Human papilloma virus nucleic acid detection quality control product as well as preparation method and application thereof

Abstract

The invention discloses a human papilloma virus nucleic acid detection quality control product as well as a preparation method and application thereof. The preparation method comprises the following steps: cloning an HPV whole genome into a vector in two segments to obtain an HPV-A recombinant plasmid and an HPV-B recombinant plasmid; transfecting cells with the two recombinant plasmids and lentivirus plasmids respectively, and packaging to obtain an HPV-A lentivirus and an HPV-B lentivirus; the method comprises the following steps: infecting a host cell with HPV-A lentivirus to obtain an HPV-A cell line; and infecting the HPV-A cell line with HPV-B lentivirus, and screening to obtain a cell line containing HPV-A and HPV-B at the same time, namely the quality control product. According to the preparation method disclosed by the invention, the HPV whole genome lentivirus transfection can be simply and efficiently realized, the background of the prepared quality control product is close to that of a real clinical sample, and the prepared quality control product is high in stability and easy to transport, store and passage; moreover, the system is wide in coverage range, is suitable for multi-purpose research, and is compatible with HPV detection of various platforms.

Description

Technical Field

[0001] The present application relates to the technical field of human papillomavirus detection, and in particular to a quality control product for human papillomavirus nucleic acid detection, and a preparation method and application thereof. Background Art

[0002] Human papilloma virus (HPV) is a non-enveloped double-stranded circular small DNA virus that is tropistic for epithelial tissues. The viral genome is about 8kb and can be divided into early genes, late genes, and non-coding upstream regulatory regions (URRs). Among them, early genes contain open reading frames E6, E7, E1, E8, E2, E4, and E5, late genes contain open reading frames L2 and L1, and the non-coding upstream regulatory region is also called the long control region (LCR).

[0003] At present, more than 200 types of HPV have been discovered, isolated and identified, and divided into high-risk and low-risk types according to their potential to induce cancer. Persistent infection with high-risk HPV has been confirmed as the main cause of cervical lesions. The "Guidelines for Human Papillomavirus (HPV) Nucleic Acid Detection and Genotyping, and Reagent Technical Review" lists 13 genotypes, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, as high-risk HPV, and 5 genotypes, including HPV26, 53, 66, 73, and 82, as medium-risk types. In the "Prevention of Cervical Cancer: Guidelines for Screening and Treatment of Cervical Precancerous Lesions" released by WHO in 2021, it is pointed out that high-risk HPV testing usually includes 14 types: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. At the same time, HPV DNA testing is recommended as the preferred method for cervical cancer screening. The latest expert consensus on human papillomavirus nucleic acid testing for cervical cancer screening (2022), issued in 2023, also clearly emphasizes the recommendation of HPV nucleic acid testing as the main method for cervical cancer screening. In addition to causing cervical cancer, HPV infection also causes approximately 35,000 cases of anal cancer, 13,000 cases of penile cancer, 8,500 cases of vulvar cancer, 12,000 cases of vaginal cancer, 29,000 cases of oropharyngeal cancer, 4,400 cases of oral cancer, and 3,800 cases of laryngeal cancer each year worldwide, accounting for 88.0%, 50.0%, 24.9%, 78.0%, 30.8%, 2.2%, and 2.4% of the global new cases of their respective cancers. Benign lesions caused by low-risk HPV infection are mainly genital warts. The annual incidence of genital warts worldwide is 160 / 100,000 to 289 / 100,000. For new genital warts, the incidence is 103 / 100,000 to 168 / 100,000 for men and 76 / 100,000 to 191 / 100,000 for women. For recurrent genital warts, the incidence is 163 / 100,000 for men and 110 / 100,000 for women.

[0004] At present, the commonly used technologies for HPV nucleic acid detection are mainly nucleic acid amplification and its derivative technologies, including: constant temperature amplification method, PCR-fluorescent probe method, PCR-melting curve method, PCR-microfluidic chip method, PCR-reverse dot hybridization / flow fluorescence hybridization method, etc. Common target sequences include L1, E6, E7 and E1 regions, and the target sequence of some kits is the entire HPV genome. However, there are many types of HPV typing detection reagents with different principles and characteristics. In the actual detection process, the analytical sensitivity, specificity and other performance indicators of the detection are very dependent on factors such as the efficiency of target nucleic acid extraction in the sample, the specificity of primer and probe design, and the rationality of enzyme amplification reaction condition design. Therefore, standard substances or quality control products with stable performance and clear characteristics for quality control of HPV nucleic acid detection are indispensable.

[0005] Standard substances or quality control products with a certain value can be used to calibrate different test kits and improve the accuracy of the test. In order to improve the accuracy and consistency of HPV test results, WHO has successively constructed recombinant plasmid DNA containing the whole genome sequence for high-risk HPV16, 18, 31, 33, 45, 52, 58 and other types since 2008. As nucleic acid standard materials, the qualitative and quantitative test results of HPV from different laboratories in different countries were compared. However, international standard materials are not easy to purchase, and it is difficult to meet the huge demand for metrological calibration. At the same time, they are easy to cause aerosol contamination during use. In addition, there are certain problems with the stability and storage conditions of existing quality control products, which may lead to false negative or false positive results in the actual detection process. In order to overcome the limitations of the application scope of quality control products in the form of recombinant plasmid DNA and realize quality control of the entire process including DNA extraction and purification, PCR amplification and detection, researchers have developed standard materials in the form of pseudoviruses for the four common high-risk subtypes of HPV16, 18, 52, and 58. However, pseudovirus packaging technology has certain limitations. It is unable to encapsulate the full-length genome sequence, and the quality control products are of a single or limited type, which makes it difficult to cover all high-risk HPV types.

[0006] In order to achieve quality control of the entire process including DNA extraction and purification, PCR amplification and detection, researchers have developed quality control products in the form of cell lines for the fourteen common high-risk subtypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68, and have begun to apply for patent protection, such as application publication number CN 117625845 A. The preparation method includes constructing the HPV whole genome into a recombinant plasmid, and transfecting the recombinant plasmid into a human cervical cancer cell line by electrofection; then single clone selection and identification are performed to obtain a human cervical cancer cell line that has been successfully transfected with the recombinant plasmid. However, the electrofection method is prone to cause massive cell death, and the scheme has certain limitations. It cannot cover low-risk HPV and is therefore limited in application.

[0007] Therefore, developing a quality control product with a wide coverage of HPV genotypes and is relatively stable remains a research focus in the field of HPV quality control products. Summary of the invention

[0008] The purpose of this application is to provide a new human papillomavirus nucleic acid detection quality control product and its preparation method and application.

[0009] This application adopts the following technical solutions: One aspect of the present application discloses a method for preparing a quality control product for human papillomavirus nucleic acid detection, comprising the following steps: The HPV genome was divided into two segments, labeled as HPV-A and HPV-B, and HPV-A and HPV-B were cloned into vectors to obtain HPV-A recombinant plasmid and HPV-B recombinant plasmid respectively; HPV-A recombinant plasmid and HPV-B recombinant plasmid were used to transfect cells with lentiviral plasmids, respectively, to obtain HPV-A lentivirus and HPV-B lentivirus by packaging; Host cells were infected with HPV-A lentivirus, and HPV-A cell lines were obtained through drug screening and monoclonal screening; The HPV-A cell line was infected with the HPV-B lentivirus, and a cell line containing both HPV-A and HPV-B was obtained through drug screening and monoclonal screening, namely, the human papillomavirus nucleic acid detection quality control product of the present application.

