Cervical cancer minimal residual focus detection method based on ctHPV marker

Abstract

The invention discloses a cervical cancer minimal residual focus detection method based on a ctHPV marker, and belongs to the technical field of medical examination. The method relates to a group of 14 high-risk ctHPV primers and probe sequences, specific sequences among different HPV types are selected in the design of the primer and probe sequences, the selected primer and probe design areas are E6 and E7, multiple pairs of primers are designed for some common high-risk HPV types, the nucleotide sequences of the primers are shown as SEQ ID NO.1-5, 7-8, 10, 12-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32 and 34-35, and the nucleotide sequences of the primers are shown as SEQ ID NO.1-5, 7-8, 10, 12-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32 and 34-35. The nucleotide sequences of the probes are as shown in SEQ ID NO.6, 9, 11, 15, 18, 21, 24, 27, 30, 33 and 36. The primer and the probe provided by the invention can be used for high-sensitivity and high-specificity detection of cervical cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing technology, and in particular relates to a method for detecting tiny residual lesions of cervical cancer based on ctHPV markers. Background Art

[0002] Cervical cancer is the fourth most common malignant tumor in women. In 2020, there will be approximately 600,000 new cases and 350,000 deaths worldwide. The main cause of cervical cancer is repeated infection with high-risk HPV, which integrates into the human genome and replicates in large quantities to drive cell malignant changes, causing cervical cancer. WHO and the National Health Commission have clearly confirmed 14 high-risk types: including HPV16 / 18 / 31 / 33 / 35 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 66 / 68; among them, HPV-16 has the highest carcinogenicity, and 55-60% of the causes of cervical cancer are related to it; HPV-18 ranks second, and is associated with 10-15% of cervical cancer. HR-HPV integrated into the human genome will be released into the blood in the form of ctDNA (ctHPV). A large number of literature reports show that ctHPV can be detected in the blood of most patients with cervical cancer stages I-IV, with a detection rate of 63.3% to 100% in advanced patients, and a high consistency rate between peripheral blood ctHPV and tissue HPV detection. Multiple studies have shown that: 1) Patients who reach the treatment endpoint and test positive for ctHPV during monitoring have shorter PFS / DFS or OS; 2) The time of ctHPV detection for possible recurrence is an average of 10 months (2 to 15 months) earlier than the time of clinical confirmation of recurrence; ctHPV can be used as a potential marker for monitoring minimal residual disease (MRD) in cervical cancer. The so-called molecular residual disease (MRD) refers to the fact that after a tumor patient has achieved complete remission after treatment, the presence of tumor lesions cannot be detected by imaging or conventional laboratory methods, but tumor cells or tumor-specific nucleic acid molecules (such as free tumor DNA, ctDNA) can be detected in blood and other body fluids by molecular biological methods, thereby indicating the presence of residual lesions. MRD monitoring based on ctHPV markers has important clinical significance for the evaluation of the treatment effect of cervical cancer patients and early intervention before recurrence.

[0003] Digital PCR (ddPCR) is a technology that can directly and accurately quantify templates. It has many advantages such as high sensitivity, simple operation, and direct quantification of HPV virus templates. It is suitable for ctHPV-based monitoring technology. At present, there is no research in China that relies on ctHPV and cervical cancer-related genes as biological targets for prognosis and recurrence monitoring. Summary of the invention

[0004] The present invention provides a kit for ctHPV monitoring, the kit comprising 14 high-risk ctHPV primers and probe sequences. The primer probe sequence design selects sequences that are specific to different HPV types, and the selected primer probe design regions are E6 and E7. Multiple pairs of primers are designed for some common high-risk HPV types to ensure the sensitivity of detection. The primer probes used in the present invention are detailed in Table 1. The probe can be a Taqman probe or an MGB probe; wherein the taqman probe quenching group can be BHQ1, BHQ2, etc., and the fluorescent group can be FAM, CY5, ROX, HEX / VIC, etc.; the MGB probe quenching group needs to be MGB.

