for the ROX channel, it indicates that the sample is positive for HPV types 51, 52, and 35. If one or more of the following types are positive: 6, 66, then mix HPV PCR reaction solution A with HPV primer-probe mixture 3 for a second test; if the test result is positive for the CY5 channel, it indicates that the sample is positive for one or more of the following HPV types: 18, 45, 68, then mix HPV PCR reaction solution A with HPV primer-probe mixture 3 and HPV primer-probe mixture 4 respectively for a second test; if the test result is positive for the Quasar 705 channel, it indicates that the sample is positive for one or more of the following HPV types: 39, 59, then mix HPV PCR reaction solution A with HPV primer-probe mixture for a second test.

[0103] In the embodiments provided by this invention, the sources of the instruments and reagents used are shown in the table below:

[0104] Table 2

[0105]

[0106] The raw materials and reagents used in the high-risk human papillomavirus nucleic acid typing detection kit provided by this invention can all be purchased from the market.

[0107] The present invention will be further illustrated below with reference to the embodiments:

[0108] Example 1

[0109] To demonstrate the detection effectiveness of the primer-probe combination and kit of this invention, clinical samples were tested using the kit of this invention, and compared with the Jiangsu Tongke Human Papillomavirus Nucleic Acid Detection Kit.

[0110] 1.1 Sample Information

[0111] Sample source: 35 clinical samples of cervical exfoliated cells from Zhengzhou Sainote Medical Laboratory;

[0112] Sample number: LC001-LC035.

[0113] 1.2 Sources of instruments and reagents used in the experiment

[0114] The sources of the instruments and reagents used in this embodiment are shown in Table 2, and the specific primer and probe sequences used for human papillomavirus typing detection are shown in Table 1.

[0115] 1.3 Experimental Methods

[0116] 1.3.1 Reagent Preparation

[0117] Human papillomavirus (HPV) PCR reaction solution A and HPV primer-probe mixture 1 were mixed as follows: 12.5 μL of HPV PCR reaction solution A and 7.5 μL of HPV primer-probe mixture 1 were added per person per reaction. A total of 37 reactions were prepared. After preparation, the mixture was vortexed and mixed thoroughly. 20 μL of each reaction solution was aliquoted into PCR reaction tubes. The reaction system is as follows:

[0118] Table 3

[0119] Components Dosage Human papillomavirus PCR reaction solution A 12.5μL Human papillomavirus primer-probe mixture 1 7.5μL template 5μL

[0120] 1.3.2 Sample Processing

[0121] Thirty-five clinical samples were shaken and mixed thoroughly. Nucleic acid was extracted from the clinical samples using nucleic acid extraction reagents from Henan Sainote Biotechnology Co., Ltd. and a fully automated nucleic acid extractor from Jifan Biotechnology Co., Ltd.

[0122] 1.3.3 Sample loading and instrumentation

[0123] Add 5 μL each of the extracted clinical sample nucleic acid, negative control and positive control to PCR reaction tubes containing reaction solution, mix and centrifuge, and perform amplification and detection on the SLAN-96S / 48P fully automated medical PCR analysis system. The reaction program is as follows: (1) 25℃, 5-10 min; (2) 95℃, 1-5 min; (3) 95℃, 5-15 sec, 60℃, 20-30 sec, for 40-45 cycles.

[0124] 1.4 Results Analysis

[0125] Samples LC001-LC035 were tested according to the above experimental steps. The results of the first test are as follows:

[0126] Table 4

[0127]

[0128]

[0129] The test results showed that the FAM channel of samples LC001-LC006 was positive, indicating that LC001-LC006 contained one or more of human papillomavirus types 16, 31, and 35. The human papillomavirus PCR reaction solution A and human papillomavirus primer and probe mixture 2 were repeated according to the experimental method in 1.3 above to confirm the positive type of the sample.

[0130] The HEX channel of sample LC007-LC012 was positive in the test results, indicating that LC007-LC012 contains one or more of human papillomavirus types 33, 52, and 58. The human papillomavirus PCR reaction solution A was repeated with human papillomavirus primer and probe mixture 2 and human papillomavirus primer and probe mixture 4 according to the experimental method in 1.3 above to confirm the positive type of the sample.

[0131] The ROX channel of sample LC013-LC018 was positive in the test results, indicating that LC013-LC018 contains one or more of human papillomavirus types 51, 56, and 66. The human papillomavirus PCR reaction solution A and human papillomavirus primer and probe mixture 3 were repeated according to the experimental method in 1.3 above to confirm the positive type of the sample.

