A nanopore sequencing-based system and method for detecting HPV-mRNA-E6 / E7 in the reproductive tract.

Abstract

This invention belongs to the field of biological detection technology, and particularly relates to a reproductive tract HPV-mRNA-E6 / E7 detection system and method based on nanopore sequencing. The system includes a sample pretreatment module, an RNA extraction and purification module, a reverse transcription module, a nanopore sequencing module, and a bioinformatics analysis module. The method includes the following steps: (1) reproductive tract sample collection and pretreatment; (2) extraction and purification of HPV-mRNA in the sample; (3) reverse transcription of the purified HPV-mRNA into cDNA; (4) nanopore sequencing of the cDNA to obtain raw sequencing data; (5) filtering, comparing, and annotating the sequencing data through the bioinformatics analysis module to achieve HPV type identification and quantitative analysis of E6 / E7 gene expression levels. This invention utilizes the advantages of nanopore sequencing, such as long read length and real-time detection, combined with the specificity of E6 / E7 mRNA as a biomarker of active infection. It can directly detect latent and active HPV infection, improve detection sensitivity, accurately distinguish infection types, and shorten the detection cycle. It enables rapid, accurate, and high-throughput detection of active HPV infection in the genital tract, providing reliable technical support for early screening, disease monitoring, and prognostic assessment of HPV-related diseases such as cervical cancer.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a detection technology for human papillomavirus (HPV) in the reproductive tract. More specifically, it relates to a system and method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing technology, which is suitable for early screening, diagnosis and disease monitoring of HPV-related malignancies such as cervical cancer. Background Technology

[0002] Current methods for detecting HPV in the genital tract mainly include nucleic acid detection and protein detection at the DNA level. HPV is a non-enveloped double-stranded DNA virus with a high affinity for epithelial cells, primarily infecting the stratified squamous epithelial cells of human skin and mucous membranes. Genital HPV infection is a major cause of cervical cancer in women, with 99.7% of cervical cancer cases associated with persistent infection with high-risk HPV. It is also associated with various malignant tumors such as anal cancer and vulvar cancer. HPV infection is extremely common, but most infections are transient and can be cleared by the human immune system within 1-2 years. Only when high-risk HPV causes persistent infection does the E6 and E7 oncogenes in the viral genome become activated and continuously expressed. The proteins they encode disrupt the function of tumor suppressor proteins such as p53 and pRb in host cells, leading to malignant transformation of cells and ultimately developing into cancer. This process usually takes several years or even decades. Therefore, the core requirement of HPV testing is not only to identify HPV types, but more importantly, to distinguish between latent and active infections. The expression level of HPV-mRNA-E6 / E7 is a key biomarker reflecting active HPV infection, and its detection results can more accurately assess the risk of cancer and provide a scientific basis for clinical intervention.

[0003] Currently, nanopore sequencing has been applied to HPV genotyping, but a dedicated detection system and standardized method for HPV-mRNA-E6 / E7 in the reproductive tract have not yet been developed. Problems exist, such as poor sample processing specificity, low RNA extraction efficiency, inaccurate interpretation of sequencing data, and inability to achieve quantitative analysis of E6 / E7 gene expression, making it difficult to meet the clinical demand for accurate detection of active HPV infection. Summary of the Invention

[0004] To address the shortcomings of existing HPV detection methods, such as difficulty in distinguishing between latent and active infections, low sensitivity, inaccurate genotyping, long detection cycles, and complex operations, the inventors have developed a genital HPV-mRNA-E6 / E7 detection system and method based on nanopore sequencing. This system leverages the long read lengths and real-time detection advantages of nanopore sequencing, combined with the specificity of E6 / E7 mRNA as a biomarker of active infection, to achieve rapid, accurate, and high-throughput detection of active HPV infection in the genital tract. Simultaneously, it enables HPV genotyping and quantitative analysis of E6 / E7 gene expression levels, providing reliable technical support for early screening, disease monitoring, and prognostic assessment of HPV-related diseases such as cervical cancer.

