Gene Panel sequencing method for intrahepatic cholestasis in gestation period

Abstract

The invention relates to the technical field of biology, and particularly discloses a gene Panel sequencing method for intrahepatic cholestasis in a gestation period, which comprises the following steps: collecting saliva, performing DNA extraction and purification on a sample, breaking the purified sample, treating the broken sample to obtain a supernatant A, performing linker connection and purification on the supernatant A to obtain a supernatant B, and sequencing the supernatant B to obtain the gene Panel of intrahepatic cholestasis in a gestation period. The invention relates to a Panel detection technology for detecting 186 cholestasis genes covering bile acid metabolism, which comprises the following steps: preparing a supernatant B, carrying out pre-PCR and purification treatment on the supernatant B to obtain a pre-PCR product, carrying out liquid phase hybridization and capture, configuring a PCR reaction system, carrying out Post-PCR reaction and purification to obtain a supernatant C, quantifying the supernatant C, screening ICP susceptible genes through mixed sample sequencing, collecting DNA (Deoxyribonucleic Acid) through a saliva sampling mode, and detecting the 186 cholestasis genes covering bile acid metabolism. Other hereditary intrahepatic cholestasis diseases can be excluded, and ICP genetics susceptibility genes are defined.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene panel sequencing method for intrahepatic cholestasis of pregnancy. Background Art

[0002] Intrahepatic cholestasis of pregnancy (ICP) is a common obstetric condition, with a global prevalence of 1% to 27.6%. The composite incidence of adverse perinatal outcomes, including preterm birth, perinatal fetal death, neonatal respiratory distress syndrome, and NICU admission, is 27%. Severe ICP is defined as a serum total bile acid level ≥40 μmol / L. Serum total bile acid levels, particularly ≥100 μmol / L, are significantly associated with the adverse outcome of perinatal fetal death in severe ICP.

[0003] In the existing technology, the cause of ICP is not yet fully understood. It may be the result of the combined effects of genetic, hormonal and environmental factors. The genes related to the onset of ICP include mutations in the gene ABCB4 encoding the capillary bile duct membrane phospholipid translocation protein multidrug resistance protein 3 (MDR3), and genes encoding other capillary bile duct transport proteins or their regulatory factors (ABCB11, ATP8B1, ABCC2 and NR1H4). Normal bile acids are synthesized from cholesterol in liver cells, secreted into the capillary bile ducts and stored in the gallbladder. After eating, the gallbladder contracts and bile enters the duodenum. After the bile acids in the intestine play a fat emulsification role, about 95% of the conjugated bile acids are actively reabsorbed in the terminal ileum, and about 5% are unbound. Synthesized bile acids are reabsorbed by passive diffusion in the jejunum and colon. The bile acids reabsorbed into the blood return to the liver through the portal vein. The liver takes up bile acids in the portal vein, directly synthesizes bile, and then re-secretes it into the gallbladder. This efficient enterohepatic circulation ensures that bile acids are reused after digestion of fat, reducing the burden of resynthesis on the liver and maintaining the stability of the bile acid pool. Each step of the bile acid cycle is finely regulated by different genes, involving multiple links of bile acid synthesis, transport, reabsorption and signal transduction, and is mainly composed of nuclear receptors, transport proteins and rate-limiting enzyme genes. Different bile acid metabolism regulatory genes and site mutations are prone to ICP of varying severity under the action of high levels and long-term estrogen during pregnancy.

[0004] However, the existing technology lacks a sequencing method for panels of genes that are susceptible to severe, relapsed, and refractory ICP. Summary of the Invention

[0005] The purpose of the present invention is to provide a gene panel sequencing method for intrahepatic cholestasis of pregnancy, aiming to solve the technical problem in the prior art of the lack of a panel sequencing method for severe, recurrent and refractory ICP susceptibility genes.

[0006] To achieve the above objectives, the present invention adopts a gene panel sequencing method for intrahepatic cholestasis of pregnancy, comprising the following steps: Conduct sample collection; DNA extraction and purification of collected samples; Shear the purified genome and concentrate the sheared sample; The concentrated sheared samples were subjected to end-filling and 3' A addition to obtain supernatant A; The supernatant A is connected to a linker and purified to obtain supernatant B; Perform pre-PCR and purification on supernatant B to obtain pre-PCR products; Perform liquid phase hybridization and capture on pre-PCR products; Prepare a PCR reaction system for Post-PCR reaction and purification to obtain supernatant C; 1.0 μL of supernatant C was taken as the library, and the library was quantified using the Qubit dsDNA HS Assay Kit. The library concentration was recorded, and mixed sequencing was immediately performed to screen for ICP susceptibility genes.

[0007] The sample collection comprises the following steps: Use sterile individually packaged test tubes to collect 3 mL of saliva to be tested according to the operating instructions; After collection, the samples are stored in preservation solution and transported using ice packs.

[0008] The DNA extraction and purification of the collected samples includes the following steps: Use the kit to extract and purify the DNA in the saliva to be tested; Use nanodrop to preliminarily test the concentration and purity of DNA; Use Qubit 3.0 to accurately detect DNA concentration; The integrity of the DNA was checked by agarose gel electrophoresis.

