A quality control product for human papilloma virus molecular detection and a preparation method thereof

Abstract

The application discloses a quality control product for human papillomavirus molecular detection and a preparation method thereof, and belongs to the technical field of biological products. After synthesizing 25 types of HPV full genomes of high-risk types (HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV82) and low-risk types (HPV6, HPV11, HPV42, HPV43, HPV44, HPV81, HPV83), the HPV full genomes are connected into a pcDNA3.1(+) skeleton vector system, the HPV full genomes are integrated into the genome of a human ovarian cancer cell line ovcar3 respectively, and 25 strains of human ovarian cancer cell lines stably integrating the HPV full genomes are obtained. The new quality control product or kit of various types of simulated clinical samples such as genomic DNA, cell precipitate and cell suspension can be prepared and is suitable for various detection platforms. The human cell line quality control product stably integrating the full-length genomes of the 25 HPV types can more accurately determine the viral load and control the whole process of HPV detection.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically, it relates to a quality control product for molecular detection of human papillomavirus and its preparation method. Background Technology

[0002] Human papillomavirus (HPV) belongs to the Papillomaviridae family. It is a non-enveloped circular double-stranded DNA virus composed of a DNA core and a protein capsid. The capsid is composed of a major capsid protein (L1) and a minor capsid protein (L2). The genome is approximately 8000 base pairs (bp) long and is divided into three functional regions: the early transcription region (E region, encoding early proteins such as e1, e2, e4, e5, e6, and e7, which are involved in viral replication, transcription, translation regulation, and transformation); the late transcription region (L region, encoding capsid proteins L1 and L2); and the non-transcriptional region (long control region, LCR, containing the origin of DNA replication and regulatory elements required for expression, regulating viral transcription and replication). More than 200 species of HPV have been reported and can infect humans through direct or indirect contact with contaminated objects or sexual transmission. Based on the severity of HPV infection, HPV is classified into high-risk types (HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV82), low-risk types (HPV6, HPV11, HPV42, HPV43, HPV44, HPV81, HPV83), and other subtypes. Epidemiological studies show that persistent infection with high-risk HPV in normal cervical epithelial cells is one of the main causes of cervical and rectal cancer, while low-risk types are associated with warts, flat warts, and plantar warts.

[0003] Early HPV detection, especially accurate genotyping, is crucial for the early detection, prevention, early warning, and treatment of cervical cancer. Clinical HPV testing primarily relies on viral nucleic acid detection, with specific methods including PCR-reverse dot blot hybridization, PCR-fluorescent probe method, PCR-flow hybridization, and hybridization capture-chemiluminescence. A variety of testing reagents exist, each with different principles and characteristics, and varying performance indicators such as analytical sensitivity and specificity. To improve the reliability and consistency of laboratory test results and those from different reagent kits, inter-laboratory quality assessment should be conducted to compare HPV testing or genotyping results, monitor the testing capabilities of different platforms and laboratories, and improve the accuracy of result reporting.

[0004] Calibrators and quality control materials are essential tools for achieving accurate and consistent results in in vitro diagnostic clinical testing and monitoring, and also serve as physical metrological standards to ensure the transfer of measurement values. The international HPV standard material is derived from recombinant HPV genomic plasmids diluted with HPV-negative genomic DNA extracted from human cervical cell lines. Meanwhile, currently available HPV quality control materials are also plasmid-derived, some obtained by diluting plasmids with human genomic DNA. These standards cannot monitor sample processing procedures and exhibit significant matrix differences from clinical samples, resulting in a limited range of forms. Furthermore, in clinical testing, HPV viral load is a crucial reference indicator for evaluating the severity of cervical lesions, further reflecting the true degree of viral infection (HPV viral load is the number of HPV copies per unit of infected epithelial cells). Studies have shown that different HPV genotypes and viral loads are important predictors of CIN2+ and CIN3+. Identifying patients with a high risk of CIN2+ and CIN3+ based on HPV genotype and viral load is crucial for developing individualized triage and follow-up strategies. Quality control samples prepared in plasmid form do not have the ability to detect viral load. Summary of the Invention