[0005] The present invention is achieved through the following technical solution: On the one hand, a reproductive tract HPV-mRNA-E6 / E7 detection system based on nanopore sequencing is provided, the detection system including a sample pretreatment module, an RNA extraction and purification module, a reverse transcription module, a nanopore sequencing module and a bioinformatics analysis module;

[0006] The sample pretreatment module is used for the collection, preservation and pretreatment of reproductive tract samples, removing impurities such as cell debris, proteins, and nucleases from the samples, protecting HPV-mRNA from degradation, and preparing for subsequent RNA extraction. The RNA extraction and purification module includes an extraction reagent, a centrifuge column, and an elution buffer. The extraction reagent includes a lysis buffer, a binding buffer, and a washing buffer. The lysis buffer works synergistically with the preservation solution in the sample pretreatment module to further lyse cells and release mRNA. The binding buffer allows mRNA to specifically bind to the silica membrane of the centrifuge column. The washing buffer removes impurities, and the elution buffer is RNase-free deionized water. The mRNA is eluted from the silica membrane to obtain purified HPV-mRNA. The reverse transcription module contains specific primers designed based on the conserved regions of the HPV E6 / E7 gene, which can specifically bind to HPV-mRNA and reverse transcribe the purified HPV-mRNA into cDNA, ensuring the specificity of reverse transcription and facilitating sequencing by the nanopore sequencing module.

[0007] The nanopore sequencing module includes a nanopore sequencing chip, a sequencer, and sequencing reagents. The nanopore sequencing chip contains multiple nanopore channels, each capable of single-molecule sequencing. The sequencer can acquire the electrical signals of the nanopore channels in real time and convert them into base sequences. This invention preferably uses the all-carbon nanopore QPreasy platform to achieve rapid and efficient sequencing, with a detection limit of 1 copies / μL for HPV16 / 18 and a genotyping accuracy exceeding 95%. The bioinformatics analysis module includes a data filtering unit, a sequence alignment unit, a type identification unit, and a quantitative analysis unit. The data filtering unit removes low-quality sequences, adapter sequences, and repetitive sequences with a Q value < 7 from the raw sequencing data to obtain high-quality valid sequences, ensuring the accuracy of subsequent analysis. The sequence alignment unit compares the high-quality valid sequences with the HPV reference genome database to screen for sequences homologous to the HPV E6 / E7 gene sequences. The database contains common genital HPV types, such as high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, and low-risk types 6 and 11. The HPV reference genome database contains the complete E6 / E7 gene sequences for each of the above HPV types, enabling accurate alignment. Through the above technical solutions, this invention can accurately distinguish between active and latent infections; accurately differentiate between high-risk and low-risk HPV types, improving the sensitivity and specificity of existing methods; the total time from sample collection to report output is no more than 82 hours, greatly shortening the detection cycle; it can directly sequence RNA and process multiple samples simultaneously, achieving high-throughput detection; by integrating sample pretreatment, RNA extraction and purification, reverse transcription, sequencing, and bioinformatics analysis into a single system, the operation process is simplified, the requirements for professional technicians and laboratory environment are reduced, making HPV detection simple, cost-effective, and easy to popularize.

[0008] On the other hand, a method for detecting reproductive tract HPV-mRNA-E6 / E7 based on nanopore sequencing is provided. This method is implemented based on the aforementioned detection system and specifically includes the following steps: Step 1) Sample collection and pretreatment: Collect reproductive tract samples using a sterile reproductive tract sampling swab, place them in a centrifuge tube containing sample preservation solution, and lyse the cells by shaking; centrifuge and filter to obtain the pretreated sample solution; Step 2) Extraction and purification of HPV-mRNA: Add the obtained sample solution to a centrifuge column, add lysis buffer, binding buffer and washing buffer in sequence, centrifuge to remove impurities, add elution buffer, and collect the purified HPV-mRNA; Step 3) Reverse transcription to synthesize cDNA: Add the purified HPV-mRNA to the reverse transcription reaction system to carry out the reverse transcription reaction and obtain cDNA product; Step 4) Nanopore sequencing: Mix the obtained cDNA product with sequencing reagents to prepare a sequencing library; load the obtained sequencing library onto a nanopore sequencing chip, start sequencing, and obtain raw sequencing data; Step 5) Bioinformatics analysis and result determination: Filter, compare, identify and quantify the obtained raw sequencing data to determine the HPV type and infection status; Step 6) Output the test report.