[0005] Table 1 All primer and probe sequences

[0006]

[0007]

[0008] 1. Establishment of digital PCR reaction system: The single-type ddPCR primer probe composition is a combination of primers and probes of the same type with different targets. The detection reagents in the single-type PCR system have the following appropriate primer and probe concentrations: primer concentration 400nM~1600nM, probe concentration 200nM~600nM; the optimized preparation of each type of primer mix is ​​shown in Table 2.

[0009] Table 2 Dosages of primers and probes for 14 high-risk ctHPV

[0010]

[0011]

[0012] The reaction system components were optimized on the DQ24 digital PCR machine produced by Suzhou Sinafo, and the 4×dPCR Probe Master Mix (cy5.5) produced by the machine was used to prepare the system. The reaction system was determined after optimization as shown in Table 3:

[0013] Table 3 Digital PCR system components

[0014] Serial number Reaction components Amount added per reaction (ul) 1 4×dPCRProbeMasterMix(cy5.5) 6 2 Probe Mixture 1 3 DNA samples 5-10 4 Reaction enhancer 5 5 Nuclease-free water Fill up to 22

[0015] The reaction enhancer is a commonly used reagent in the art, such as formamide, DMSO, etc.

[0016] Amplification detection was performed using the DQ24 digital PCR all-in-one machine, and the amplification program is shown in Table 4:

[0017] Table 4 Digital PCR amplification program

[0018]

[0019] 2. Data analysis: Use the analysis software provided with the DQ24 digital PCR machine for analysis, with the FAM channel threshold set to 15,000. Collect the results displayed by the software in copies.

[0020] 3. The kit is used for monitoring of minimal residual lesions of cervical cancer:

[0021] 1) Determine clinical typing: Determine clinical HPV typing based on the patient's known clinical test results or using an HPV sharing test kit;

[0022] 2) Determine the detection baseline; use the corresponding type of digital PCR system to detect the cfDNA sample extracted from 10mL of the patient's peripheral blood sample, and calculate the baseline ctHPV index based on the test results: ctHPV content (copies / mL) = FAM channel copy number / peripheral blood volume.

[0023] 3) Continuous monitoring after treatment: The recommended monitoring frequency is 1 to 3 months within 1 year after treatment; 3 to 6 months within 2 years after treatment; and 6 months within 6 years after treatment. Blood samples should be collected for testing.

[0024] The method for detecting micro-residual lesions of cervical cancer based on ctHPV markers adopts digital PCR technology, including primer probe design, kit system and amplification program optimization, and the kit is used for monitoring micro-residual lesions of cervical cancer. The kit is developed based on the Sinafo DQ24 digital PCR all-in-one machine, but in the case of unchanged primers and probes, by fine-tuning the reaction system components, it can be compatible with different digital PCR instrument platforms, including Bio-rad QX200, etc.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) Developed using digital PCR technology, it has high sensitivity and specificity. The detection limits of the 14 types of HPV described in Table 2 are as low as 1 to 3 copies per reaction to ensure the detection of low-level ctHPV content;

[0027] 2) In particular, multiple target primers and probes are designed for the common HPV16 and 18 subtypes, and a strategy of sharing detection primers and probes is adopted for some rare types of HPV to optimize the detection cost;

[0028] 3) The detection speed is fast, the experimental operation is simple, and the reporting cycle and detection cost are better than HPV-seq detection, which makes it more universal and accessible in clinical applications;

[0029] 4) The test sample is peripheral blood, which is non-invasive: it avoids the trauma and complication risks of traditional tissue biopsy. Combined with the test results of quantitative indicators, multiple tests can achieve real-time monitoring of changes in patients' MRD levels, which is helpful for adjusting treatment plans and prognosis assessment.

[0030] The present invention has developed a kit for detecting ctHPV in cervical cancer based on the ddPCR technology platform. The kit can specifically detect ctHPV in the peripheral blood of patients with cervical cancer, and use ctHPV as a biological target for MRD monitoring. Compared with the high-throughput sequencing detection of HPV, which has complicated operation steps and requires complex bioinformatics interpretation, it is more suitable for convenient clinical applications. The present invention can not only realize the quantitative detection of ctHPV in the blood, but also realize the effective dynamic monitoring of MRD, evaluate the prognosis of patients with cervical cancer, and provide a convenient and low-cost MRD monitoring technology for patients with cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the design of HPV18 type primers and probes. Two sets of primers and probes are designed for the E6 and E7 regions respectively, and the probes are labeled with a unified fluorescent group.