[0005] 1. The problem to be solved

[0006] To address the limitations of the aforementioned standards / quality control products in practical applications, this invention proposes a novel quality control product: a human cell line quality control product with stable integration of the full-length genomes of 25 HPV genotypes. This product can more accurately determine viral load and control the entire HPV detection process. Furthermore, the quality control product and kit prepared for HPV genotyping can be prepared in various forms such as gDNA, cell precipitate, and cell suspension, making it suitable for multiple detection platforms.

[0007] 2. Technical Solution

[0008] To solve the above problems, the present invention adopts the following technical solution.

[0009] A quality control product for molecular detection of human papillomavirus, wherein the quality control product is prepared from a human ovarian cancer cell line containing the complete HPV genome.

[0010] Of the quality control materials described above for the molecular detection of human papillomavirus,

[0011] The human ovarian cancer cell line containing the complete HPV genome was obtained by integrating the complete HPV genome into the genome of the human ovarian cancer cell line ovcar3.

[0012] Of the quality control materials described above for the molecular detection of human papillomavirus,

[0013] The HPV whole genome is linked into the pcDNA3.1(+) backbone vector system.

[0014] Of the quality control materials described above for the molecular detection of human papillomavirus,

[0015] The HPV genome includes both high-risk and low-risk HPV genomes.

[0016] Of the quality control materials described above for the molecular detection of human papillomavirus,

[0017] The high-risk HPV genome includes HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82.

[0018] Of the quality control materials described above for the molecular detection of human papillomavirus,

[0019] The low-risk HPV genome includes HPV6, HPV11, HPV42, HPV43, HPV44, HPV81, and HPV83.

[0020] Of the quality control materials described above for the molecular detection of human papillomavirus,

[0021] The quality control materials include genomic DNA, cell pellets, and cell suspensions.

[0022] A method for preparing a quality control sample for molecular detection of human papillomavirus includes the following steps:

[0023] (1) Synthesis of HPV genome sequence;

[0024] (2) Transfection of HPV plasmids;

[0025] (3) Screening and identification of HPV monoclonal antibodies;

[0026] (4) Preparation of quality control samples with different HPV copy number gradients.

[0027] In the above-described method for preparing quality control materials for molecular detection of human papillomavirus,

[0028] In step (1), restriction enzyme sites are added to both ends of the HPV genome sequence, with NheI at the 5' end and PmeI at the 3' end.

[0029] The transfection method in step (2) is as follows:

[0030] By ligating NheI and PmeI restriction enzyme sites into the pcDNA3.1(+) backbone vector, 25 HPV genotyping plasmids, HPVx-pcDNA3.1(+), were obtained, where x represents a specific HPV genotype. These 25 plasmids were then transfected into ovcar3 cells using the following transfection system:

[0031] buffer: 75μL

[0032] HPVx-pcDNA3.1(+): 1 μg,

[0033] reagent: 3.6 μL

[0034] Mix the above-mentioned system thoroughly and let it stand at room temperature for 10 minutes. Add the mixed system to a 12-well plate pre-inoculated with ovcar3 cell line and incubate in an incubator for 4-6 hours, then replace with normal culture medium.

[0035] In the above-described method for preparing quality control materials for molecular detection of human papillomavirus,

[0036] The specific methods for monoclonal screening and identification in step (3) are as follows:

[0037] 24 hours after transfection, the cell culture medium was changed to G418 medium containing antibiotics for cell selection. After selection, the remaining cells were cloned into single cells. The cells were digested and collected, and the cells were accurately counted according to the serial dilution method. The viable cells were diluted with culture medium to 5 cells / mL, and the cells were thoroughly mixed. 10 mL of cell suspension was taken and evenly distributed into a 96-well plate, 0.1 mL per well. The cells were placed in a CO2 incubator and cultured at 37°C. When cell clones formed, the wells marked with clones were digested with trypsin. A portion of the cells was used as amplification templates, and the clones were amplified and verified by Sanger sequencing using primers. The remaining cells were expanded and cultured for later use.