[0009] The shearing of the purified genome and concentrating the sheared sample comprises the following steps: Mix genomic DNA with nuclease-free water to a total volume of 40uL, transfer to a low-adsorption centrifuge tube, mix thoroughly, and centrifuge briefly; Turn on the ultrasonic fragmentation instrument Bioruptor Pico in advance. After the cold cycler temperature reaches 4°C, set the parameters to ON 30s, OFF 30s as one cycle. Perform 30 cycles of fragmentation on whole blood DNA, 20 cycles on tissue samples, and 20 cycles on paraffin-embedded DNA. Place each group of fragmented samples on an oscillator to mix thoroughly. Take 1.2 μL of sample and add 8.8 μL of DNA buffer for fragment detection using QSEP 100. After fragmentation, the main peak of the sample detection is between 150 bp and 200 bp; The fragmented sample was concentrated to a volume of 25 μL using a vacuum freeze concentrator.

[0010] The concentrated sheared sample is subjected to end-filling and 3' A addition to obtain supernatant A, comprising the following steps: Add 8 μL of End Repair-A Tailing Buffer and 2 μL of End Repair-A Tailing Enzyme Mix to 25 μL of the disrupted sample and vortex. The vortexed solution was centrifuged and then incubated in a Thermomixer at 20°C for 15 min, 72°C for 15 min, and extended at 4°C. Add 56 μL of Inlighten magnetic beads to the solution tube and mix by pipetting several times; Incubate in the greenhouse for 2 min, and place the PCR tube containing the solution on a magnetic stand for 3 min until clear; Remove the supernatant and place the PCR tube on the magnetic stand. Add 200 μL of 80% ethanol solution to the PCR tube and wash the surface of the magnetic beads with a pipette. Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Let it stand in the greenhouse for 5 minutes to allow the residual ethanol to evaporate completely; Add 35.5 μL of nuclease-free water, remove the PCR tube from the magnetic stand, resuspend the magnetic beads by pipetting, and let it stand in the room for 5 minutes; Place the PCR tube on the magnetic stand for 2 minutes, use a pipette to aspirate 35 μL of the supernatant, and transfer it to a new PCR tube to obtain supernatant A.

[0011] The step of connecting the supernatant A to a linker and purifying the supernatant to obtain the supernatant B comprises the following steps: Add 11.5 μL of Ligation Buffer, 1 μL of T4 DNA Ligase, and 2.5 μL of Adaptor Oligo Mix to 35 μL of supernatant A and vortex. The vortexed solution was centrifuged and then incubated at 20°C in a Thermomixer for 30 min. Add 45 μL of Inlighten magnetic beads to the PCR tube and use a pipette to mix the solution by pipetting several times; The solution in the PCR tube was incubated in a greenhouse for 5 min, and the PCR tube was placed on a magnetic stand for 3 min; Remove the supernatant from the PCR tube, place the PCR tube on the magnetic stand, add 200 μL of 80% ethanol solution to the PCR tube, and wash the surface of the magnetic beads with a pipette; Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Place in a greenhouse and let it stand for 5 minutes to allow the residual ethanol to evaporate completely; Add 35.5 μL of Nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by pipetting. Let it stand at room temperature for 5 minutes. Place the PCR tube on a magnetic stand for 2 minutes, use a pipette to aspirate 35 μL of supernatant, and transfer it to a new PCR tube to prepare supernatant B.

[0012] The step of performing pre-PCR and purification on the supernatant B to obtain a pre-PCR product comprises the following steps: Prepare 35 μL of supernatant B, 10 μL of 5× Herculase II Reaction Buffer, 0.5 μL of 100 mM dNTP Mix, 1.5 μL of Forward Primer, 1 μL of Herculase II Fusion DNA Polymerase, and 2 μL of SureSelect XT Low Input Index Primer in a PCR tube; Use a pipette to mix gently by pipetting 6 times, then centrifuge briefly; Place the centrifuged product on a PCR instrument and start the PCR program to process the product; After the PCR program is completed, 2 μL is taken for gel running test. The agarose gel concentration in the gel running test is 2%, the electric field strength is 170 V, and the electrophoresis time is 20 min. Add 48 μL of Inlighten magnetic beads to the PCR tube and mix well by pipetting several times; Incubate in the greenhouse for 5 min, then place the PCR tube on a magnetic stand for 3 min; Remove the supernatant and place the PCR tube on the magnetic stand. Add 200 μL of 80% ethanol solution to the PCR tube and use a pipette to wash the surface of the magnetic beads. Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Let it stand in the greenhouse for 5 minutes to allow the residual ethanol to evaporate completely; Add 35 μL of Nuclease-free water, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by pipetting. Let it stand at room temperature for 5 minutes. Place the PCR tube on a magnetic stand for 2 minutes; Use a pipette to aspirate 35 μL of the supernatant and transfer it to a new 1.5 mL tube to obtain the pre-PCR product.