[0010] It should be noted that the present application creatively divides the HPV genome into two segments of about 4000bp, namely HPV-A and HPV-B, and performs lentiviral transfection separately to obtain cell lines containing both HPV-A and HPV-B; not only does it overcome the problem that the efficiency of lentiviral transfection of the whole genome is very low and difficult to succeed; but also, the background of the prepared stable cell line is closer to the real clinical sample, with high stability, easy transportation, storage and passage. In one implementation of the present application, 41 HPV full-length gene stable cell lines of 39 HPV types were prepared by the preparation method of the present application. As a quality control product, it can not only more accurately measure the viral load, but also has a wide coverage range, and can be applied to a variety of research purposes, and is compatible with HPV detection methods on multiple platforms, such as PCR-reverse dot hybridization, fluorescent PCR, flow-through hybridization, sequencing, etc.

[0011] It should also be noted that HPV-A and HPV-B are just the numbers of two sequences, so as to facilitate the description of the corresponding recombinant plasmids, lentiviruses and cell lines later; therefore, when the host cells are infected with lentiviruses, the host cells can be infected with HPV-B lentiviruses first, and the HPV-B cell lines can be screened, and then the HPV-A lentiviruses can be used to infect the HPV-B cell lines to screen and obtain cell lines containing both HPV-A and HPV-B. However, considering the efficiency of lentivirus transfection and the quality of quality control products, it is recommended to first infect the host cells with the lentivirus corresponding to the longer sequence of HPV-A and HPV-B, screen and obtain the corresponding cell lines, and then use another lentivirus to infect the cell lines obtained by screening, and finally screen and obtain cell lines containing both HPV-A and HPV-B; of course, one of the lentiviruses can also be used to infect the host cells at will, screen and obtain the corresponding cell lines, and then use another lentivirus to infect the cell lines obtained by screening, and finally screen and obtain cell lines containing both HPV-A and HPV-B.

[0012] In one implementation of the present application, the HPV whole genome includes 39 HPV genotypes, specifically, 18 HPV high-risk types HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82; and 21 HPV low-risk types HPV 6, 11, 30, 34, 40, 42, 43, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, 81, 83, 84, 89; and, for HPV54 and 68, there are two subtypes, a and b.

[0013] It should be noted that the preparation method of the present application can be applied to HPV6, HPV11, HPV16, HPV18, HPV26, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54a, HPV54b, HPV55, HPV56, HPV Quality control products of 41 HPV full-length genes of 39 HPV types, including HPV58, HPV59, HPV61, HPV62, HPV66, HPV67, HPV68a, HPV68b, HPV69, HPV70, HPV71, HPV72, HPV73, HPV81, HPV82, HPV83, HPV84 and HPV89, are prepared, so that 41 types of HPV full-length gene quality control products can be obtained, covering a wider range.

[0014] In one implementation of the present application, HPV-A and HPV-B are combined into the HPV full genome.

[0015] In one implementation of the present application, HPV-A contains early genes, and HPV-B contains other sequences in the HPV genome except HPV-A; wherein the early genes include, for example, E6, E7, E1, E2, E4 and E5. For example, the HPV-A sequence is about 4000bp, and the HPV-B sequence is about 3600bp; HPV-B contains late genes and a long control region, or HPV-B contains late genes, a long control region, an X gene and a Y gene. Among them, late genes are such as L2 and L1. In addition, X gene and Y gene are some HPV types that also contain X gene and Y gene after L2 and L1, such as HPV43 and HPV82. In short, HPV-B mainly contains late genes and most of the long control region.

[0016] It should be noted that the key to this application is to divide the HPV whole genome of about 8kb into two segments for lentiviral transfection; in the preferred scheme, while ensuring the transfection efficiency, the first transfected segment is made as long as possible, and then the relatively shorter sequence is transfected, so that the efficiency and quality of the two transfections can be better guaranteed. It can be understood that HPV-A and HPV-B are just the numbers of two sequences. In principle, the first transfected segment only needs to be about 4000bp and the second transfected segment only needs to be about 3600bp; of course, while ensuring the transfection efficiency, the HPV whole genome can also be directly divided into two segments on average.

[0017] In one implementation of the present application, the HPV-A recombinant plasmid and the HPV-B recombinant plasmid use different screening genes.

[0018] In one implementation of the present application, HPV-A and HPV-B are constructed into PCDH-mcherry-zeocin and PCDH-CMV-EGFP-puro, respectively.

[0019] In one implementation of the present application, HPV-A is constructed into PCDH-mcherry-zeocin, and HPV-B is constructed into PCDH-CMV-EGFP-puro.

[0020] It should be noted that HPV-A and HPV-B were constructed into two different vectors respectively, mainly to facilitate subsequent drug screening. The two vectors can be interchanged, for example, HPV-A was constructed into PCDH-CMV-EGFP-puro, and HPV-B was constructed into PCDH-mcherry-zeocin.

[0021] In one implementation of the present application, the lentiviral plasmid includes psPAX2 and pMD2G.

[0022] It should be noted that the psPAX2 vector contains the gag gene, which encodes the main structural protein of the virus; the pol gene, which encodes a virus-specific enzyme; and the rev gene, which encodes a regulatory factor that regulates the expression of the gag and pol genes. The pMD2G vector contains the vsvg gene, which provides the envelope protein required for virus packaging. The target plasmid (the HPV-A recombinant plasmid or the HPV-B recombinant plasmid in this application) and two lentiviral plasmids are co-transfected into the packaging cells using a transfection reagent, and the transcribed target gene RNA and the proteins translated from the psPAX2 and pMD2G genes can be assembled into a lentivirus.

[0023] In one implementation of the present application, the host cell is a human cervical cancer cell line or a human ovarian cancer cell line.

[0024] In one implementation of the present application, the HPV-A cell line and the cell line containing both HPV-A and HPV-B are screened and screened using different drugs.

[0025] In one implementation of the present application, the HPV-A cell line is screened and the cell line containing both HPV-A and HPV-B is screened, and the screening drugs used for both are non-repetitive and are selected from at least one of phleomycin, puromycin, neomycin, hygromycin B and blasticidin.

[0026] In one implementation of the present application, the preparation method of the present application further comprises performing Sanger sequencing on a cell line containing both HPV-A and HPV-B to ensure that the entire HPV genome is successfully recombined into the cell line.

[0027] In one implementation of the present application, the preparation method of the present application also includes using specific primers and probes to quantitatively detect at least one gene in the HPV genome in the quality control product, and using the quantitative detection result of the gene as the concentration of the quality control product.

[0028] In one implementation of the present application, the L1 gene in the HPV whole genome is quantitatively detected, and the copy number of the L1 gene is used as the concentration of the quality control product.

[0029] In one implementation of the present application, the upstream primers for quantitatively detecting HPV6, HPV11, HPV16, HPV18, HPV26, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54a, HPV54b, HPV55, HPV56, HPV58, HPV59, HPV61, HPV62, HPV66, HPV67, HPV68a, HPV68b, HPV69, HPV70, HPV71, HPV72, HPV73, HPV81, HPV82, HPV83, HPV84 and HPV89 are the sequences shown in SEQ ID No.1-41 in sequence, and the downstream primers are the sequences shown in SEQ ID No. The sequences shown in SEQ ID No.42-82, and the probes are the sequences shown in SEQ ID No.83-123 in sequence.