[0038] The preparation method in step (4) is as follows:

[0039] Clones validated by Sanger sequencing of the entire HPV genome were selected, and the clones were expanded and cultured. gDNA was extracted, and the copy number of HPV in the five selected clones was detected by digital PCR. Gradient quality control samples with different copy numbers were prepared with ddPCR as a reference.

[0040] Using the five types of HPV-integrated cells from step (4) above, cell suspensions, cell pellets, or gDNA-based quality control products were prepared, with HPV copy numbers of 1E+02, 1E+03, 1E+04, and 1E+05, respectively.

[0041] 3. Beneficial effects

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] Currently available HPV quality control products are all plasmid-derived, with a few obtained by diluting plasmids with human genome. These quality control products differ significantly from clinical samples and do not require nucleic acid extraction, thus failing to provide quality control over the entire HPV testing process. In contrast, the quality control product provided in this invention has the following advantages:

[0044] (1) Achieve integration of the entire HPV genome into the human genome. The HPV genome is stably integrated into human cell lines, posing no risk of biological infection. Stable integration into immortalized cells can simulate clinical samples, including sample preservation and nucleic acid extraction, enabling quality control of the entire HPV testing process;

[0045] (2) Contains HPV whole genome information, covering all detection sites of the kit, enabling full HPV genotyping detection and achieving qualitative and quantitative detection;

[0046] (3) The product contains 25 subtypes, with a wide copy number coverage, and can prepare different copy number gradients to meet the testing and daily quality control needs of the detection limit;

[0047] (4) It can prepare quality control products in the form of gDNA, cell precipitate or cell solution, and is compatible with a variety of detection platforms. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the HPV transgenic plasmid in this invention;

[0049] Figure 2 This is a linear analysis graph of Ct values ​​for different copy number gradient quality control samples in the detection platform of Anbiping Human Papillomavirus Nucleic Acid Detection Kit - HPV18 Fluorescent PCR Detection Kit of the present invention.

[0050] Figure 3 This is a linear analysis graph showing the Ct values ​​of different copy number gradient quality control samples in this invention and their corresponding values ​​on the Shanghai Transview Human Papillomavirus Nucleic Acid Detection and 16 / 18 Genotyping Kit (Fluorescent PCR Method) detection platform. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments.

[0052] The comprehensive technical solution of the present invention is as follows:

[0053] A quality control product for molecular detection of human papillomavirus, wherein the quality control product is prepared from a human ovarian cancer cell line containing the complete HPV genome.

[0054] Of the quality control materials mentioned above for the molecular detection of human papillomavirus,

[0055] The human ovarian cancer cell line containing the complete HPV genome was obtained by integrating the complete HPV genome into the genome of the human ovarian cancer cell line ovcar3.

[0056] Of the quality control materials mentioned above for the molecular detection of human papillomavirus,

[0057] The HPV whole genome is linked into the pcDNA3.1(+) backbone vector system.

[0058] Of the quality control materials mentioned above for the molecular detection of human papillomavirus,

[0059] The HPV genome includes both high-risk and low-risk HPV genomes.

[0060] Of the quality control materials mentioned above for the molecular detection of human papillomavirus,

[0061] The high-risk HPV genome includes HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82.

[0062] Of the quality control materials mentioned above for the molecular detection of human papillomavirus,

[0063] The low-risk HPV genome includes HPV6, HPV11, HPV42, HPV43, HPV44, HPV81, and HPV83.

[0064] Of the quality control materials mentioned above for the molecular detection of human papillomavirus,

[0065] The quality control materials include genomic DNA, cell pellets, and cell suspensions.