[0009] Preferably, in step 1), the temperature of the sample preservation solution is 4 ℃, the sample preservation time is 24 h; the centrifugation speed is 8000 r / min, and the time is 5 min; the filtration uses a filter membrane with a pore size of 0.22 μm.

[0010] Preferably, in step 2), the purity of the purified HPV-mRNA is detected using a Nanodrop instrument to determine that the A260 / A280 value of the obtained mRNA is between 1.8 and 2.0.

[0011] Preferably, in step 3), the reverse transcription reaction system consists of: 10 U / μL reverse transcriptase, 0.1 μmol / L specific primers, 0.5 mmol / L dNTPs, and 0.5 U / μL RNase inhibitor; the reverse transcription reaction conditions are: reaction temperature of 42 ℃, time of 30 min, and inactivation temperature of reverse transcriptase of 95 ℃ for 5 min.

[0012] Preferably, in step 4), the sequencing temperature is 37 ℃ and the time is 30-60 min. In the raw sequencing data, sequences with a Q value ≥ 7 are considered high-quality, valid sequences.

[0013] Preferably, in step 5), the criteria for type identification are as follows: if the homology between the compared sequence and the E6 / E7 gene sequence of a certain type of HPV is ≥95%, it is determined to be an infection of that type; if the E6 / E7 gene sequences of multiple types of HPV are detected at the same time, it is determined to be a multiple infection.

[0014] Preferably, in step 5), the criteria for determining the infection status are: if the expression level of E6 / E7 mRNA is higher than a set threshold, it is determined to be an active infection; if it is lower than the threshold, it is determined to be a latent infection; if no HPV E6 / E7 gene sequence is detected, it is determined to be no infection; the threshold is determined by clinical sample calibration.

[0015] Beneficial effects Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses HPV-mRNA-E6 / E7 as the detection target. The expression level of the E6 / E7 gene can directly reflect the carcinogenic potential of HPV, accurately distinguishing between active and latent infections, avoiding overtreatment of patients with latent infections, and timely detection of patients with active infections, providing a basis for early intervention and reducing the risk of cervical cancer and other malignant diseases.

[0016] This invention optimizes sample preservation solutions, specific primer design, and nanopore sequencing parameters. The detection limit for both HPV16 and HPV18 types reaches 1 copy / μL, with a sensitivity of over 97.5% and a specificity of 100% for high-risk HPV active infection. It can effectively detect samples with low viral loads, reducing false negatives. Simultaneously, the specific primers avoid non-specific amplification, and using ≥95% homology as the criterion effectively avoids false positives and accurately distinguishes between high-risk and low-risk HPV types, thus improving detection sensitivity and specificity.

[0017] After optimizing the sequencing process, the total time from sample collection to report output is no more than 8 hours, which is much shorter than that of next-generation sequencing and traditional PCR testing. This allows for rapid reference in clinical diagnosis and is especially suitable for large-scale screening scenarios.

[0018] The HPV reference genome database of this invention covers 18 high-risk HPV types and 19 low-risk HPV types, enabling simultaneous detection of multiple types with a typing accuracy of ≥95%. It can effectively detect mixed infections, with intra-batch / inter-batch coefficient of variation <5%, meeting the clinical need for precise HPV typing. At the same time, nanopore sequencing can achieve single-molecule sequencing, directly sequencing RNA without PCR amplification, and can process multiple samples simultaneously, achieving high-throughput detection.