[0013] The liquid phase hybridization and capture of the pre-PCR product comprises the following steps: Transfer 500-1000 ng of pre-PCR product into a new centrifuge tube and concentrate the product to 12 μL in a vacuum concentrator at 40°C. Add 5 μL of SureSelect XT HS and XT Low Input Blocker Mix to 12 μL of concentrated product, shake to mix, and centrifuge. The PCR program was pre-set as follows: Step 1: 95°C for 5 minutes, one cycle; Step 2: 65°C for 10 minutes, one cycle; Step 3: 65°C for 1 minute, one cycle with a pause point; Step 4: 65°C for 1 minute followed by 37°C for 3 seconds, 60 cycles; Step 5: Hold at 65°C, one cycle; Transfer 17 μL of the total mixed solution to a 200 μL PCR tube and perform the PCR reaction according to the preset PCR program. Prepare the probe mix 15 minutes before the reaction. When the Capture Library is less than 3 Mb, the probe configuration ratio is: 0.5 μL RNase Block solution, 2 μL Capture Library probe, 4.5 μL RNase-free water, and 6 μL SureSelect Fast Hybridization Buffer; when the Capture Library is ≥3 Mb, the probe configuration ratio is: 0.5 μL RNase Block solution, 5 μL Capture Library probe, 1.5 μL RNase-free water, and 6 μL SureSelect Fast Hybridization Buffer; Pause the PCR reaction at step 3, keep the DNA + Blocker on the PCR instrument, add 13 μL of probe mix to the DNA + Blocker, mix thoroughly by pipetting, centrifuge briefly, and quickly return to the PCR instrument to continue the subsequent reaction; Aliquot Wash Buffer 2 in advance and preheat it to 70°C in a ThermoMixer. Remove the magnetic beads from 4°C and resuspend by vortexing; For each sample, 50 μL of magnetic beads was placed in a new 1.5 mL centrifuge tube, placed on a magnetic stand for 1 min, and the supernatant was removed; Remove the centrifuge tube from the magnetic stand, add 200 μL of SureSelect Binding buffer and mix by pipetting several times, resuspend the magnetic beads, place on the magnetic stand for 1 minute, remove the supernatant, and repeat this step twice, for a total of three washes. Remove the centrifuge tube from the magnetic stand, add 200 μL of SureSelect Binding buffer, pipette and pipette 6 times to resuspend the magnetic beads for later use; GenMagbio fully automated purification instrument was used to capture the target gene sequence and wash the beads six times with Wash Buffer 2. When the hybridization reaction is complete and reaches the 65°C hold, remove the reaction tube from the PCR instrument, gently remove it, and place it at room temperature; Add reagents to a 96-well deep-well plate: well 1 or 7: 200ul Wash Buffer 2; well 2 or 8: 30ul hybridization product and 200ul washed MyOne T1 magnetic beads; well 3 or 9: 200ul Wash Buffer 1; well 6 or 12: 200ul Wash Buffer 2. Perform the automated purification instrument operation according to the preset program 1. After program 1 is completed, remove the deep-well plate and place it on a 96-well magnetic plate. Allow the plate to adhere for 2 minutes. Remove the used Wash Buffer 2 supernatant from the wells in columns 1 or 7 and 6 or 12. Add 200 μL of preheated Wash Buffer 2 to the wells in columns 1 or 7 and 6 or 12. Return the deep-well plate to the instrument's original position and start program 2. After the second program is finished, remove the deep-well plate and place it on a 96-well magnetic plate. Allow it to magnetically absorb for 2 minutes. Remove the used Wash Buffer 2 supernatant from the 1st or 7th column and the 6th or 12th column. Then, add 200 μL of preheated Wash Buffer 2 to the 1st or 7th column and the 6th or 12th column. At the same time, add 41 μL of Nuclease-free water to the 4th or 10th column. Then, place the deep-well plate in the machine and start the third program. After the third program is finished, remove the deep-well plate and aspirate 37.5 μL of the target gene capture product bound to magnetic beads from well 4 or 10 for post-PCR. Add 30 μL of the hybridized product to 200 μL of MyOne T1 magnetic beads, pipette up and down 6 times to mix, and place on a rotary mixer at 10 rpm / min for 30 min at room temperature; Place the centrifuge tube on a magnetic stand for 2 minutes and remove the supernatant; Add 200 μL of Wash Buffer 1 to the centrifuge tube, pipette up and down 15-20 times to mix, resuspend the magnetic beads, and place on a magnetic stand for 1 minute. Remove the supernatant. Add 200 μL of 70°C preheated Wash Buffer 2, vortex mix for 5 seconds, centrifuge, and incubate at 70°C for 5 minutes on a ThermoMixer. After a brief centrifugation, place the centrifuge tube on a magnetic stand for 2 minutes, remove the supernatant, and repeat the wash 5 times, for a total of 6 times. Finally, completely remove Wash Buffer 2 for the last time. Add 38 μL of Nuclease-free water to the centrifuge tube, remove the centrifuge tube from the magnetic stand, and pipette 6 times to resuspend the magnetic beads.