[0066] The method for preparing quality control materials for molecular detection of human papillomavirus includes the following steps:

[0067] (1) Synthesis of HPV genome sequence;

[0068] (2) Transfection of HPV plasmids;

[0069] (3) Screening and identification of HPV monoclonal antibodies;

[0070] (4) Preparation of quality control samples with different HPV copy number gradients.

[0071] In the above-described method for preparing quality control materials for molecular detection of human papillomavirus,

[0072] In step (1), restriction enzyme sites are added to both ends of the HPV genome sequence, with NheI at the 5' end and PmeI at the 3' end.

[0073] The transfection method in step (2) is as follows:

[0074] By ligating NheI and PmeI restriction enzymes into the pcDNA3.1(+) backbone vector, 25 HPV genotyping plasmids, HPVx-pcDNA3.1(+), were obtained, where x represents a specific HPV genotype. These 25 plasmids were then transfected into ovcar3 cells using the following transfection system:

[0075] buffer: 75μL

[0076] HPVx-pcDNA3.1(+): 1 μg,

[0077] reagent: 3.6 μL

[0078] Mix the above-mentioned system thoroughly and let it stand at room temperature for 10 minutes. Add the mixed system to a 12-well plate pre-inoculated with ovcar3 cell line and incubate in an incubator for 4-6 hours, then replace with normal culture medium.

[0079] In the above-described method for preparing quality control materials for molecular detection of human papillomavirus,

[0080] The specific methods for monoclonal screening and identification in step (3) are as follows:

[0081] 24 hours after transfection, the cell culture medium was changed to G418 medium containing antibiotics for cell selection. After selection, the remaining cells were cloned into single cells. The cells were digested and collected, and the cells were accurately counted according to the serial dilution method. The viable cells were diluted with culture medium to 5 cells / mL, and the cells were thoroughly mixed. 10 mL of cell suspension was taken and evenly distributed into a 96-well plate, 0.1 mL per well. The cells were placed in a CO2 incubator and cultured at 37°C. When cell clones formed, the wells marked with clones were digested with trypsin. A portion of the cells was used as amplification templates, and the clones were amplified and verified by Sanger sequencing using primers. The remaining cells were expanded and cultured for later use.

[0082] The preparation method in step (4) is as follows:

[0083] Clones validated by Sanger sequencing of the entire HPV genome were selected, and the clones were expanded and cultured. gDNA was extracted, and the copy number of HPV in the five selected clones was detected by digital PCR. Gradient quality control samples with different copy numbers were prepared with ddPCR as a reference.

[0084] Example

[0085] The complete genomes of 25 HPV genotypes (high-risk (HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, HPV82) and low-risk (HPV6, HPV11, HPV42, HPV43, HPV44, HPV81, HPV83) were chemically synthesized and then ligated into the pcDNA3.1(+) backbone vector system. The HPV genomes were then integrated into the genome of the human ovarian cancer cell line ovcar3, resulting in 25 stable human ovarian cancer cell lines with integrated HPV genomes. Novel quality control products, such as genomic DNA, cell pellets, and cell suspensions, can be prepared to simulate various clinical samples.

[0086] Specifically, the quality control products and kits prepared based on the above 25 HPV genotypes can be prepared in various forms such as gDNA, cell precipitate, and cell suspension, and are suitable for various detection platforms.

[0087] The specific experimental plan is as follows:

[0088] Genome sequences and chemical synthesis of 25 HPV subtypes

[0089] Using NCBI sequences as a reference, the following sequences were chemically synthesized: low-risk HPV11 (M14119.1), HPV42 (M73236), HPV43 (AJ620205), HPV44 (U31788.1), HPV6b (X00203.1), HPV81 (AJ620209), HPV83 (AF151983.1); high-risk HPV16 (K02718.1), HPV18 (X05015.1), HPV26 (NC_001583), HPV31 (J04353.1), HPV33 (M12732.1), and HPV35H (X7 HPV39(M62849.1), HPV45(X74479.1), HPV51(M62877.1), HPV52(X74481.1), HPV53(NC_001593.1), HPV56(X74483.1), HPV58(D90400.1), HPV59(X77858.1), HPV66(U31794), HPV68a(DQ080079.1), HPV73(X94165), HPV82(AB027021) were synthesized with restriction enzyme sites (5-NheI, 3-PmeI) added to both ends of the sequence.