[0032] Figure 2 The results of ctHPV16 detection in Example 2, wherein 1A is S10, 1C is a negative control, and 1E is a positive control.

[0033] Figure 3 This is a trend chart of the ctHPV content in the peripheral blood of the patient MRD-S1 in Example 3. The ctHPV content in the patient's peripheral blood is consistent with the patient's disease progression. DETAILED DESCRIPTION

[0034] Example 1

[0035] The ctHPV detection kit corresponding to the most common HPV16 and HPV18 types is used. The kit contains primers and probes for detecting HPV16 and HPV18 types. See Table 1 for details. See Table 2 for the dosage of primers and probes, see Table 3 for the reaction system, and see Table 4 for the reaction procedure. Use positive and negative controls as reagent quality control, and use the company reference products of each product to verify performance. Among them, negative reference products are used to verify reagent specificity, and weak positive, medium positive and positive company reference products are used to verify reagent detection capabilities. The specific steps are as follows:

[0036] (1) Prepare ctHPV16 and ctHPV18 reaction solutions according to Tables 2 and 3, and add 5 μL of quality control or reference material to each test reaction solution;

[0037] (2) According to the procedure in Table 4, complete the test in the DQ24 digital PCR machine;

[0038] (3) The analysis software provided with the DQ24 digital PCR machine was used for analysis, with the FAM channel threshold set to 15,000 to obtain the actual copy number of the sample.

[0039] The results are as follows:

[0040] Table 5 Original data of ctHPV16 and ctHPV18 kit performance verification

[0041]

[0042]

[0043] Experimental data show that: the ctHPV16 test kit passed the quality control, the reference products of each company met the qualified standards, and other HPV reference products of the same type all tested negative, achieving the purpose of performance verification. This kit has good specificity and detection ability; the ctHPV18 test kit passed the quality control, the reference products of each company met the qualified standards, and other HPV reference products of the same type all tested negative, achieving the purpose of performance verification. This kit has good specificity and detection ability.

[0044] Example 2

[0045] In order to verify the detection performance of the ctHPV reagent of the present invention for real clinical samples, peripheral blood samples of 12 HPV-related cervical cancer patients before surgery were collected and extracted and tested using the corresponding type reagent to verify the real detection performance of the reagent for clinical samples. The specific steps are as follows:

[0046] (1) Collect 10 mL of peripheral blood from the patient using a dedicated cell-free DNA collection tube, and extract cfDNA using a commercial cell-free DNA extraction kit;

[0047] (2) Prepare the corresponding ctHPV reaction solution according to Table 2 and Table 3, and add 10 μL of the extracted cfDNA sample to each test reaction solution;

[0048] (3) According to the procedure in Table 4, complete the test in the DQ24 digital PCR machine;

[0049] (4) The analysis software provided with the DQ24 digital PCR machine was used for analysis, with the FAM channel threshold set at 15,000 to obtain the actual copy number of the sample, and the ctHPV content of the peripheral blood sample was calculated based on the extracted cfDNA elution volume and the test input cfDNA volume.

[0050] The information and test results of the 612 samples are as follows:

[0051] Sample No. FIGO staging of cervical cancer HPV type Test results (dPCR) S1 Ib1 HPV16 Negative S2 Ib2 HPV16 Positive (5.75 copies / mL) S3 IIIC1 HPV16 Positive (598.24 copies / mL) S4 Ib1 HPV16 Positive (2.57 copies / mL) S5 Ib2 HPV18 Positive (8.38 copies / mL) S6 IIIC1 HPV16 Positive (387.37 copies / mL) S7 IIb HPV16 Positive (152.39 copies / mL) S8 IIIC1 HPV16 Positive (1175.32 copies / mL) S9 IIa1 HPV18 Positive (1175.32 copies / mL) S10 IIb HPV16 Positive (34.75 copies / mL) S11 Ib1 HPV18 Negative S12 IIIC1 HPV16 Positive (463.52 copies / mL)

[0052] The results of peripheral blood sample test of HPV-related cervical cancer patients before surgery show that the ctHPV content in the patient's peripheral blood is positively correlated with the FIGO stage of cervical cancer. All patients with stage II and above were tested positive for ctHPV, and 3 of the 5 stage I patients were tested positive for ctHPV. The lowest ctHPV content was 2.57 copies / mL, and the positive rate was 60%, which is consistent with the literature reports. The results show that the ctHPV reagent of the present invention has good detection performance for real clinical samples.