[0014] The configuration of the PCR reaction system for post-PCR reaction and purification to obtain supernatant C comprises the following steps: Prepare the PCR reaction system in a 0.2 mL centrifuge tube, including 37.5 μL of bead-bound target-enriched DNA samples, 10 μL of 5× Herculase II Reaction Buffer, 1 μL of Herculase II Fusion DNA Polymerase, 0.5 μL of 100 mM dNTP Mix, and 1 μL of SureSelect Post-Capture Primer Mix; Mix the prepared PCR reaction system by pipetting and pumping 6 times, and then place it on the PCR instrument; Run the PCR instrument to cycle the reaction system; After the PCR instrument finishes running, vortex the PCR product to mix it evenly, centrifuge it at a light speed, add 70 μL of Inlighten magnetic beads to the PCR product, and pipette it up and down 6 times to mix it evenly; Incubate in the greenhouse for 5 min, then place the PCR tube on a magnetic stand for 3 min; The supernatant was removed, and the PCR tube was placed on the magnetic stand again. 200 μL of 80% anhydrous ethanol was added and allowed to stand for 30 seconds. Remove the supernatant and add 200 μL of 80% anhydrous ethanol to the PCR tube. Let it stand for 30 seconds and then completely remove the supernatant. Place in the greenhouse for 5 minutes to allow the residual ethanol to evaporate; Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic stand, pipette to mix and resuspend the magnetic beads, and place in the room temperature for 2 minutes; Place the PCR tube on a magnetic stand for 2 minutes; Use a pipette to draw 24.5 μL of the supernatant into a 1.5 mL centrifuge tube to prepare supernatant C.

[0015] The present invention provides a gene panel sequencing method for intrahepatic cholestasis of pregnancy, which collects DNA through saliva sampling and covers a panel detection technology of 186 cholestasis genes involved in bile acid metabolism. This helps clinicians conduct genetic susceptibility gene testing for intrahepatic cholestasis of pregnancy, while excluding other hereditary intrahepatic cholestasis diseases and identifying ICP genetic susceptibility genes. This helps clinicians determine the cause of the disease, distinguish ICP from other hereditary intrahepatic cholestasis diseases, avoid misdiagnosis and missed diagnosis, and provide patients with targeted treatment plans. For pregnant women known to carry susceptibility genes, more accurate pregnancy health management recommendations can be provided, and a large amount of ICP-related genetic data can be accumulated to enrich the disease database. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a flowchart of the steps of a gene panel sequencing method for intrahepatic cholestasis of pregnancy of the present invention. DETAILED DESCRIPTION

[0018] See also Figure 1 The present invention provides a gene panel sequencing method for intrahepatic cholestasis of pregnancy, comprising the following steps: S1: perform sample collection; S2: DNA extraction and purification of collected samples; S3: Shear the purified genome and concentrate the sheared sample; S4: The concentrated sheared samples are subjected to end-filling and 3' A addition to obtain supernatant A; S5: Connect the linker and purify the supernatant A to obtain supernatant B; S6: Perform pre-PCR and purification on supernatant B to obtain pre-PCR products; S7: liquid phase hybridization and capture of pre-PCR products; S8: Prepare a PCR reaction system for post-PCR reaction and purification to obtain supernatant C; S9: Take 1.0 μL of supernatant C as the library, quantify the library using the Qubit dsDNA HS Assay Kit, record the library concentration, and immediately perform mixed sequencing to screen for ICP susceptibility genes.

[0019] Furthermore, the sample collection comprises the following steps: Use sterile individually packaged test tubes to collect 3 mL of saliva to be tested according to the operating instructions; After collection, the samples are stored in preservation solution and transported using ice packs.

[0020] The DNA extraction and purification of the collected samples includes the following steps: Use the kit to extract and purify the DNA in the saliva to be tested; Use nanodrop to preliminarily test the concentration and purity of DNA; Use Qubit 3.0 to accurately detect DNA concentration; The integrity of the DNA was detected by agarose gel electrophoresis.

[0021] Further, the purified genome is broken and the broken sample is concentrated, including the following steps: Mix the genomic DNA with Nuclease-free water to a total volume of 40uL, transfer to a low-adsorption centrifuge tube, mix thoroughly, and centrifuge briefly; Turn on the ultrasonic disruptor Bioruptor Pico in advance, set the parameters ON 30s, OFF 30s for one cycle, and perform 30 cycles of disruption on whole blood DNA, 20 cycles of disruption on tissue samples, and 20 cycles of disruption on DNA from paraffin-embedded sources. Mix the disrupted samples thoroughly on a shaker; Take 1.2uL of the sample and add 8.8uL of DNA buffer to use QSEP 100 for fragment detection. The main peak of the disrupted sample is between 150bp and 200bp; Use a vacuum freeze concentrator to concentrate the disrupted sample to 25uL.

[0022] Further, the broken and concentrated sample is end-repaired and 3'-A-tailed, and the supernatant A is obtained after processing, including the following steps: Add 8uL of End Repair-A Tailing Buffer and 2uL of End Repair-A Tailing Enzyme Mix to the 25uL disrupted sample and vortex; Centrifuge the vortexed solution, then incubate in a Thermomixer at 20℃ for 15min, 72℃ for 15min, and 4℃ for extension; Add 56uL of Axygen magnetic beads to the solution tube and mix thoroughly using a pipette; Incubate in a warm room for 2min, place the PCR tube containing the solution in a magnetic stand for 3min until it is clear; Remove the supernatant, continue to place the PCR tube in the magnetic stand, add 200uL of 80% ethanol solution to the PCR tube, and blow the magnetic bead surface with a pipette; Remove the supernatant, add 200uL of 80% ethanol solution to the PCR tube, and blow the magnetic bead surface with a pipette to completely remove the supernatant; Incubate in a warm room for 5min to allow the residual ethanol to evaporate completely; Add 35.5uL of Nuclease-free water, remove the PCR tube from the magnetic stand, resuspend the magnetic beads by pipetting, and incubate in a warm room for 5min; Place the PCR tube on the magnetic stand for 2 minutes, use a pipette to aspirate 35 μL of the supernatant, and transfer it to a new PCR tube to obtain supernatant A.