[0090] Construction of 25 HPV subtype plasmids

[0091] The chemically synthesized sequence was ligated into a pcDNA3.1(+) backbone vector (purchased from Invitrogen, catalog number: V79520) via 5-NheI and 3-PmeI restriction sites, respectively (vector schematic shown). Figure 1 As shown in the figure, the final vector was named HPVx-pcDNA3.1(+), where x represents the specific HPV genotype.

[0092] Transfection with 25 HPV subtypes

[0093] The constructed HPVx-pcDNA3.1(+) plasmid was transfected into the ovcar3 cell line (purchased from AT CC, catalog number: FS-0329) using liposomes. The specific procedure was as follows: Follow the instructions for use of the transfection reagent (Polyplus).

[0094] The configuration system is as follows:

[0095] buffer: 75μL

[0096] HPVx-pcDNA3.1(+): 1μg

[0097] reagent: 3.6 μL

[0098] Mix the above system thoroughly and let it stand at room temperature for 10 minutes. Add the mixed system to a 12-well plate pre-inoculated with ovcar3 cell line, incubate in an incubator for 4-6 hours, and then replace with normal culture medium.

[0099] Screening and identification of 25 HPV subtypes using monoclonal antibodies (full-length amplification)

[0100] (1) 24 hours after transfection, the cell culture medium was replaced with a medium containing antibiotics (G418, 1ug / ml) for cell screening.

[0101] (2) After screening, the remaining cells were cloned into single cells: a) Digest and collect the cells. b) Count the cells accurately according to the serial dilution method, dilute the live cells to 5 cells / mL with culture medium, and mix the cells thoroughly. c) Take 10 mL of cell suspension and evenly distribute it into a 96-well plate, 0.1 mL per well.

[0102] (3) Place the cells in a carbon dioxide incubator and incubate at 37°C. When cell clones are formed, add a small amount of trypsin (about 10uL) to the wells marked with clones to digest the cells. Take a portion of the cells as amplification templates and use the primers in Table 1 to amplify the clones and verify them with Sanger sequencing. Expand the remaining cells for later use.

[0103] Table 1 Primers for cell clone identification

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Preparation of HPV copy number gradient quality control samples (The following examples select five representative HPV genotypes to illustrate the preparation and application of quality control samples)

[0111] Clones validated by Sanger sequencing of the entire HPV genome were selected. These clones were then cultured and gDNA was extracted. Digital PCR (ddPCR) was used to determine the HPV copy number in five selected clones. Gradient quality control samples with different copy numbers were prepared based on the copy number determined by ddPCR. The ddPCR detection included the following steps:

[0112] Prepare the reaction premix as shown in Table 2:

[0113] Table 2. Preparation of ddPCR reaction system

[0114]

[0115] The primer and probe sequences used for HPV copy number identification are shown in Table 3.

[0116] Table 3 Primer and probe sequences used for HPV copy number identification

[0117]

[0118] Droplet generation

[0119] Add the premixed solution prepared above to the middle well of the droplet generating card (purchased from Bio-Rad, part number: 1864008), and add the droplet generating oil to the lower well. Cover both sides of the droplet generating card with rubber sleeves, place the droplet generating card in the droplet generator, close the cap, and wait for the droplet generation to complete.

[0120] Droplet transfer

[0121] Use a pipette to transfer the generated droplets into a 96-well PCR reaction plate, keeping the pipette tip at a certain angle to prevent the bottom of the card from clogging the tip;

[0122] sealing film

[0123] After all the samples have been transferred by droplets, place an aluminum film on the surface of the 96-well plate and seal it with a heat sealer.