[0132] The test results showed that the CY5 channel of sample LC019-LC024 was positive, indicating that LC019-LC024 contained one or more of human papillomavirus types 18, 45, and 68. The human papillomavirus PCR reaction solution A was repeated with human papillomavirus primer and probe mixture 3 and human papillomavirus primer and probe mixture 4 according to the experimental method in 1.3 above to confirm the positive type of the sample.

[0133] The Quasar 705 channel of sample LC025-LC028 was positive in the test results, indicating that LC025-LC028 contains one or more positive human papillomavirus types 39 or 59. The human papillomavirus PCR reaction solution A and human papillomavirus primer probe mixture 3 were repeated according to the experimental method in 1.3 above to confirm the positive type of the sample.

[0134] The test results for samples LC029-LC035 were negative, and no secondary testing is required.

[0135] In Table 4, the original curves of the first detection results for sample LC002 fluorescence quantitative PCR are shown below. Figure 1 As shown, the detection channel for this sample is FAM, with a Ct value of 22.91, indicating a positive result for this channel; the original curve of the LC023 fluorescence quantitative PCR detection result for this sample is shown below. Figure 2 As shown, the detection channel for this sample is CY5, with a Ct value of 30.77, indicating a positive result for this channel. The original curve of the LC007-LC012 fluorescence quantitative PCR detection results is shown below. Figure 3 As shown, the detection channel for this sample is HEX. The Ct value of sample LC007 is 25.38, and the detection result of this channel is positive; the Ct value of sample LC008 is 26.05, and the detection result of this channel is positive; the Ct value of sample LC009 is 31.34, and the detection result of this channel is positive; the Ct value of sample LC010 is 24.93, and the detection result of this channel is positive; the Ct value of sample LC011 is 22.73, and the detection result of this channel is positive; the Ct value of sample LC012 is 26.53, and the detection result of this channel is positive.

[0136] Samples LC001-LC028 were subjected to secondary testing according to the above experimental steps, and the test results are shown in Table 5.

[0137] Samples LC001-LC035 were tested according to the instructions of the Jiangsu Tongke reagent kit, and the test results were compared with those of the reagent kit of this invention. The results are as follows:

[0138] Table 5

[0139]

[0140]

[0141] Through comparative analysis, the positive and negative concordance rates of the test results of the kit of the present invention and the control kit were both 100%. The positive sample detection Ct value was used to construct a linear regression equation for linear regression analysis, and the linear function was obtained as y = 1.0756x - 1.5622. The absolute value of the correlation R between the two was 0.99.

[0142] 1.5 Conclusion

[0143] Analysis of the comparative test results shows that the test results of the present invention kit are in good agreement with those of the comparison kit, indicating that the present invention can achieve the same detection effect as the Jiangsu Tongke kit.

[0144] Example 2

[0145] To demonstrate that the final concentration of each primer and probe of each type in the kit provided by this invention has been specifically tested and optimized using clinical samples, this embodiment uses the optimal final concentration screening and optimization process of primers of types 16, 31, and 35 as an example to illustrate the optimal final concentration screening and optimization process of all primers and probes.

[0146] 2.1 Sample Information

[0147] Sample source: Three clinical samples of cervical exfoliated cells from Zhengzhou Sainote Medical Laboratory that were positive for type 16;

[0148] Sample numbers: LCBD1033-LCBD1035.

[0149] 2.2 Sources of instruments and reagents used in the experiment

[0150] The sources of the instruments and reagents used in this embodiment are shown in Table 2. The specific primers and probe sequences used for human papillomavirus typing detection are shown in SEQ ID No. 1 to 3 in Table 1.

[0151] 2.3 Experimental Methods

[0152] 2.3.1 Reagent Preparation

[0153] Reagents were prepared according to the experimental objectives. Controlled variables were used, keeping the amounts of other components constant, and only the primers were changed to screen and optimize the optimal final concentration.