[0023] Furthermore, the supernatant A is subjected to connector ligation and purification to obtain supernatant B, comprising the following steps: Add 11.5 μL of Ligation Buffer, 1 μL of T4 DNA Ligase, and 2.5 μL of Adaptor Oligo Mix to 35 μL of supernatant A and vortex. The vortexed solution was centrifuged and then incubated at 20°C in a Thermomixer for 30 min. Add 45 μL of Inlighten magnetic beads to the PCR tube and use a pipette to mix the solution by pipetting several times; The solution in the PCR tube was incubated in a greenhouse for 5 min, and the PCR tube was placed on a magnetic stand for 3 min; Remove the supernatant from the PCR tube, place the PCR tube on the magnetic stand, add 200 μL of 80% ethanol solution to the PCR tube, and wash the surface of the magnetic beads with a pipette; Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Place in a greenhouse and let it stand for 5 minutes to allow the residual ethanol to evaporate completely; Add 35.5 μL of Nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by pipetting. Let it stand at room temperature for 5 minutes. Place the PCR tube on a magnetic stand for 2 minutes, use a pipette to aspirate 35 μL of supernatant, and transfer it to a new PCR tube to prepare supernatant B.

[0024] In this embodiment, The reaction system is as follows: Furthermore, the supernatant B is subjected to pre-PCR and purification treatment to obtain a pre-PCR product, comprising the following steps: Prepare 35 μL of supernatant B, 10 μL of 5× Herculase II Reaction Buffer, 0.5 μL of 100 mM dNTP Mix, 1.5 μL of Forward Primer, 1 μL of Herculase II Fusion DNA Polymerase, and 2 μL of SureSelect XT Low Input Index Primer in a PCR tube; Use a pipette to mix gently by pipetting 6 times, then centrifuge briefly; Place the centrifuged product on a PCR instrument and start the PCR program to process the product; After the PCR program is completed, 2 μL is taken for gel running test. The agarose gel concentration in the gel running test is 2%, the electric field strength is 170 V, and the electrophoresis time is 20 min. Add 48 μL of Inlighten magnetic beads to the PCR tube and mix well by pipetting several times; Incubate in the greenhouse for 5 min, and place the PCR tube on a magnetic stand for 3 min; Remove the supernatant and place the PCR tube on the magnetic stand. Add 200 μL of 80% ethanol solution to the PCR tube and use a pipette to wash the surface of the magnetic beads. Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Let it stand in the greenhouse for 5 minutes to allow the residual ethanol to evaporate completely; Add 35 μL of Nuclease-free water, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by pipetting. Let it stand at room temperature for 5 minutes. Place the PCR tube on a magnetic stand for 2 minutes; Use a pipette to aspirate 35 μL of the supernatant and transfer it to a new 1.5 mL tube to obtain the pre-PCR product.