[0124] PCR amplification

[0125] Place the 96-well plate on the PCR instrument and close the lid tightly. Set the instrument's heating / cooling rate to 2-3℃ / s and run it according to the following procedure (Table 4):

[0126] Table 4

[0127]

[0128] Signal collection

[0129] Once the PCR program is complete, transfer the 96-well plate to the QX200 droplet reader. On the computer, open the "QuantaSoft" software, select the wells containing the samples, and make the necessary settings.

[0130] Data Analysis

[0131] After the data reading is complete, open the experimental data to be analyzed and select "Analyze" to analyze the results (as shown in Table 5).

[0132] Table 5. Copy number detection results of five HPV subtype cell lines

[0133] HPV subtype cell lines Copy number HPV16 12 HPV18 16 HPV52 28 HPV58 20 HPV44 36

[0134] Quality control products with different copy number gradients

[0135] The above five types of HPV-integrated cells were prepared into cell suspensions, cell pellets, or gDNA-based quality control products. The product specifications included 1E+02, 1E+03, 1E+04, and 1E+05 HPV gene copies, and were stored at -20℃ for later use.

[0136] HPV quality control product testing platform compatibility test

[0137] Different detection platforms use different extraction methods. Commonly used methods include boiling lysis and sample release agents. For the cell solutions and cell pellets of the different copy number gradient quality control samples prepared above, gDNA extraction was performed using the boiling lysis method or Anbiping sample release agent. Specific extraction methods are detailed in the kit instructions. The extracted gDNA was used for the corresponding downstream detection steps. Quality control samples in gDNA form were used directly without extraction.

[0138] Compatibility test of fluorescent PCR method

[0139] Fluorescent PCR DNA detection is the most effective and accurate method for early detection of cervical cancer among HPV DNA detection technologies. It has many advantages such as high sensitivity, low cost, and simple operation, and is a commonly used detection method in reagent kit design.

[0140] The quality control samples with different copy number gradients prepared above were tested using the Anbiping Human Papillomavirus Nucleic Acid Detection Kit—HPV18 Fluorescent PCR Detection Kit (Guangzhou Anbiping Pharmaceutical Technology). The results are shown in Table 6.

[0141] Table 6. Results of HPV18 Fluorescent PCR Detection Kit (Guangzhou Anbiping Pharmaceutical Technology)

[0142] Classification Concentration copies / ml Ct mean Detection rate CV, % 16 1.00E+05 30.22 4 / 4 0.28 16 1.00E+04 33.54 4 / 4 0.43 16 1.00E+03 36.69 4 / 4 1.07 16 1.00E+02 39.11 4 / 4 1.00 18 1.00E+05 28.87 4 / 4 0.45 18 1.00E+04 31.98 4 / 4 0.53 18 1.00E+03 35.84 4 / 4 2.56 18 1.00E+02 39.45 4 / 4 1.00 52 1.00E+05 30.65 4 / 4 1.56 52 1.00E+04 33.97 4 / 4 0.35 52 1.00E+03 36.98 4 / 4 0.89 52 1.00E+02 39.78 4 / 4 3.27 58 1.00E+05 29.04 4 / 4 0.34 58 1.00E+04 32.2 4 / 4 0.54 58 1.00E+03 35.72 4 / 4 1.27 58 1.00E+02 39.7 4 / 4 1.98 44 1.00E+05 28.79 4 / 4 0.53 44 1.00E+04 32 4 / 4 0.71 44 1.00E+03 35.15 4 / 4 1.96 44 1.00E+02 38.12 4 / 4 2.33

[0143] As shown in Table 6, the quality control samples at 1E+02, 1E+03, 1E+04, and 1E+05 copies / mL were all effectively detected in the fluorescence PCR detection platform of Guangzhou Anbiping Pharmaceutical Technology Co., Ltd. after DNA extraction, with a detection rate of 100%. This indicates that the quality control samples can be well used for quality control in the DNA extraction and fluorescence PCR detection process.