[0030] Another aspect of the present application discloses a quality control product for human papillomavirus nucleic acid detection prepared by the method of the present application.

[0031] It should be noted that the human papillomavirus nucleic acid detection quality control product of this application contains 41 HPV full-length genes of 39 HPV types, which not only covers a wider range, but also has a stable cell line background closer to the real clinical sample, high stability, and is easy to transport, store and pass. In addition, the quality control product of this application can more accurately measure the viral load, is suitable for multi-purpose research, and is compatible with HPV detection on multiple platforms.

[0032] Another aspect of the present application discloses the use of the human papillomavirus nucleic acid detection quality control product of the present application in the preparation of an HPV nucleic acid detection kit.

[0033] Another aspect of the present application discloses an HPV nucleic acid detection kit containing the human papillomavirus nucleic acid detection quality control product of the present application.

[0034] It should be noted that the HPV nucleic acid detection kit of the present application, because it contains the human papillomavirus nucleic acid detection quality control product of the present application, also has the advantages of wide coverage, being closer to real clinical samples, high stability, easy transportation, storage and propagation, etc. It can also be suitable for multi-purpose research and is compatible with HPV detection on multiple platforms.

[0035] The beneficial effects of this application are: The applicant's method for preparing quality control products for papillomavirus nucleic acid detection can simply and efficiently achieve lentiviral transfection of the HPV whole genome. The obtained stably transfected cell lines are used as quality control products. The background is close to that of real clinical samples, and they are highly stable and easy to transport, store and propagate. The quality control products prepared by the present application cover 41 full-length HPV genes of 39 HPV types, have a wide coverage range, are suitable for multi-purpose research, and are compatible with HPV detection on multiple platforms. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the result of fluorescence microscopy observation 48 hours after HPV45-A plasmid transfection in the embodiment of the present application; Figure 2 This is the result of fluorescence microscopy observation 48 hours after HPV45-B plasmid transfection in the example of this application; Figure 3 This is the result of fluorescence microscopy observation 48 hours after HPV45-A virus infection in the example of this application; Figure 4 This is the result of fluorescence microscopy observation 48 hours after HPV45-B virus infection in the example of this application; Figure 5 It is the partial sequencing result of HPV45 in the example of this application; Figure 6 It is the HPV45 sequencing comparison result in the example of this application; Figure 7 This is a graph of the HPV45 digital PCR results in the examples of this application; Figure 8 This is a fluorescence PCR test result diagram in the embodiment of the present application; Fig. 9 It is the test result of HPV6 to HPV39 quality control products by different test kits in the examples of this application; Fig.10 It is the test result of HPV40 to HPV54 quality control products by different test kits in the examples of this application; Fig.11 It is the test result of HPV55 to HPV68 quality control products by different test kits in the examples of this application; Fig.12These are the test results of HPV69 to HPV89 quality control products by different test kits in the examples of this application. DETAILED DESCRIPTION

[0037] The problem to be solved by this application is that the existing HPV whole genome quality control products have a small number of HPV types, a single structure of plasmid standard materials, low stability, and low complexity, and cannot truly reflect the true situation of the genome. Therefore, this application has developed and provided a human cervical cancer cell line quality control product with stable integration of HPV typing full-length genome, which covers 39 types, has high stability, can more accurately measure viral load, quality control the entire process of HPV detection, and is suitable for multiple platforms and its preparation method.

[0038] The present application study found that the length of 41 HPV full-length genes of 39 HPV types, including HPV6, HPV11, HPV16, HPV18, HPV26, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54a, HPV54b, HPV55, HPV56, HPV58, HPV59, HPV61, HPV62, HPV66, HPV67, HPV68a, HPV68b, HPV69, HPV70, HPV71, HPV72, HPV73, HPV81, HPV82, HPV83, HPV84 and HPV89, are all around 8kb, and are generally less than 8kb. Therefore, the present application creatively divides these full-length HPV genes into two segments, and constructs them into vectors respectively, i.e., obtains HPV-A recombinant plasmid and HPV-B recombinant plasmid; uses HPV-A recombinant plasmid and HPV-B recombinant plasmid to transfect cells with lentiviral plasmid respectively, and packages to obtain HPV-A lentivirus and HPV-B lentivirus; then, first infects host cells with HPV-A lentivirus, and screens to obtain HPV-A cell line; then, infects HPV-A cell line with HPV-B lentivirus, and screens to obtain stable cell line containing both HPV-A and HPV-B, which is used as quality control product for human papillomavirus nucleic acid detection in the present application.

[0039] In one implementation of the present application, specifically, the HPV whole genome DNA is divided into two sequences, HPV-A and HPV-B, wherein the HPV-A sequence is about 4000 bases long and contains E6, E7, E1, E2, E4 or E5 genes; the HPV-B sequence is about 3600 bases long and contains L2, L1, X or Y genes. The expression vectors are PCDH-mcherry-zeocin and PCDH-CMV-EGFP-puro, respectively. Specifically, the HPV-A fragment sequence is constructed into the PCDH-mcherry-zeocin vector, and the HPV-B fragment sequence is constructed into the PCDH-CMV-EGFP-puro vector. Its function is: HPV-A expresses with the mcherry red fluorescent protein tag, and HPV-B expresses with the GFP green fluorescent protein tag, so that cell imaging can be distinguished by a microscope during the culture process; HPV-A expresses with zeocin, and HPV-A expresses with puromycin (puro), which can be screened with different drug combinations during the cell drug screening process.

[0040] Then, the 82 HPV typing recombinant plasmids obtained by the above synthesis were transfected into HEK293T cells with the lentiviral plasmids psPAX2 and pMD2G under the conditions of LentiFit liposome transfection reagent. Taking the 6-well plate culture cells as an example, the specific steps are as follows: a. Culture 293T cells in a 6-well plate and replace the culture medium with fresh medium when the cells grow to 70%-90%.

[0041] b. Prepare the three-plasmid mixture in tube 1 and the LentiFit transfection reagent in tube 2 separately, let stand for 5 minutes; then gently mix tube 1 and tube 2, and incubate at room temperature for 15 minutes.

[0042] Tube 1: 4 μg of target plasmid, 3 μg of psPAX2, 1 μg of pMD2G, 70 μL of serum-free DMEM.

[0043] Tube 2: LentiFit reagent 13 μL, serum-free DMEM 70 μL.

[0044] c. Add the transfection mixture dropwise into the 6-well plate, gently shake the culture dish to mix the cells and the transfection mixture, and then place it in the incubator for culture. Replace the cells with fresh culture medium 8-12 hours after transfection. Observe the mcherry or GFP fluorescence under a confocal microscope 48 hours after transfection to determine the transfection effect, collect the virus supernatant, and obtain HPV-A lentivirus and HPV-B lentivirus.