[0053] Example 3

[0054] In order to further verify the feasibility of the present invention for the detection of molecular residual lesions of cervical cancer, peripheral blood was collected from a patient with HPV16 type cervical cancer (MRD-S1) multiple times before and after surgery to dynamically monitor the changes in the ctHPV content in the peripheral blood, and the patient was followed up regularly. The specific steps are as follows:

[0055] (1) Collect 10 mL of peripheral blood from the patient using a dedicated cell-free DNA collection tube, and extract cfDNA using a commercial cell-free DNA extraction kit;

[0056] (2) Prepare the corresponding ctHPV16 reaction solution according to Table 2 and Table 3, and add 10 μL of the extracted cfDNA sample to each test reaction solution;

[0057] (3) According to the procedure in Table 4, complete the test in the DQ24 digital PCR machine;

[0058] (4) The analysis software provided with the DQ24 digital PCR machine was used for analysis, with the FAM channel threshold set at 15,000 to obtain the actual copy number of the sample, and the ctHPV content of the peripheral blood sample was calculated based on the extracted cfDNA elution volume and the test input cfDNA volume.

[0059] The patient's postoperative follow-up information and ctHPV test results are as follows:

[0060] Table 7

[0061] Postoperative days (d) ctHPV content (copy / mL) Follow-up -5 1253 7 532 30 45 61 126 105dPD 124 436 Adjuvant chemotherapy started on 131d 151 154 183 64 195d to end adjuvant chemotherapy 208 15 246 0 PR 302 0

[0062] The trend chart of the patient's peripheral blood ctHPV content monitored multiple times is shown in the attached figure. Figure 3As shown, the ctHPV content in the patient's peripheral blood is consistent with the patient's disease progression. Two months after surgery, ctHPV first decreased and then increased, and clinical detection at 105 days indicated that the patient's disease progressed (PD). After adjuvant chemotherapy, the ctHPV content decreased significantly, and clinical detection at 246 days indicated that the patient's disease was in remission (PR). Through the dynamic monitoring of ctHPV in the patient's peripheral blood, the feasibility of the present invention for the detection of molecular residual lesions of cervical cancer was preliminarily verified, and it has value in prognosis evaluation and recurrence monitoring.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer probe for detecting cervical cancer, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NOs. 1-5, 7-8, 10, 12-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, and the nucleotide sequences of the probes are shown in SEQ ID NOs. 6, 9, 11, 15, 18, 21, 24, 27, 30, 33, 36.

2. The primer probe according to claim 1, characterized in that Also included are primer probes for detecting an internal reference gene.

3. A reagent for detecting cervical cancer, characterized in that: The reagent contains the primer probe according to claim 1 or 2.

4. A kit, characterized in that The kit comprises a PCR reaction system having at least the primer probe as claimed in claim 1 or 2.

5. The kit according to claim 4, characterized in that The concentrations of the primers and probes in the PCR reaction system meet the following requirements: the primer concentration is 400nM to 1600nM, and the probe concentration is 200nM to 600nM.

6. The primer probe according to claim 1 or 2, characterized in that The probe is a taqman probe or an MGB probe.

7. The primer probe according to claim 6, characterized in that The quenching group in the probe is BHQ1 or BHQ2, and the fluorescent group is FAM, CY5, ROX, HEX or VIC.

8. Use of the primer probe according to claim 1 or 2 in the preparation of a product for detecting cervical cancer.

9. The use according to claim 8, characterized in that The product is a reagent or a kit.

10. The use according to claim 8 or 9, characterized in that: The test sample is peripheral blood.

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