[0019] This invention integrates sample pretreatment, RNA extraction and purification, reverse transcription, sequencing, and bioinformatics analysis into a single system, simplifying the operation process and reducing the requirements for professional technicians and laboratory environment. The nanopore sequencing device can be miniaturized, with a lower cost than second-generation sequencing, and the cost of sample processing and sequencing reagents is controllable, making it easy for primary healthcare institutions to widely adopt it.

[0020] The sample pretreatment module of this invention can effectively process various reproductive tract samples such as cervical exfoliated cells and vaginal secretions. The sample preservation solution can effectively protect HPV-mRNA from degradation. Even low-quality samples such as FFPE samples with nucleic acid degradation can be effectively detected, thus expanding the scope of applicable samples for detection. Attached Figure Description Figure 1 This is a schematic diagram of the detection system of the present invention. Figure 2 This is a flowchart illustrating an exemplary detection method of the present invention. Detailed Implementation To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] Example 1 Step 1) Construction of sample preprocessing module Sterile cervical swabs were used as sample collectors; a sample preservation solution was prepared containing 10 U / μL RNase inhibitor, 4 mol / L guanidine isothiocyanate, 25 mmol / L sodium citrate, and 0.5% sodium dodecyl sarcosinate, with the pH adjusted to 7.0; a high-speed refrigerated centrifuge with a speed range of 0-15000 r / min was used as the centrifugation device; and a 0.22 μm filter membrane was used as the filtration device, forming a sample pretreatment module.

[0022] Step 2) Construction of RNA extraction and purification module Extraction reagents were prepared, including lysis buffer, binding buffer, and washing buffer. The lysis buffer had the same composition as the sample preservation solution. The binding buffer consisted of 6 mol / L guanidine hydrochloride and 20 mmol / L Tris-HCl at pH 7.5. The washing buffer consisted of 70% ethanol and 20 mmol / L Tris-HCl at pH 7.5. A silica gel centrifuge column was used as the centrifuge column. RNase-free deionized water was used as the elution buffer. This assembled the RNA extraction and purification module.

[0023] Step 3) Construction of the reverse transcription module 200 U / μL M-MLV reverse transcriptase was used; specific primers targeting the conserved regions of the HPV E6 / E7 gene were designed; 10 mmol / L dNTPs were used; a reverse transcription buffer containing 50 mmol / L Tris-HCl, 75 mmol / L KCl, and 3 mmol / L MgCl2 was prepared at pH 8.3; and 10 U / μL RNase inhibitors were used to assemble a reverse transcription module. The specific primer sequences for HPV mRNA-E6 / E7 are shown in the table below.

[0024] Step 4) Construction of nanopore sequencing module The QiCan nanopore gene sequencer was selected as the sequencer; the QiCan QCell-6k nanopore sequencing chip, which contains 6000 nanopore channels, was selected; sequencing reagents were prepared, including sequencing buffer consisting of 10 mmol / L Tris-HCl and 50 mmol / L NaCl, pH 8.0, and 10 U / μL phi29 DNA polymerase, and the nanopore sequencing module was assembled.

[0025] Step 5) Construction of Bioinformatics Analysis Module A bioinformatics analysis program was developed using Python, including data filtering, sequence alignment, type identification, and quantitative analysis units. An HPV reference genome database was constructed, containing the complete E6 / E7 gene sequences of 18 high-risk HPV types and 19 low-risk HPV types. The high-risk HPV types are HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 73. The study included 82 types of HPV; low-risk HPV types consisted of HPV6, 11, 34, 40, 42, 43, 44, 54, 57, 61, 67, 69, 70, 71, 72, 81, 83, 84, and 89; the internal reference gene was the ACTB gene, whose sequence was obtained from the NCBI database, with GenBank accession number NM_001101.5; the analysis program was installed on a computer and connected to a nanopore sequencer to form a bioinformatics analysis module.

[0026] Step 6) Connect the above 5 modules sequentially to ensure smooth signal and sample transmission between modules, thus completing the nanopore sequencing-based reproductive tract HPV-mRNA-E6 / E7 detection system. A schematic diagram of the detection system is shown below. Figure 1 As shown.