[0045] In one implementation of the present application, the method of infecting host cells with a lentivirus is as follows: HPV-A lentivirus and C-33A cells with a density of 30~40% in a 6-well plate are inoculated at a volume ratio of 1:1 (the number of cells is about 1 million), and 1000× Polybrene infection reagent is added at the same time, and the cells are gently mixed and placed in a 37°C incubator for culture; zeocin screening is performed during the culture process. After the screening is completed, the cells are digested with Trypsin, diluted with culture medium, and then plated into a 96-well plate, and a well with only one monoclonal clone is selected, and the culture is continued, and then the monoclonal culture product is sequenced and verified to confirm that it contains the HPV-A fragment gene, thereby obtaining a stable HPV-A-C33A cell line, that is, an HPV-A cell line.

[0046] Furthermore, the HPV-B lentivirus and cells of the HPV-A-C33A cell line were inoculated at a volume ratio of 1:1 (the number of cells was approximately 1 million), and 1000× polybrene infection reagent was added, gently mixed, and cultured in a 37°C incubator; puromycin selection was performed during the culture process. After the screening, the cells were digested with Trypsin, diluted with culture medium, and then plated into a 96-well plate. Wells with only one monoclonal clone were selected and cultured continuously. The monoclonal culture product was then sequenced and verified to confirm that it contained the HPV-A fragment gene and the HPV-A fragment gene, thereby obtaining the stable HPV-C33A cell line of the present application, that is, the stably transformed cell line containing both HPV-A and HPV-B of the present application, which is used as the quality control product for human papillomavirus nucleic acid detection of the present application.

[0047] In one implementation of the present application, it also includes quantitative detection of the prepared quality control product, for example, using a digital PCR method to quantitatively detect at least one gene in the HPV genome, and using the copy number of the gene as the concentration value of the quality control product.

[0048] Specifically, in one implementation of the present application, the L1 gene is quantitatively detected, and the copy number of L1 is used as the concentration value of the quality control product. The upstream primers for quantitative detection of HPV6, HPV11, HPV16, HPV18, HPV26, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54a, HPV54b, HPV55, HPV56, HPV58, HPV59, HPV61, HPV62, HPV66, HPV67, HPV68a, HPV68b, HPV69, HPV70, HPV71, HPV72, HPV73, HPV81, HPV82, HPV83, HPV84 and HPV89 are shown in sequence as SEQ ID No.1-41, and the downstream primers are shown in sequence as SEQ ID The sequences shown in SEQ ID No.42-82, and the probes are the sequences shown in SEQ ID No.83-123 in sequence.

[0049] Compared with the prior art, the preparation method of the present application and the HPV full-length genome quality control product prepared therefrom have the following beneficial effects: 1. This application adopts the lentiviral infection method to prepare HPV full-length gene stably transfected cell line quality control products, the background is close to the real clinical samples, the stability is high, and it is easy to transport, store and passage.

[0050] 2. The HPV full-length genome quality control products provided in this application cover 39 types, with a wide coverage range and can be applied to various research purposes.

[0051] 3. The HPV full-length genome quality control product provided in this application can more accurately measure the viral load. The quality control HPV detection includes the entire process of DNA extraction and purification, PCR amplification and detection.

[0052] 4. The HPV full-length genome quality control product provided in this application is compatible with HPV detection methods on various platforms, such as PCR-reverse dot hybridization, fluorescent PCR, flow-through hybridization, sequencing, etc.

[0053] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application. Example

[0054] 1. Synthesis of HPV full-length genome sequence and vector construction Using the sequence of NCBI as a reference, the HPV whole genome DNA was divided into two sequences, HPV-A and HPV-B. 39 HPV type sequences were synthesized by chemical synthesis, including 18 HPV high-risk types: HPV16, HPV18, HPV26, HPV 31, HPV33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 53, HPV 56, HPV 58, HPV 59, HPV 66, HPV68, HPV 73, and HPV 82; 21 HPV low-risk types: HPV 6, HPV 11, HPV 30, HPV 34, HPV 40, HPV 42, HPV43, HPV 44, HPV 54, HPV 55, HPV 61, HPV 62, HPV 67, HPV HPV 69, HPV 70, HPV 71, HPV 72, HPV81, HPV 83, HPV 84, HPV 89, and distinguish subtypes a and b for HPV54 and 68. The specific information of recombinant plasmids constructed for each HPV typing is shown in Table 1. The 82 fragments in this example were synthesized by General Bio (Anhui) Co., Ltd.

[0055] The chemically synthesized sequence HPV-A was connected to the PCDH-mcherry-zeocin vector, and the HPV-B sequence was connected to the PCDH-CMV-EGFP-puro vector to construct 82 plasmids of 39 types. The plasmid construction was completed by General Biotechnology (Anhui) Co., Ltd.

[0056] Table 1 HPV complete genome and HPV-A and HPV-B sequences Type Serial Number Whole genome (bp) HPV-A fragment location HPV-B fragment location HPV6 JN252323.1 7996 1-4337 4338-7996 HPV11 HE574705.1 7933 1-4370 4371-7933 HPV16 JQ004096.1 7905 1-4101 4102-7905 HPV18 NC_001357.1 7857 1-4157 4158-7857 HPV26 NC_001583.1 7855 1-4188 4189-7855 HPV30 KF436843.1 7843 1-4270 4271-7843 HPV31 J04353.1 7912 1-4070 4071-7912 HPV33 M12732.1 7909 1-4081 4082-7909 HPV34 NC_001587.1 7723 1-4105 4106-7723 HPV35 M74117.1 7851 1-4038 4039-7851 HPV39 M62849.1 7833 1-4176 4177-7833 HPV40 LR862047.1 7905 1-4384 4385-7905 HPV42 M73236.1 7917 1-4206 4207-7917 HPV43 AJ620205.1 7975 1-4378 4379-7975 HPV44 U31788.1 7833 1-4324 4325-7833 HPV45 KC470260.1 7849 1-4235 4236-7849 HPV51 KU298905.1 7811 1-4134 4135-7811 HPV52 X74481.1 7942 1-4262 4263-7942 HPV53 X74482.1 7856 1-4282 4283-7856 HPV54a KF436890.1 7760 1-4214 4215-7760 HPV54b KF436893.1 7717 1-4193 4194-7717 HPV55 U31791.1 7822 1-4324 4325-7822 HPV56 EF177176.1 7866 1-4222 4223-7866 HPV58 D90400.1 7824 1-4122 4123-7824 HPV59 X77858.1 7896 1-4129 4130-7896 HPV61 KX514419.1 7989 1-4326 4327-7989 HPV62 KU298924.1 8092 1-4276 4277-8092 HPV66 U31794.1 7824 1-4271 4272-7824 HPV67 LC664138.1 7803 1-4121 4122-7803 HPV68a DQ080079.1 7822 1-4051 4052-7822 HPV68b FR751039.1 7836 1-4071 4072-7836 HPV69 OP711971.1 7705 1-4157 4158-7705 HPV70 U21941.1 7905 1-4145 4146-7905 HPV71 MT250602.1 8037 1-4331 4332-8037 HPV72 X94164.1 7988 1-4408 4409-7988 HPV73 X94165.1 7700 1-4082 4083-7700 HPV81 AJ620209.1 8070 1-4461 4462-8070 HPV82 AB027021.1 7871 1-4167 4168-7871 HPV83 AF151983.1 8104 1-4348 4349-8104 HPV84 AF293960.1 7948 1-4262 4263-7948 HPV89 AF436128.1 8078 1-4351 4352-8078 2. Preparation of HPV Whole Genome Quality Control In this example, the method of constructing a lentiviral stable cell line was used to first transfect the recombinant plasmid and the lentiviral plasmid into HEK293T cells (American Type Culture Collection (ATCC), Catalog Number: CRL-3216) to obtain HPV-A lentivirus and HPV-B lentivirus, and then the HPV-A lentivirus was used to infect C-33A cells to obtain stable HPV-AC-33A cells. Finally, the HPV-B lentivirus was used to infect HPV-AC-33A cells to obtain a stable HPV whole genome quality control product.