[0154] The purpose of this embodiment is to screen the optimal concentration range of primers. Therefore, the contents of type 16, 31, and 35 probes and ddH2O are kept constant, while the amount of primers added is changed to prepare primer-probe mixtures ①-⑤ for types 16, 31, and 35, respectively. The preparation methods for primer-probe mixtures ①-⑤ are as follows:

[0155] Table 6

[0156] Table 7

[0157]

[0158] Table 8

[0159]

[0160] Table 9

[0161]

[0162] Table 10

[0163]

[0164] Prepare primer-probe mixtures ① to ⑤ according to the methods shown in Tables 6 to 10. After preparation, prepare 5 corresponding reagents according to Yisheng's PCR amplification MIX system, with 5 doses of each reagent prepared for 5 people. The systems are as follows:

[0165] Table 11

[0166] Components Dosage per person Human papillomavirus PCR reaction solution A 12.5μL Primer-probe mixture ①~⑤ 7.5μL

[0167] 2.3.2 Sample Processing

[0168] Three clinical samples were shaken and mixed thoroughly, and nucleic acid was extracted from the clinical samples using nucleic acid extraction reagents from Henan Sainote Biotechnology Co., Ltd. and a fully automated nucleic acid extractor from Jifan Biotechnology Co., Ltd.

[0169] 2.3.3 Sample loading and instrumentation

[0170] Add 5 μL each of the extracted clinical sample nucleic acid, negative control and positive control to PCR reaction tubes containing reaction solution, mix and centrifuge, and perform amplification and detection on the SLAN-96S / 48P fully automated medical PCR analysis system. The reaction program is as follows: (1) 25℃, 5-10 min; (2) 95℃, 1-5 min; (3) 95℃, 5-15 sec, 60℃, 20-30 sec, for 40-45 cycles.

[0171] 2.4 Results Analysis

[0172] 2.4.1 The test results are as follows:

[0173] Table 12

[0174]

[0175] 2.4.2 Conclusion

[0176] Analysis of the detection results showed that the detection sensitivity was good when the final concentration of the primers was between 0.1 μmol / L and 0.5 μmol / L.

[0177] Example 3

[0178] The second application scenario of this patent application is when the hospital's daily sample volume does not exceed 31 cases. To save medical staff's time, a two-stage testing strategy can be directly used. Human papillomavirus (HPV) PCR reaction solution A is mixed with HPV primer-probe mixture 2, HPV primer-probe mixture 3, and HPV primer-probe mixture 4 in specific proportions, then amplified. High-risk HPV typing can be performed directly in a single experiment, achieving a simple, rapid, and efficient result. This embodiment will detail the implementation process of this detection method.

[0179] 3.1 Sample Information

[0180] Sample source: 17 clinical samples of cervical exfoliated cells from Zhengzhou Sainote Medical Laboratory;

[0181] Sample number: LC036-LC052.

[0182] 3.2 Sources of instruments and reagents used in the experiment

[0183] The sources of the instruments and reagents used in this embodiment are shown in Table 2. The specific primers and probe sequences used for human papillomavirus typing detection are shown in Table 1 as SEQ ID No. 16-60.

[0184] 3.3 Experimental Methods

[0185] 3.3.1 Reagent Preparation

[0186] Human papillomavirus (HPV) PCR reaction solution A was mixed with HPV primer-probe mixture 2, HPV primer-probe mixture 3, and HPV primer-probe mixture 4 respectively to prepare three reagents ①, ②, and ③. Each reagent was used to prepare 19 reactions. For reagent ①, the amount of HPV PCR reaction solution A added per person was 12.5 μL, and the amount of HPV primer-probe mixture 2 added per person was 7.5 μL. For reagent ②... For each human papillomavirus (HPV) PCR reaction, add 12.5 μL of HPV primer-probe mixture 3 per person. For reagent ③, add 12.5 μL of HPV PCR reaction solution A per person and 7.5 μL of HPV primer-probe mixture 4 per person. After preparing the three reagents, vortex to mix well, and aliquot 20 μL into each PCR reaction tube. The reaction system is as follows:

[0187] Table 13

[0188] Reagent ① Components Dosage Human papillomavirus PCR reaction solution A 12.5μL Human papillomavirus primer-probe mixture 2 7.5μL

[0189] Table 14

[0190] Reagent ② Component Dosage Human papillomavirus PCR reaction solution A 12.5μL Human papillomavirus primer-probe mixture 3 7.5μL

[0191] Table 15

[0192] Reagent ③ Components Dosage Human papillomavirus PCR reaction solution A 12.5μL Human papillomavirus primer-probe mixture 4 7.5μL

[0193] 3.3.2 Sample Processing

[0194] Seventeen clinical samples were shaken and mixed, and nucleic acid was extracted from the clinical samples using nucleic acid extraction reagents from Henan Sainote Biotechnology Co., Ltd. and a fully automated nucleic acid extractor from Jifan Biotechnology Co., Ltd.