[0144] Figure 2 In the study, a strong linear correlation was observed between the concentration of the quality control sample and the Ct value of the fluorescent PCR, meaning that a higher concentration of the quality control sample resulted in a lower Ct value for the PCR. These results indicate that the quality control sample exhibits excellent performance and can effectively control the DNA extraction and fluorescent PCR detection processes.

[0145] The human papillomavirus (HPV) nucleic acid detection and 16 / 18 genotyping kit (fluorescent PCR method) (Shanghai TransGen Biotech Co., Ltd.) was used to detect the different copy number gradient quality control samples prepared above. The results are as follows (Table 7):

[0146] Table 7. Results of Human Papillomavirus Nucleic Acid Detection and 16 / 18 Genotyping Kit (Fluorescent PCR Method)

[0147]

[0148]

[0149] As shown in Table 7, the quality control samples at 1E+02, 1E+03, 1E+04, and 1E+05 copies / mL were all effectively detected in the fluorescent PCR detection platform of Shanghai TransGen Biotech Co., Ltd. after DNA extraction, with a detection rate of 100%. This indicates that the quality control samples can be well used for quality control in the DNA extraction and fluorescent PCR detection process.

[0150] The results showed that different copy number gradient quality control samples were linearly correlated with the results of quantitative fluorescence CT value determination. Figure 2 , Figure 3 The reported results showed no difference between different test kits. Furthermore, a low concentration (1.00E+02 copies) of the quality control sample could control the limit of detection of the test kit.

[0151] PCR reverse dot blot compatibility test

[0152] PCR-reverse dot blot hybridization is a method for genotyping and detecting gene mutations by hybridizing biotin-labeled specific PCR amplification products with probes immobilized on a membrane and developing a colorimetric result. Using a human papillomavirus genotyping kit (PCR-reverse dot blot hybridization) (KAP Biotechnology), and following the kit's instructions, the results are shown in Table 8 below:

[0153] Table 8

[0154]

[0155]

[0156] The test results show that the test results on different platforms are consistent with the expected design, indicating that the HPV integrated cell line quality control product has multi-platform compatibility.

[0157] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for preparing a quality control sample for molecular detection of human papillomavirus, characterized in that, Includes the following steps: (1) Synthesize the HPV genome sequence and add restriction enzyme sites to both ends of the HPV genome sequence, wherein the restriction enzyme site at the 5' end is NheI and the restriction enzyme site at the 3' end is PmeI; (2) The HPV genome sequence was ligated into the pcDNA3.1(+) backbone vector through the restriction enzyme sites NheI and PmeI to obtain the HPV genotyping plasmid. The HPV genotyping plasmid was then transfected into the human ovarian cancer cell line ovcar3. The transfection system was configured as follows: jetPRIME® buffer: 75 µL HPV genotyping plasmid: 1 µg jetPRIME® reagent: 3.6 µL, Mix the above-mentioned system thoroughly, let it stand at room temperature for 10 minutes, add the mixed system to a 12-well plate pre-inoculated with ovcar3 cell line, incubate in an incubator for 4-6 hours, and then replace with normal culture medium. (3) 24 hours after transfection, the cell culture medium was changed to G418 medium containing antibiotics for cell screening. After screening, the remaining cells were cloned into single cells. When the single cell clones were formed, the wells marked with clones were added to digest the cells with trypsin. The digested cells were divided into two parts. One part of the cells was used as amplification templates. Primers were used to amplify the single clones and Sanger sequencing was performed to verify whether the HPV genome was successfully integrated. The other part of the cells was expanded and cultured for later use. (4) Select single-cell clones that have successfully integrated the HPV whole genome by Sanger sequencing, expand the clones for culture, extract gDNA, and use digital PCR to detect the copy number of HPV in the selected clones; and use ddPCR to identify the copy number as a reference to prepare gradient quality control products with different copy numbers; wherein the quality control products are in the form of cell suspension, cell pellet or gDNA.

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