[0057] Taking the construction of HPV45 quality control products as an example, the specific steps are as follows: (1) HPV45-A and HPV45-B lentiviral packaging Culture 293T cells in a 6-well plate. When the cells grow to 70%-90%, replace with fresh culture medium, which is 90% volume of DMEM medium, 10% fetal bovine serum (FBS) and 1% double antibody (penicillin / streptomycin); prepare the three-plasmid mixture in tube 1 and the LentiFit transfection reagent in tube 2 respectively, and let stand for 5 minutes; then gently mix tubes 1 and 2 and incubate at room temperature for 15 minutes.

[0058] Tube 1: 4 μg of target plasmid, 3 μg of psPAX2 (Addgene, Catalog No. 12260), 1 μg of pMD2G (Addgene, Catalog No. 12259), 70 μL of serum-free DMEM.

[0059] Tube 2: LentiFit reagent 13 μL, serum-free DMEM 70 μL.

[0060] Add the transfection mixture dropwise into the 6-well plate, shake the culture dish to mix the cells and transfection mixture, and culture in a 5% CO2, 37°C constant temperature incubator. Replace the cells with fresh culture medium 8-12 hours after transfection. Observe the mcherry or GFP fluorescence under a confocal microscope 48 hours after transfection. The results are as follows: Figure 1 and 2 shown.

[0061] Figure 1 and Figure 2 The results showed that the transfection effects of HPV45-A lentivirus and HPV45-B lentivirus were good, and HPV-A lentivirus and HPV-B lentivirus were obtained by collecting the viral supernatant.

[0062] (2) Construction of HPV45-A-C33A stable cell line Prepare C-33A cells (ATCC, HTB-31): Wash the cells in a T75 culture flask with 1 mL of PBS, then add 1 mL of trypsin for 2 min, then add twice the volume of culture medium to terminate the digestion, transfer to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. After centrifugation, remove the supernatant and add 1 mL of culture medium to resuspend.

[0063] Cell counting: Take 10 μL of cell suspension and add 10 μL of trypan blue. After mixing, take 10 μL and add it to the cell counting plate for counting. When the cell density of the 6-well plate is 30-40%, the infection experiment is carried out and the number of cells is about 1 million.

[0064] Virus infection: Add 1 mL of culture medium to the 6-well plate as the bottom layer, then add the cell suspension calculated above for inoculation, and then add HPV45-A virus supernatant in a 1:1 volume. At the same time, add 1000× polybrene, shake gently and put the 6-well plate back into the incubator.

[0065] Drug screening: After 24h and 48h of culture, fresh culture medium was replaced and phleomycin was added for drug screening. After 72-96h of culture, fresh culture medium was replaced and phleomycin was removed. The infection status of cells was observed under a fluorescence microscope. Figure 3 The results of fluorescence microscopy observation 48 hours after infection.

[0066] Monoclonal selection: After drug screening is completed, continue to culture the remaining cells, then digest the cells with 0.25% trypsin, count the number of cells, and then adjust the cell concentration to 5 cells / mL with cell culture medium containing 10% FBS, and spread them to a 96-well plate with a volume of 100μL per well. Place the 96-well plate in a CO2 incubator and culture for about 7 to 14 days. Monoclonal formation can be seen under a microscope. Select the wells with only one monoclonal cell population and remove the remaining multiple clones or cell-free wells. Continue to culture the monoclonal wells, digest the cells after the cell population is formed, and transfer them to a 24-well plate for expansion culture. Take part of the expanded cultured cells for identification.

[0067] (3) Construction of HPV45-AB-C33A stable cell line Consistent with the above-mentioned construction of the HPV45-A-C33A stable cell line, the HPV45-B virus was infected with the identified HPV45-A-C33A cell line, and then puromycin was used for drug screening. Figure 4 The fluorescence microscopy results are shown 48 hours after infection. The cells were cultured continuously and single clones were selected. The selected cells were cultured extensively and some were taken for identification.

[0068] 3. Sanger sequencing identification The cell lines collected above were identified by Sanger sequencing. The DNA of the collected cells was extracted using the "Nucleic Acid Extraction Reagent" (Model: MEB-16C; Registration Number: Guangdong Shenxiebei 20200318) of Yaneng Biotechnology (Shenzhen) Co., Ltd. and sent to Sangon Biotechnology (Shanghai) Co., Ltd. for Sanger sequencing, using the instrument: 3730xl DNA Analyzer.

[0069] The sequence returned by sequencing was compared with the NCBI sequence to calculate its consistency and evaluate whether the HPV gene was successfully transferred into the cell line. Figure 5 As shown, the HPV45 sequencing comparison results are as follows Figure 6 All sequencing alignment results of 41 quality control products are shown in Table 2.

[0070] Table 2 Sequencing alignment results Cell Name genotype Total sequence length Compare Sequence IDs consistency Cell Name genotype Total sequence length Compare Sequence IDs consistency C-33A-H6 HPV6 7996 JN252323.1 100% C-33 A-H55 HPV55 7822 U31791.1 100% C-33A-H11 HPV11 7933 HE574705.1 100% C-33A-H56 HPV56 7866 EF177176.1 99% C-33A-H16 HPV16 7905 JQ004096.1 100% C-33A-H58 HPV58 7824 D90400.1 100% C-33A-H18 HPV18 7857 NC_001357.1 100% C-33A-H59 HPV59 7896 X77858.1 99% C-33A-H26 HPV26 7855 NC_001583.1 99% C-33A-H61 HPV61 7989 KX514419.1 100% C-33 A-H30 HPV30 7843 KF436843.1 99% C-33A-H62 HPV62 8092 KU298924.1 100% C-33A-H31 HPV31 7912 J04353.1 99% C-33A-H66 HPV66 7824 U31794.1 100% C-33 A-H33 HPV33 7909 M12732.1 99% C-33A-H67 HPV67 7803 LC664138.1 100% C-33A-H34 HPV34 7723 NC_001587.1 100% C-33A-H68a HPV68 7822 DQ080079.1 100% C-33A-H35 HPV35 7851 M74117.1 99% C-33A-H68b HPV68 7836 FR751039.1 99% C-33 A-H39 HPV39 7833 M62849.1 99% C-33 A-H69 HPV69 7705 OP711971.1 100% C-33 A-H40 HPV40 7905 LR862047.1 100% C-33 A-H70 HPV70 7905 U21941.1 100% C-33 A-H42 HPV42 7917 M73236.1 100% C-33 A-H71 HPV71 8037 MT250602.1 100% C-33 A-H43 HPV43 7975 AJ620205.1 99% C-33 A-H72 HPV72 7988 X94164.1 100% C-33 A-H44 HPV44 7833 U31788.1 100% C-33 A-H73 HPV73 7700 X94165.1 99% C-33 A-H45 HPV45 7849 KC470260.1 99% C-33 A-H81 HPV81 8070 AJ620209.1 99% C-33 A-H51 HPV51 7811 KU298905.1 99% C-33 A-H82 HPV82 7871 AB027021.1 100% C-33 A-H52 HPV52 7942 X74481.1 100% C-33 A-H83 HPV83 8104 AF151983.1 100% C-33 A-H53 HPV53 7856 X74482.1 100% C-33 A-H84 HPV84 7948 AF293960.1 100% C-33 A-H54a HPV54 7760 KF436890.1 100% C-33 A-H89 HPV89 8078 AF436128.1 99% C-33 A-H54b HPV54 7717 KF436893.1 100% The results in Table 2 show that the consistency of the 39 types is >99%, indicating that the HPV cell line was successfully constructed.