[0027] Example 2 Sterile cervical swabs were used as sample collectors; a sample preservation solution was prepared containing 10 U / μL RNase inhibitor, 4 mol / L guanidine isothiocyanate, 25 mmol / L sodium citrate, and 0.5% sodium dodecyl sarcosinate, with the pH adjusted to 7.0; a high-speed refrigerated centrifuge with a speed range of 0-15000 r / min was used as the centrifugation device; and a 0.22 μm filter membrane was used as the filtration device, forming a sample pretreatment module.

[0028] According to the following table L9 (3) 4 Orthogonal experiments were conducted to optimize and validate the concentrations of each component in the preservation solution.

[0029] The results showed that: too low a concentration of guanidine isothiocyanate resulted in insufficient lysis, while too high a concentration easily crystallized and inhibited the sequencing reaction; too low a concentration of RNase inhibitor resulted in severe degradation of E6 / E7 mRNA, while too high a concentration offered no additional protective benefit; deviations from the optimal concentrations of sodium dodecyl sarcosinate and sodium citrate both led to decreased sample clarity and distorted nucleic acid quantification; the optimized ratio of this invention can achieve sufficient lysis of cell viruses, complete and stable preservation of HPV E6 / E7 mRNA, and efficient removal of impurities, ensuring the accuracy and reliability of subsequent sequencing expression level detection, thereby achieving accurate differentiation between active and latent infections, with significant optimization effects.

[0030] Application Example 1 One hundred clinical reproductive tract samples were selected, including 20 cases clinically diagnosed with active HPV16 infection, 20 cases with active HPV18 infection, 15 cases with active HPV52 infection, 10 cases with multiple infections of HPV16+18, 15 cases with latent infection, and 20 cases without infection. The detection system constructed in Examples 1-2 was used for testing. A schematic diagram of the detection method is shown below. Figure 2 As shown, the specific steps are as follows: Step 1) Sample collection and preprocessing Cervical exfoliated cells were collected from the subjects using sterile cervical swabs. The swabs were placed in centrifuge tubes containing 2 mL of sample preservation solution and shaken thoroughly for 30 s to allow for complete cell lysis. The centrifuge tubes were then stored at 4 ℃ for subsequent processing within 6 h. The preserved samples were then centrifuged at 8000 r / min for 5 min in a high-speed refrigerated centrifuge to remove the precipitate and collect the supernatant. The supernatant was then filtered through a 0.22 μm filter membrane to remove impurities, yielding the pretreated sample solution.

[0031] Step 2) Extraction and purification of HPV-mRNA Take 1 mL of pretreated sample solution and add it to a silica gel centrifuge column. Add 500 μL of lysis buffer and let it stand at room temperature for 5 min. Add 1 mL of binding buffer, invert and mix well, and let it stand at room temperature for 10 min. Centrifuge at 12000 r / min for 1 min and discard the waste liquid. Add 700 μL of washing buffer, centrifuge at 12000 r / min for 1 min and discard the waste liquid. Repeat the washing once, centrifuge at 12000 r / min for 2 min to remove residual washing buffer. Add 50 μL of RNase-free deionized water, let it stand at room temperature for 2 min, centrifuge at 12000 r / min for 1 min, and collect the eluent. Use a micro-volume UV-Vis spectrophotometer (model: NanoDrop 2000c, purchased from Thermo Fisher Scientific) to detect the sample. The A260 / A280 of mRNA in the eluent was 1.85-1.95, and the concentration was 50-200 ng / μL, which met the requirements for subsequent experiments.

[0032] Step 3) Reverse transcription to synthesize cDNA Take 10 μL of purified HPV-mRNA and add it to the reverse transcription reaction system (total volume 20 μL). The system composition is: 1 μL M-MLV reverse transcriptase, 2 μL specific primers, 2 μL dNTPs, 5 μL reverse transcription buffer, 1 μL RNase inhibitor, and 10 μL HPV-mRNA. Place the reaction system in a PCR instrument and set the reaction conditions as follows: reverse transcription at 42 ℃ for 30 min, followed by inactivation at 95 ℃ for 5 min to obtain cDNA product. Store the cDNA product at -20 ℃ for later use.