[0195] 3.3.3 Sample loading and instrumentation

[0196] Add 5 μL each of the extracted clinical sample nucleic acid, negative control and positive control to PCR reaction tubes containing reaction solution, mix and centrifuge, and perform amplification and detection on the SLAN-96S / 48P fully automated medical PCR analysis system. The reaction program is as follows: (1) 25℃, 5-10 min; (2) 95℃, 1-5 min; (3) 95℃, 5-15 sec; 60℃, 20-30 sec, for 40-45 cycles.

[0197] 3.4 Test Results:

[0198] The detection results of the reagent kit of this invention are shown in Table 16. Samples LC036-LC052 were tested using the Jiangsu Tongke reagent kit according to the instructions, and the detection results of the two reagents were compared and analyzed. The results are as follows:

[0199] Table 16

[0200]

[0201] Comparative analysis showed that the positive and negative concordance rates of the kit and the control kit were both 100%. Linear regression analysis was performed using a linear regression equation to construct a linear regression equation from the Ct values ​​of the positive samples, yielding a linear function y = 1.0649x - 1.5071. The absolute value of the correlation R between the two kits was 0.99 (results are shown below). Figure 5 (As shown).

[0202] 3.5 Conclusion

[0203] Analysis of the comparative detection results shows that the test results of the kit of this invention are consistent with those of the control kit. Therefore, the second application scenario of this patent application is: when the daily sample volume of a hospital does not exceed 31 cases, in order to save the time of medical staff, a secondary testing strategy can be directly used, which can be successfully implemented. This strategy is fast and efficient when the sample volume is less than 31 cases.

[0204] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer-probe combination, characterized in that, The primer-probe combination includes mixture 1; The primer-probe combination further includes one or more of mixture 2, mixture 3, or mixture 4; The mixture 1 includes primer and probe combinations as shown in combination 1, combination 2, combination 3, combination 4, combination 5, and combination 6; The mixture 2 includes primer and probe combinations as shown in combinations 7, 8, 9, 10, 11, and 6. The mixture 3 includes primer and probe combinations as shown in combinations 13, 14, 15, 16, 17, and 6. The mixture 4 includes primer and probe combinations as shown in combinations 12, 18, 19, 20, and 6. Combination X: I. The nucleotide sequence of the upstream primer is shown in SEQ ID No. (3X-2); II. The nucleotide sequence of the downstream primer is shown in SEQ ID No. (3X-1); III. The nucleotide sequence of the probe is shown in SEQ ID No. 3X; Where X is any integer selected from 1 to 20; When X=1, human papillomavirus types 16, 31, and 35 are detected. When X=2, human papillomavirus types 33, 52, and 58 are detected. When X=3, human papillomavirus types 51, 56, and 66 are detected. When X=4, human papillomavirus types 18, 45, and 68 are detected. When X=5, human papillomavirus types 39 and 59 are detected; When X=6, the intrinsic parameter is detected; When X=7, human papillomavirus type 16 is detected; When X=8, human papillomavirus type 31 is detected; When X=9, human papillomavirus type 35 is detected; When X=10, human papillomavirus type 33 is detected; When X=11, human papillomavirus type 52 is detected; When X=12, human papillomavirus type 58 is detected; When X=13, human papillomavirus type 51 is detected; When X=14, human papillomavirus type 56 is detected; When X=15, human papillomavirus type 66 is detected; When X=16, human papillomavirus type 18 is detected; When X=17, human papillomavirus type 45 was detected; When X=18, human papillomavirus type 68 is detected; When X=19, human papillomavirus type 39 is detected; When X=20, human papillomavirus type 59 was detected.

2. The use of the primer-probe combination as described in claim 1 in the preparation of reagents or kits for detecting human papillomavirus.

3. The application as described in claim 2, characterized in that, The human papillomaviruses mentioned include high-risk human papillomaviruses 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

4. A detection reagent, characterized in that, It includes the primer-probe combination as described in claim 1 and acceptable auxiliaries.

5. The use of the detection reagent as described in claim 4 in the preparation of a kit for detecting human papillomavirus.

6. The application as described in claim 5, characterized in that, The human papillomaviruses mentioned include high-risk human papillomaviruses 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

7. A reagent kit, characterized in that, Includes the primer-probe combination as described in claim 1 or the detection reagent as described in claim 4.

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