[0071] 4. Digital PCR Quantification Select a monoclonal cell line verified by Sanger sequencing, extract cell gDNA, and use digital PCR to perform quantitative detection to determine the concentration of HPV DNA in each quality control product. In this case, the digital PCR method is used to perform quantitative detection on the L1 gene of HPV, and the copy number concentration of the L1 gene in the quality control product is used as its value. Digital PCR quantitative detection includes the following steps: (1) Digital PCR primers and probes were designed according to the HPV 39 types. The sequences of digital PCR primers and probes for HPV 39 types are shown in Table 3. The 5' end of probe P was labeled with the reporter fluorescent group FAM, and the 3' end was labeled with MGB. All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0072] (2) The reaction system of digital PCR is: 2×ddPCR mix (Mg 2+ , dNTP plus, Taq, ROX, buffer) 10μL, 10μM upstream primer 0.2μL, 10μM downstream primer 0.2μL, 10μM fluorescent probe 0.2μL, 4μL DNA template, add water to make up to 20μL system.

[0073] (3) Follow the procedure to use the Sinafo Digital PCR Instrument DQ24 all-in-one to perform the reaction: 60°C stabilization droplet for 5 min, 95°C for 5 min, and then 45 cycles: 95°C denaturation for 15 sec, 60°C annealing and extension for 45 sec.

[0074] Table 3 Digital PCR primers / probes SEQ ID No. Primer / Probe Name Specific Nucleic Acid Sequence (5'-3') 1 H6-F GCCCAGGGACATAACAATGG 2 H11-F GGCTCAGGGACATAACAATGG 3 H16-F GCACAGGGCCACAATAATGG 4 H18-F GCACAGGGTCATAACAATGGT 5 H26-F CGTGCACAGGGTCATAATAATG 6 H30-F CAGGGACACAATAATGGCATTTG 7 H31-F GCTCAGGGACACAATAATGG 8 H33-F CACAAGGTCATAATAATGGTAT 9 H34-F GGCCCAGGGACAAAACAATG 10 H35-F GTGCACAAGGCCATAATAATG 11 H39-F GCCCAGGGCCACAACAATGG 12 H40-F GGCCCAGGGCCATAACAATG 13 H42-F GCACAAGGACACAATAATGG 14 H43-F GGCCCAGGGACATAATAATG 15 H44-F GCGCAGGGCCACAATAATGG 16 H45-F GGCCCAGGGCCATAACAATG 17 H51-F GCGCAGGGTCACAATAATGGC 18 H52-F GCGCAGGGCCACAATAATGGC 19 H53-F TGCCCAGGGACATAATAATGG 20 H54a-F GGCCCAGGGTCAAAACAATGG 21 H54b-F GGCCCAGGGTCAGAACAATGG 22 H55-F GCGCAGGGCCACAATAATGG 23 H56-F GCCCAAGGCCATAATAATG 24 H58-F TGCACAAGGTCATAACAATG 25 H59-F TAAACCATATTGGCTGCAC 26 H61-F ATATTGGCTGCAACGTG 27 H62-F TAAGCCATATTGGCTGCAG 28 H66-F TGCACAGGGCCATAATAAT 29 H67-F GCACAAGGTCATAACAATG 30 H68a-F AAGGCACAGGGACACAA 31 H68b-F GGCACAGGGACACAACAAT 32 H69-F TGCCCAGGGTCATAATAAT 33 H70-F GGCCCAGGGACACAATAA 34 H71-F GCACAAACAATGGCATTTG 35 H72-F AGGGTCACAACAATGGC 36 H73-F GGCACAGGGACAAAATAAT 37 H81-F GCACAGGGCCATAATAAT 38 H82-F ACAATAATGGCATTTGCTGG 39 H83-F TGCCCAGGGACATAATAAT 40 H84-F AAAGGCCCAGGGTCATAA 41 H89-F GCACAGGGCCATAATAAT 42 H6-R TCCCAAAGGATACTGATCCA 43 H11-R TCCAAGGGGAAACTGATCTA 44 H16-R CCTAAGGAAACTGATCTAGGT 45 H18-R GTCCAAGGGGATATTGATCT 46 H26-R CGCCCTAGTGGAAATTGAT 47 H30-R CCAGTGGGAATTGGTCAA 48 H31-R GCGACCCAGTGGAAACTGAT 49 H33-R GCGTCCCAAAGGAAACTGAT 50 H34-R CCTAAGGCAAACTGATCTAA 51 H35-R GGAAATTGATCTAAGTCTGCAG 52 H39-R GGGAATTGATCAAGTTCCAAAC 53 H40-R CCTAATGGAAATTGATCTAATTGG 54 H42-R CTACTAAAGGAAATTGACTTAAAT 55 H43-R GGGAAACTGGGTAAGTTGTGC 56 H44-R CTACCAAGGGGATATTGATC 57 H45-R CGACCAAGGGGATATTGATCC 58 H51-R TGCAACAAAAACTTTCGACC 59 H52-R CTACCTAAAGGAAACTGATCT 60 H53-R CCTGCCAAGAGGAAACTGATCC 61 H54a-R CTTGCGACCCAAGGGAAACTGG 62 H54b-R CTTGCGACCCAAGGGAAACTGG 63 H55-R TCCTACCAAGGGGATATTGATC 64 H56-R CCCAGTGGAAATTGATCCA 65 H58-R CTTTCGTCCCAAAGGAAA 66 H59-R TTTACGTCCCAAAGGAAAC 67 H61-R TTGCGCCCCAAAGGAAA 68 H62-R CTTACGCCCCAAAGGAAA 69 H66-R CTACCCAAAGGAAACTGA 70 H67-R TCTACCTAAGGGAAATTGATCT 71 H68a-R AATTGGTCCAGTTCAGAAC 72 H68b-R ACTGGTCCAGTTCAGAAC 73 H69-R CAGTGGAAATTGATCTAAATCAAT 74 H70-R CCAAAGGAAACTGATCTAGTT 75 H71-R GACCCAATGGAAACTGATC 76 H72-R CCTAAAGGAAACTGATCCAAG 77 H73-R AAAGGAAACTGGTCTAATTCT 78 H81-R CGACCCAAAGGAAACTGA 79 H82-R AAAGAAAAGCGTTCCTTAAGG 80 H83-R CTTACGGCCCAAAGGAAA 81 H84-R TTGCGACCCAAGGGATAC 82 H89-R CCCAATTGTAATAAAAACTTGCG 83 H6-P GAAAAATAAATTGTAAATCATACTCTTC 84 H11-P GTTACTGTGGTAGATACCACACGC 85 H16-P GTGCTGCCATATCTACTTCAGAAA 86 H18-P GTTACTGTGGTAGATACCACTCGC 87 H26-P GGAGTGGATGCAGATGCTGCAGA 88 H30-P GCAACCACACAAACGTTATCC 89 H31-P GTGCTGCAATTGCAAACAGTGATA 90 H33-P TGTCACTAGTTACTTGTGTGC 91 H34-P GTGTAGGTACACAATCCACAAGTA 92 H35-P CTGTGTGTTCTGCTGTGTCTACTA 93 H39-P CTCTATAGAGTCTTCCATACCTTCTA 94 H40-P GGTGTGGGGGACTGTGTGGC 95 H42-P GCCACTGCAACATCTGGTG 96 H43-P ACCCTACTGTGCCCAGTACA 97 H44-P GCCACTACACAGTCCCCTCC 98 H45-P GCCTCTACACAAAATCCTGTGC 99 H51-P ACTATTAGCACTGCCACTGC 100 H52-P GCACTAACATGACTTTATGTGCTGA 101 H53-P TCTGCAACCACACAGTCTATGTC 102 H54a-P GCATCCACGCAGGATAGCTTTAATA 103 H54b-P CCTCGCAGGATACGTTTAATAATGCTA 104 H55-P GTGCTGCTACAACTCAGTCTCCATC 105 H56-P CATGACTATTAGTACTGCTACAGAACAG 106 H58-P CTCGTAGCACTAATATGACATTATGCAC 107 H59-P CCTCCACATGTCTGGCATATTC 108 H61-P CTACATCCCCCCCTGTATCTG 109 H62-P AACTTTAGGGAATTTTTGCGACACA 110 H66-P GCAGCTAAAAGCACATTAACTAAATATG 111 H67-P CAGAGGCTACATACAAAAATGAAAAC 112 H68a-P CTACTGTACCAGCTGTGTATGATTCTA 113 H68b-P GGTACAGCTGATTCAGTAGTAGTAGAC 114 H69-P AGTACTGTATCTGCACAATCTGC 115 H70-P GTCTGCCTGCACCGAAACG 116 H71-P GAGTCTACATATAAAGCCTCTAGTTTCATG 117 H72-P GCTTCTAATTTTCGTGAGTATCTTCGCC 118 H73-P GCTAGTAGCTCTACTACAACGTATGCC 119 H81-P CTGCTGCAGAATACAAGGCCTCTAAC 120 H82-P ATTAGCACTGCTGTTACTCCATCTGTT 121 H83-P ACACAGGCTAATGAATACACAGCCTC 122 H84-P CCAACACCGAATCAGAATATAAACCTACC 123 H89-P CTGGCACAGAATACAGTTCTACACGC After the reaction, the instrument takes a picture of each chip, and then uses the software SightPro for digital analysis. The HPV45 digital PCR results are as follows: Figure 7 As shown, the copy number quantification results of 41 quality control products using digital PCR absolute quantification method are shown in Table 4.