[0033] Step 4) Nanopore sequencing Take 5 μL of cDNA product and mix it with 10 μL of sequencing buffer and 1 μL of phi29 DNA polymerase to prepare a sequencing library; load the sequencing library onto a nanopore sequencing chip, place the chip into the QPreasy sequencer, set the sequencing parameters: sequencing temperature 37 ℃, sequencing time 40 min, and start sequencing; the sequencer collects electrical signals in real time, converts them into raw sequencing data, and transmits them to the bioinformatics analysis module.

[0034] Step 5) Bioinformatics analysis and result determination The data analysis unit removes low-quality sequences, adapter sequences, and repetitive sequences with a Q value < 7 from the raw sequencing data to obtain high-quality effective sequences, with an effective sequence recovery rate of 85%-92%. The sequence alignment unit compares high-quality, valid sequences with the HPV reference genome database. Sequences with a similarity of ≥90% are considered valid alignment sequences. Based on the comparison results, if the effective matched sequence has ≥95% homology with the E6 / E7 gene sequence of a certain HPV type, the type identification unit determines that it is infected with that type; multiple infected samples can detect effective matched sequences corresponding to multiple types; Quantitative analysis and result determination: The quantitative analysis unit calculates the sequencing depth of the E6 / E7 gene sequence and, combined with the expression level of the ACTB gene, calculates the relative expression level of E6 / E7 mRNA; the threshold is set to 1.0 through calibration with clinical active infection samples. A relative expression level >1.0 is determined to be an active infection, ≤1.0 is determined to be a latent infection, and no effective alignment sequence is detected is determined to be no infection.

[0035] Step 6) Output the results Output a test report that clearly identifies the HPV type, E6 / E7 mRNA expression level, and infection status of the sample.

[0036] The test results for each sample are shown in the table below: Comparison table of test results of 100 clinical reproductive tract samples

[0037] In summary, comparing the detection results of Example 5 with the clinical diagnosis results, out of 100 samples, 98 cases had detection results consistent with the clinical diagnosis results, with an accuracy rate of 98%. Among them, 2 latent infection samples were misclassified as not infected because the HPV-mRNA concentration in the samples was extremely low, less than 50 mg / L. The detection rate was ng / μL. In the detection of active HPV types 16, 18, and 52, 58 out of 60 samples were detected, with a sensitivity of 97.5%. All multiple infections were detected with a sensitivity of 100%. All 20 uninfected samples were detected with a specificity of 100% and no false positives were observed. Low-risk HPV infections were accurately distinguished from high-risk types. The total time from sample collection to report output for a single sample was less than 2 hours, while next-generation sequencing required 1-3 days and traditional PCR testing required 4-6 hours. It accurately identified 18 high-risk and 19 low-risk HPV types with a typing accuracy ≥95%, effectively detecting mixed infections missed by Sanger sequencing. The intra-batch / inter-batch coefficient of variation was <5%, and the 150 bp target sequence coverage reached 100%. Therefore, the detection system and method of this invention have high accuracy, sensitivity, and specificity, a short detection cycle, and accurate typing, effectively distinguishing between active and latent HPV infections, meeting clinical testing needs.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reproductive tract HPV-mRNA-E6 / E7 detection system based on nanopore sequencing, characterized in that, It includes a sample pretreatment module, an RNA extraction and purification module, a reverse transcription module, a nanopore sequencing module, and a bioinformatics analysis module; The detection system determines whether an infection has occurred based on the expression level of the HPV E6 / E7 gene. Furthermore, the clinical active infection sample calibration threshold is set at 100 copies / reaction. A relative expression level >100 copies / reaction is considered an active infection, ≤100 copies / reaction is considered a latent infection, and no effective alignment sequence is detected is considered no infection.