[0075] Table 4 Digital PCR quantitative results Cell Name genotype Dilution multiple Fixed value results Cell Name genotype Dilution multiple Fixed value results C-33A-H6 HPV6 1000 499 copies / μL C-33 A-H55 HPV55 1000 356 copies / μL C-33A-H11 HPV11 1000 802 copies / μL C-33A-H56 HPV56 1000 354 copies / μL C-33A-H16 HPV16 1000 238 copies / μL C-33A-H58 HPV58 1000 139 copies / μL C-33A-H18 HPV18 1000 166 copies / μL C-33 A-H59 HPV59 1000 417 copies / μL C-33 A-H26 HPV26 1000 226 copies / μL C-33 A-H61 HPV61 1000 223 copies / μL C-33 A-H30 HPV30 1000 826 copies / μL C-33 A-H62 HPV62 1000 153 copies / μL C-33 A-H31 HPV31 1000 842 copies / μL C-33 A-H66 HPV66 1000 394 copies / μL C-33 A-H33 HPV33 1000 684 copies / μL C-33 A-H67 HPV67 1000 245 copies / μL C-33 A-H34 HPV34 1000 218 copies / μL C-33 A-H68a HPV68 1000 444 copies / μL C-33 A-H35 HPV35 1000 541 copies / μL C-33 A-H68b HPV68 1000 212 copies / μL C-33 A-H39 HPV39 1000 128 copies / μL C-33 A-H69 HPV69 1000 226 copies / μL C-33 A-H40 HPV40 1000 223 copies / μL C-33 A-H70 HPV70 1000 149 copies / μL C-33 A-H42 HPV42 1000 122 copies / μL C-33 A-H71 HPV71 1000 299 copies / μL C-33 A-H43 HPV43 1000 185 copies / μL C-33 A-H72 HPV72 1000 425 copies / μL C-33 A-H44 HPV44 1000 616 copies / μL C-33 A-H73 HPV73 1000 539 copies / μL C-33 A-H45 HPV45 1000 169 copies / μL C-33 A-H81 HPV81 1000 793 copies / μL C-33 A-H51 HPV51 1000 684 copies / μL C-33 A-H82 HPV82 1000 658 copies / μL C-33 A-H52 HPV52 1000 261 copies / μL C-33 A-H83 HPV83 1000 283 copies / μL C-33 A-H53 HPV53 1000 668 copies / μL C-33 A-H84 HPV84 1000 356 copies / μL C-33 A-H54a HPV54 1000 505 copies / μL C-33 A-H89 HPV89 1000 461 copies / μL C-33 A-H54b HPV54 1000 243 copies / μL 5. Application of HPV quality control products In this case, the human papillomavirus genotyping (type 23) detection kit (PCR-reverse dot hybridization method) (registration certificate number: National Medical Device No. 20193401918, manufacturer: Yaneng Biotechnology (Shenzhen) Co., Ltd.), human papillomavirus genotyping detection kit (PCR-reverse dot hybridization method) (registration certificate number: National Medical Device No. 20173400638, manufacturer: Guangzhou Anping Pharmaceutical Technology Co., Ltd.) and human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method) (registration certificate number: National Medical Device No. 20173404681, manufacturer: Yaneng Biotechnology (Shenzhen) Co., Ltd.) were used to test the quality control products prepared above to evaluate their application effects.

[0076] (1) Test each type of quality control product according to the instructions of the Yaneng Bio Kit Human Papillomavirus Genotyping (Type 23) Detection Kit (PCR-Reverse Dot Blot Method) and the Anbiping Kit Human Papillomavirus Genotyping Detection Kit (PCR-Reverse Dot Blot Method). The test results are as follows: Figures 9 to 12 And as shown in Table 5.

[0077] Table 5 PCR reverse dot hybridization test results Cell name Genotype Yanen result Anbiping result Cell name genotype Asian Energy Results Anbi Ping Results C-33A-H6 HPV6 + + C-33 A-H55 HPV55 / / C-33A-H11 HPV11 + + C-33A-H56 HPV56 + + C-33A-H16 HPV16 + + C-33A-H58 HPV58 + + C-33A-H18 HPV18 + + C-33A-H59 HPV59 + + C-33A-H26 HPV26 / + C-33A-H61 HPV61 / + C-33 A-H30 HPV30 / / C-33A-H62 HPV62 / / C-33A-H31 HPV31 + + C-33A-H66 HPV66 + + C-33 A-H33 HPV33 + + C-33A-H67 HPV67 / / C-33A-H34 HPV34 / / C-33A-H68a HPV68 + + C-33A-H35 HPV35 + + C-33A-H68b HPV68 + + C-33A-H39 HPV39 + + C-33A-H69 HPV69 / / C-33 A-H40 HPV40 / + C-33A-H70 HPV70 / / C-33A-H42 HPV42 + + C-33A-H71 HPV71 / / C-33A-H43 HPV43 + / C-33A-H72 HPV72 / / C-33A-H44 HPV44 / / C-33A-H73 HPV73 + + C-33A-H45 HPV45 + + C-33A-H81 HPV81 + + C-33A-H51 HPV51 + + C-33A-H82 HPV82 + + C-33A-H52 HPV52 + + C-33A-H83 HPV83 + + C-33 A-H53 HPV53 + + C-33A-H84 HPV84 / / C-33A-H54a HPV54 / + C-33A-H89 HPV89 / / C-33 A-H54b HPV54 / + In the table, “+” represents a positive test result and “ / ” represents a result that is not within the detection range of the kit.

[0078] Figures 9 to 12 The results in Table 5 show that it can be observed with the naked eye that each subtype of the quality control product can be detected within the detection range of the kit.

[0079] (2) Test the reference material according to the instructions of the Human Papillomavirus Nucleic Acid Detection Kit (PCR-fluorescent probe method), using the human papillomavirus whole genome typing reference material HPV16, HPV18 and HPV45 developed by the China Food and Drug Inspection Institutes as the control, and test three concentrations, 10 4 copies / mL, 10 5 copies / mL, 10 6 The corresponding subtype quality control products were tested, and each subtype was repeated 3 times. The fluorescence curve results were as follows: Figure 8 The average Ct values ​​of each test sample are shown in Table 6.

[0080] Table 6 Summary results of fluorescence PCR detection Cell Name genotype Average Ct value Cell Name genotype Average Ct value C-33A-H16 HPV16 26.26 C-33A-H52 HPV52 26.24 C-33A-H18 HPV18 26.85 C-33 A-H53 HPV53 25.32 C-33A-H26 HPV26 26.53 C-33A-H56 HPV56 25.98 C-33A-H31 HPV31 25.11 C-33A-H58 HPV58 26.97 C-33 A-H33 HPV33 25.39 C-33A-H59 HPV59 25.80 C-33A-H35 HPV35 25.62 C-33A-H66 HPV66 25.83 C-33A-H39 HPV39 26.83 C-33A-H68a HPV68 25.72 C-33A-H45 HPV45 26.90 C-33A-H68b HPV68 26.59 C-33A-H51 HPV51 25.37 C-33A-H82 HPV82 25.34 The results showed that the human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method) can effectively detect the corresponding subtypes with an accuracy rate of 100%.

[0081] From the above test results, it can be seen that the detection results of different platforms are consistent with the expected design, indicating that the HPV whole genome stably transformed cell line quality control product prepared in this example has multi-platform compatibility.

[0082] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A method for preparing a quality control product for human papillomavirus nucleic acid detection, characterized in that: The steps include: The whole HPV genome was divided into two segments, namely HPV-A and HPV-B, and HPV-A and HPV-B were cloned into vectors to obtain HPV-A recombinant plasmid and HPV-B recombinant plasmid respectively; HPV-A recombinant plasmid and HPV-B recombinant plasmid were used to transfect cells with lentiviral plasmids, respectively, to obtain HPV-A lentivirus and HPV-B lentivirus by packaging; Host cells were infected with HPV-A lentivirus, and HPV-A cell lines were obtained through drug screening and monoclonal screening; The HPV-A cell line is infected with the HPV-B lentivirus, and a cell line containing both HPV-A and HPV-B is obtained through drug screening and monoclonal screening, namely the human papillomavirus nucleic acid detection quality control product.

2. The preparation method according to claim 1, characterized in that: The HPV full genome includes 39 HPV genotypes, specifically, 18 HPV high-risk types HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82; and 21 HPV low-risk types HPV 6, 11, 30, 34, 40, 42, 43, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, 81, 83, 84, 89; and, for HPV54 and 68, there are two subtypes, a and b.

3. The preparation method according to claim 1, characterized in that: The HPV-A and HPV-B are combined into the HPV full genome, the HPV-A contains early genes, and the HPV-B contains at least other sequences in the HPV full genome except HPV-A.

4. The preparation method according to claim 1, characterized in that: The HPV-A recombinant plasmid and the HPV-B recombinant plasmid use different screening genes.

5. The preparation method according to claim 1, characterized in that: HPV-A and HPV-B were constructed into PCDH-mcherry-zeocin and PCDH-CMV-EGFP-puro, respectively.

6. The preparation method according to claim 1, characterized in that: The lentiviral plasmid includes psPAX2 and pMD2G.

7. The preparation method according to claim 1, characterized in that: The host cell is a human cervical cancer cell line or a human ovarian cancer cell line.

8. The preparation method according to claim 1, characterized in that: Screening for HPV-A cell lines and screening for cell lines containing both HPV-A and HPV-B are performed using different drugs.

9. The preparation method according to claim 8, characterized in that: The HPV-A cell line is screened and the cell line containing both HPV-A and HPV-B is screened, and the screening drugs used in the two are non-repetitive and are selected from at least one of phleomycin, puromycin, neomycin, hygromycin B and blasticidin.

10. The preparation method according to any one of claims 1 to 9, characterized in that: The method also includes performing Sanger sequencing on the cell line containing both HPV-A and HPV-B to ensure that the entire HPV genome is successfully recombined into the cell line.

11. The preparation method according to any one of claims 1 to 9, characterized in that: It also includes using specific primers and probes to quantitatively detect at least one gene in the HPV genome of the quality control product, and using the quantitative detection result of the gene as the concentration of the quality control product.

12. The preparation method according to claim 11, characterized in that: The L1 gene in the HPV whole genome was quantitatively detected, and the copy number of the L1 gene was used as the concentration of the quality control product.

13. The preparation method according to claim 12, characterized in that: The upstream primers for quantitative detection of HPV6, HPV11, HPV16, HPV18, HPV26, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54a, HPV54b, HPV55, HPV56, HPV58, HPV59, HPV61, HPV62, HPV66, HPV67, HPV68a, HPV68b, HPV69, HPV70, HPV71, HPV72, HPV73, HPV81, HPV82, HPV83, HPV84 and HPV89 are shown in SEQ ID No.1-41 in sequence, and the downstream primers are shown in SEQ ID No.1-41 in sequence. The sequences shown in SEQ ID No.42-82, and the probes are the sequences shown in SEQ ID No.83-123 in sequence.

14. A quality control product for human papillomavirus nucleic acid detection obtained by the preparation method according to any one of claims 1 to 13.

15. Use of the human papillomavirus nucleic acid detection quality control product according to claim 14 in the preparation of an HPV nucleic acid detection kit.

16. An HPV nucleic acid detection kit comprising the human papillomavirus nucleic acid detection quality control product according to claim 14.

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