2. The reproductive tract HPV-mRNA-E6 / E7 detection system based on nanopore sequencing according to claim 1, characterized in that, The sample pretreatment module includes a sample preservation solution containing an RNase inhibitor and a lysis buffer of guanidine isothiocyanate. The concentration range of the RNase inhibitor is 8–12 U / μL and the concentration range of the guanidine isothiocyanate is 3–6 mol / L.

3. The reproductive tract HPV-mRNA-E6 / E7 detection system based on nanopore sequencing according to claim 1, characterized in that, The reverse transcription module uses specific primers to reverse transcribe the purified HPV-mRNA into cDNA for sequencing in the nanopore sequencing module. The specific primers are designed based on the conserved regions of the HPV E6 / E7 gene, and their forward and reverse primer sequences are shown in the table below.

4. The reproductive tract HPV-mRNA-E6 / E7 detection system based on nanopore sequencing according to claim 1, characterized in that, The nanopore sequencing module includes a nanopore sequencing chip, a sequencer, and sequencing reagents; The sequencer is a carbon nanopore QPreasy platform; the sequencing reagents include sequencing buffer and DNA polymerase; the sequencing buffer contains 10 mmol / L Tris-HCl and 50 mmol / L NaCl, and the pH value is 8.0; the concentration of DNA polymerase added is 10 U / μL.

5. A method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing, characterized in that, Based on the detection system according to any one of claims 1-4, the following steps are included: Step 1) Sample collection and preprocessing; Reproductive tract samples were collected using sterile reproductive tract sampling swabs and placed in centrifuge tubes containing sample preservation solution. Cells were lysed by shaking. The samples were then centrifuged and filtered to obtain pretreated sample solution. Step 2) Extraction and purification of HPV-mRNA; Add the obtained sample solution to a centrifuge column, then add lysis buffer, binding buffer and washing buffer in sequence. Centrifuge to remove impurities, add elution buffer and collect the purified HPV-mRNA. Step 3) Reverse transcription to synthesize cDNA; The purified HPV-mRNA was added to the reverse transcription reaction system to perform reverse transcription and obtain cDNA product. Step 4) Nanopore sequencing; The obtained cDNA product was mixed with sequencing reagents to prepare a sequencing library; the sequencing library was loaded onto a nanopore sequencing chip, sequencing was started, and raw sequencing data were obtained. Step 5) Bioinformatics analysis and result determination; The obtained raw sequencing data were filtered, compared, identified, and quantitatively analyzed to determine the HPV type and infection status.

6. The method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing according to claim 5, characterized in that, In step 1), the temperature of the sample preservation solution is 4 ℃, and the sample preservation time is 24 h; the centrifugation speed is 8000 r / min, and the time is 5 min; the filtration uses a filter membrane with a pore size of 0.22 μm.

7. The method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing according to claim 5, characterized in that, In step 2), the purity of the purified HPV-mRNA was detected using a Nanodrop instrument, and the A260 / A280 value of the obtained mRNA was determined to be between 1.8 and 2.

0.

8. The method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing according to claim 5, characterized in that, In step 3), the system for the reverse transcription reaction is as follows: 10 U / μL reverse transcriptase, 0.1 μmol / L specific primer, 0.5 mmol / L dNTPs, and 0.5 U / μL RNase inhibitor; the conditions for the reverse transcription reaction are: reaction temperature of 42 ℃, time of 30 min, and inactivation temperature of the reverse transcriptase of 95 ℃ for 5 min.

9. The method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing according to claim 5, characterized in that, In step 4), the sequencing temperature is 37 ℃ and the time is 30-60 min; in the raw sequencing data, sequences with a Q value ≥ 7 are high-quality valid sequences.

10. The method for detecting HPV-mRNA-E6 / E7 in the reproductive tract based on nanopore sequencing according to claim 5, characterized in that, In step 5), the criteria for type identification are as follows: if the homology between the compared sequence and the E6 / E7 gene sequence of a certain type of HPV is ≥95%, it is determined to be an infection of that type; if the E6 / E7 gene sequences of multiple types of HPV are detected at the same time, it is determined to be a multiple infection.

Images