[0025] In this embodiment, The reaction system was configured as follows: The PCR procedure is as follows: PCR amplification cycle parameters are as follows: Furthermore, the liquid phase hybridization and capture of the pre-PCR product comprises the following steps: Transfer 500-1000 ng of pre-PCR product into a new centrifuge tube and concentrate the product to 12 μL in a vacuum concentrator at 40°C. Add 5 μL of SureSelect XT HS and XT Low Input Blocker Mix to 12 μL of concentrated product, shake to mix, and centrifuge. The PCR program was pre-set as follows: Step 1: 95°C for 5 minutes, one cycle; Step 2: 65°C for 10 minutes, one cycle; Step 3: 65°C for 1 minute, one cycle with a pause point; Step 4: 65°C for 1 minute followed by 37°C for 3 seconds, 60 cycles; Step 5: Hold at 65°C, one cycle; Transfer 17 μL of the total mixed solution to a 200 μL PCR tube and perform the PCR reaction according to the preset PCR program. Prepare the probe mix 15 minutes before the reaction. When the Capture Library is less than 3 Mb, the probe configuration ratio is: 0.5 μL RNase Block solution, 2 μL Capture Library probe, 4.5 μL RNase-free water, and 6 μL SureSelect Fast Hybridization Buffer; when the Capture Library is ≥3 Mb, the probe configuration ratio is: 0.5 μL RNase Block solution, 5 μL Capture Library probe, 1.5 μL RNase-free water, and 6 μL SureSelect Fast Hybridization Buffer; Pause the PCR reaction at step 3, keep the DNA + Blocker on the PCR instrument, add 13 μL of probe mix to the DNA + Blocker, mix thoroughly by pipetting, centrifuge briefly, and quickly return to the PCR instrument to continue the subsequent reaction; Aliquot Wash Buffer 2 in advance and preheat it to 70°C in a ThermoMixer. Remove the magnetic beads from 4°C and resuspend by vortexing; For each sample, 50 μL of magnetic beads was placed in a new 1.5 mL centrifuge tube, placed on a magnetic stand for 1 min, and the supernatant was removed; Remove the centrifuge tube from the magnetic stand, add 200 μL of SureSelect Binding buffer and mix by pipetting several times, resuspend the magnetic beads, place on the magnetic stand for 1 minute, remove the supernatant, and repeat this step twice, for a total of three washes. Remove the centrifuge tube from the magnetic stand, add 200 μL of SureSelect Binding buffer, pipette and pipette 6 times to resuspend the magnetic beads for later use; GenMagbio fully automated purification instrument was used to capture the target gene sequence and wash the beads six times with Wash Buffer 2. When the hybridization reaction is complete and reaches the 65°C hold, remove the reaction tube from the PCR instrument, gently remove it, and place it at room temperature; Add reagents to a 96-well deep-well plate: well 1 or 7: 200ul Wash Buffer 2; well 2 or 8: 30ul hybridization product and 200ul washed MyOne T1 magnetic beads; well 3 or 9: 200ul Wash Buffer 1; well 6 or 12: 200ul Wash Buffer 2. Perform the automated purification instrument operation according to the preset program 1. After program 1 is completed, remove the deep-well plate and place it on a 96-well magnetic plate. Allow the plate to adhere for 2 minutes. Remove the used Wash Buffer 2 supernatant from the wells in columns 1 or 7 and 6 or 12. Add 200 μL of preheated Wash Buffer 2 to the wells in columns 1 or 7 and 6 or 12. Return the deep-well plate to the instrument's original position and start program 2. After the second program is finished, remove the deep-well plate and place it on a 96-well magnetic plate. Allow it to magnetically absorb for 2 minutes. Remove the used Wash Buffer 2 supernatant from the 1st or 7th column and the 6th or 12th column. Then, add 200 μL of preheated Wash Buffer 2 to the 1st or 7th column and the 6th or 12th column. At the same time, add 41 μL of Nuclease-free water to the 4th or 10th column. Then, place the deep-well plate in the machine and start the third program. After the third program is finished, remove the deep-well plate and aspirate 37.5 μL of the target gene capture product bound to magnetic beads from well 4 or 10 for post-PCR. Add 30 μL of the hybridized product to 200 μL of MyOne T1 magnetic beads, pipette up and down 6 times to mix, and place on a rotary mixer at 10 rpm / min for 30 min at room temperature; Place the centrifuge tube on a magnetic stand for 2 minutes and remove the supernatant; Add 200 μL of Wash Buffer 1 to the centrifuge tube, pipette up and down 15-20 times to mix, resuspend the magnetic beads, and place on a magnetic stand for 1 minute. Remove the supernatant. Add 200 μL of 70°C preheated Wash Buffer 2, vortex mix for 5 seconds, centrifuge, and incubate at 70°C for 5 minutes on a ThermoMixer. After a brief centrifugation, place the centrifuge tube on a magnetic stand for 2 minutes, remove the supernatant, and repeat the wash 5 times, for a total of 6 times. Finally, completely remove Wash Buffer 2 for the last time. Add 38 μL of Nuclease-free water to the centrifuge tube, remove the centrifuge tube from the magnetic stand, and pipette 6 times to resuspend the magnetic beads.

[0026] In this embodiment, The preset PCR program is as follows: The hybridization probe Mix configuration reaction system is as follows: Procedure 1 is as follows: Procedure 2 is as follows: Furthermore, the configuration of the PCR reaction system for post-PCR reaction and purification to obtain supernatant C comprises the following steps: Prepare the PCR reaction system in a 0.2 mL centrifuge tube, including 37.5 μL of bead-bound target-enriched DNA samples, 10 μL of 5× Herculase II Reaction Buffer, 1 μL of Herculase II Fusion DNA Polymerase, 0.5 μL of 100 mM dNTP Mix, and 1 μL of SureSelect Post-Capture Primer Mix; Mix the prepared PCR reaction system by pipetting and pumping 6 times, and then place it on the PCR instrument; Run the PCR instrument to cycle the reaction system; After the PCR instrument finishes running, vortex the PCR product to mix it evenly, centrifuge it at a light speed, add 70 μL of Inlighten magnetic beads to the PCR product, and pipette it up and down 6 times to mix it evenly; Incubate in the greenhouse for 5 min, then place the PCR tube on a magnetic stand for 3 min; The supernatant was removed, and the PCR tube was placed on the magnetic stand again. 200 μL of 80% anhydrous ethanol was added and allowed to stand for 30 seconds. Remove the supernatant and add 200 μL of 80% anhydrous ethanol to the PCR tube. Let it stand for 30 seconds and then completely remove the supernatant. Place in the greenhouse for 5 minutes to allow the residual ethanol to evaporate; Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic stand, pipette to mix and resuspend the magnetic beads, and place in the room temperature for 2 minutes; Place the PCR tube on a magnetic stand for 2 minutes; Use a pipette to draw 24.5 μL of the supernatant into a 1.5 mL centrifuge tube to prepare supernatant C.

[0027] In this embodiment, Configure the PCR reaction system as follows: The PCR instrument operation program is as follows: The above disclosure is only one embodiment of the present invention, and certainly cannot be used to limit the scope of the rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A gene panel sequencing method for intrahepatic cholestasis of pregnancy, characterized in that: The steps include: Conduct sample collection; DNA extraction and purification of collected samples; Shear the purified genome and concentrate the sheared sample; The concentrated sheared samples were subjected to end-filling and 3' A addition to obtain supernatant A; The supernatant A is connected to a linker and purified to obtain supernatant B; Perform pre-PCR and purification on supernatant B to obtain pre-PCR products; Liquid phase hybridization and capture of pre-PCR products; Prepare a PCR reaction system for Post-PCR reaction and purification to obtain supernatant C; 1.0 μL of supernatant C was taken as the library, and the library was quantified using the Qubit dsDNA HS Assay Kit. The library concentration was recorded, and mixed sequencing was immediately performed to screen for ICP susceptibility genes.

2. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein the sample collection is performed, The steps include: Use sterile individually packaged test tubes to collect 3 mL of saliva to be tested according to the operating instructions; After collection, the samples are stored in preservation solution and transported using ice packs.

3. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein DNA extraction and purification of the collected samples is performed, The steps include: Use the kit to extract and purify the DNA in the saliva to be tested; Use nanodrop to preliminarily test the concentration and purity of DNA; Use Qubit 3.0 to accurately detect DNA concentration; The integrity of the DNA was checked by agarose gel electrophoresis.

4. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein the purified genome is fragmented and the fragmented samples are concentrated, wherein: The steps include: Mix genomic DNA with nuclease-free water to a total volume of 40uL, transfer to a low-adsorption centrifuge tube, mix thoroughly, and centrifuge briefly; Turn on the ultrasonic disruptor Bioruptor Pico in advance. After the cold cycler temperature reaches 4°C, set the parameters to ON 30s, OFF 30s as one cycle. Perform 30 cycles of disruption for whole blood DNA, 20 cycles for tissue samples, and 20 cycles for paraffin-embedded DNA. Place each group of disrupted samples on an oscillator to mix thoroughly. Take 1.2 μL of sample and add 8.8 μL of DNA buffer for fragment detection using QSEP 100. After fragmentation, the main peak of the sample detection is between 150 bp and 200 bp; The fragmented sample was concentrated to a volume of 25 μL using a vacuum freeze concentrator.

5. A gene panel sequencing method for intrahepatic cholestasis of pregnancy according to claim 1, wherein the concentrated fragmented sample is subjected to end-filling and 3' A addition to obtain supernatant A, characterized in that: The steps include: Add 8 μL of End Repair-A Tailing Buffer and 2 μL of End Repair-A Tailing Enzyme Mix to 25 μL of the disrupted sample and vortex. The vortexed solution was centrifuged and then incubated in a Thermomixer at 20°C for 15 min, 72°C for 15 min, and extended at 4°C. Add 56 μL of Inlighten magnetic beads to the solution tube and mix by pipetting several times; Incubate in the greenhouse for 2 min, and place the PCR tube containing the solution on a magnetic stand for 3 min until clear; Remove the supernatant and place the PCR tube on the magnetic stand. Add 200 μL of 80% ethanol solution to the PCR tube and wash the surface of the magnetic beads with a pipette. Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Let it stand in the greenhouse for 5 minutes to allow the residual ethanol to evaporate completely; Add 35.5 μL of nuclease-free water, remove the PCR tube from the magnetic stand, resuspend the magnetic beads by pipetting, and let it stand in the room for 5 minutes; Place the PCR tube on the magnetic stand for 2 minutes, use a pipette to aspirate 35 μL of the supernatant, and transfer it to a new PCR tube to obtain supernatant A.

6. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein the supernatant A is subjected to linker ligation and purification to obtain supernatant B, wherein: The steps include: Add 11.5 μL of Ligation Buffer, 1 μL of T4 DNA Ligase, and 2.5 μL of Adaptor Oligo Mix to 35 μL of supernatant A and vortex. The vortexed solution was centrifuged and then incubated at 20°C in a Thermomixer for 30 min. Add 45 μL of Inlighten magnetic beads to the PCR tube and use a pipette to mix the solution by pipetting several times; The solution in the PCR tube was incubated in a greenhouse for 5 min, and the PCR tube was placed on a magnetic stand for 3 min; Remove the supernatant from the PCR tube, place the PCR tube on the magnetic stand, add 200 μL of 80% ethanol solution to the PCR tube, and wash the surface of the magnetic beads with a pipette; Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Place in a greenhouse and let it stand for 5 minutes to allow the residual ethanol to evaporate completely; Add 35.5 μL of Nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by pipetting. Let it stand at room temperature for 5 minutes. Place the PCR tube on a magnetic stand for 2 minutes, use a pipette to aspirate 35 μL of supernatant, and transfer it to a new PCR tube to prepare supernatant B.

7. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein the supernatant B is subjected to pre-PCR and purification to obtain a pre-PCR product, wherein: The steps include: In a PCR tube, prepare 35 μL of supernatant B, 10 μL of 5× Herculase II Reaction Buffer, 0.5 μL of 100 mM dNTP Mix, 1.5 μL of Forward Primer, 1 μL of Herculase II Fusion DNA Polymerase, and 2 μL of SureSelect XT Low Input Index Primer; Use a pipette to mix gently by pipetting 6 times, then centrifuge briefly; Place the centrifuged product on a PCR instrument and start the PCR program to process the product; After the PCR program is completed, 2 μL is taken for gel running test. The agarose gel concentration in the gel running test is 2%, the electric field strength is 170 V, and the electrophoresis time is 20 min. Add 48 μL of Inlighten magnetic beads to the PCR tube and mix well by pipetting several times; Incubate in the greenhouse for 5 min, then place the PCR tube on a magnetic stand for 3 min; Remove the supernatant and place the PCR tube on the magnetic stand. Add 200 μL of 80% ethanol solution to the PCR tube and use a pipette to wash the surface of the magnetic beads. Remove the supernatant and add 200 μL of 80% ethanol solution to the PCR tube. Use a pipette to wash the surface of the magnetic beads and completely remove the supernatant. Let it stand in the greenhouse for 5 minutes to allow the residual ethanol to evaporate completely; Add 35 μL of Nuclease-free water, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by pipetting. Let it stand at room temperature for 5 minutes. Place the PCR tube on a magnetic stand for 2 minutes; Use a pipette to aspirate 35 μL of the supernatant and transfer it to a new 1.5 mL tube to obtain the pre-PCR product.

8. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein the pre-PCR product is subjected to liquid phase hybridization and capture, wherein: The steps include: Transfer 500-1000 ng of pre-PCR product into a new centrifuge tube and concentrate the product to 12 μL in a vacuum concentrator at 40°C. Add 5 μL of SureSelect XT HS and XT Low Input Blocker Mix to 12 μL of concentrated product, shake to mix, and centrifuge. The PCR program was pre-set as follows: Step 1: 95°C for 5 minutes, one cycle; Step 2: 65°C for 10 minutes, one cycle; Step 3: 65°C for 1 minute, one cycle with a pause point; Step 4: 65°C for 1 minute followed by 37°C for 3 seconds, 60 cycles; Step 5: Hold at 65°C, one cycle; Transfer 17 μL of the total mixed solution into a 200 μL PCR tube and perform the PCR reaction according to the preset PCR program. Prepare the probe mix 15 min before the reaction. When Capture Library is less than 3Mb, the probe configuration ratio is: 0.5 μL RNase Block solution, 2 μL Capture Library probe, 4.5 μL RNase-free water, and 6 μL SureSelect FastHybridization Buffer. When the Capture Library is ≥3 Mb, the probe configuration ratio is: 0.5 μL RNase Block solution, 5 μL Capture Library probe, 1.5 μL RNase-free water, and 6 μL SureSelectFast Hybridization Buffer; Pause the PCR reaction at step 3, keep the DNA + Blocker on the PCR instrument, add 13 μL of probe mix to the DNA + Blocker, mix thoroughly by pipetting, centrifuge briefly, and quickly return to the PCR instrument to continue the subsequent reaction; Aliquot Wash Buffer 2 in advance and preheat it to 70°C in a ThermoMixer. Remove the magnetic beads from 4°C and resuspend by vortexing; For each sample, 50 μL of magnetic beads was placed in a new 1.5 mL centrifuge tube, placed on a magnetic stand for 1 min, and the supernatant was removed; Remove the centrifuge tube from the magnetic stand, add 200 μL of SureSelect Binding buffer and mix by pipetting several times, resuspend the magnetic beads, place on the magnetic stand for 1 minute, remove the supernatant, and repeat this step twice, for a total of three washes. Remove the centrifuge tube from the magnetic stand, add 200 μL of SureSelect Binding buffer, pipette and pipette 6 times to resuspend the magnetic beads for later use; GenMagbio fully automated purification instrument was used to capture the target gene sequence and wash the beads six times with Wash Buffer 2. When the hybridization reaction is complete and reaches the 65°C hold, remove the reaction tube from the PCR instrument, gently remove it, and place it at room temperature; Add reagents to a 96-well deep-well plate: well 1 or 7: 200ul Wash Buffer 2; well 2 or 8: 30ul hybridization product and 200ul washed MyOne T1 magnetic beads; well 3 or 9: 200ul Wash Buffer 1; well 6 or 12: 200ul Wash Buffer 2. Perform the automated purification instrument operation according to the preset program 1. After program 1 is completed, remove the deep-well plate and place it on a 96-well magnetic plate. Allow the plate to adhere for 2 minutes. Remove the used Wash Buffer 2 supernatant from the wells in columns 1 or 7 and 6 or 12. Add 200 μL of preheated Wash Buffer 2 to the wells in columns 1 or 7 and 6 or 12. Return the deep-well plate to the instrument's original position and start program 2. After the second program is finished, remove the deep-well plate and place it on a 96-well magnetic plate. Allow it to magnetically absorb for 2 minutes. Remove the used Wash Buffer 2 supernatant from the 1st or 7th column and the 6th or 12th column. Then, add 200 μL of preheated Wash Buffer 2 to the 1st or 7th column and the 6th or 12th column. At the same time, add 41 μL of Nuclease-free water to the 4th or 10th column. Then, place the deep-well plate in the machine and start the third program. After the third program is finished, remove the deep-well plate and aspirate 37.5 μL of the target gene capture product bound to magnetic beads from well 4 or 10 for post-PCR. Add 30 μL of the hybridized product to 200 μL of MyOne T1 magnetic beads, pipette up and down 6 times to mix, and place on a rotary mixer at 10 rpm / min for 30 min at room temperature; Place the centrifuge tube on a magnetic stand for 2 minutes and remove the supernatant; Add 200 μL of Wash Buffer 1 to the centrifuge tube, pipette up and down 15-20 times to mix, resuspend the magnetic beads, and place on a magnetic stand for 1 minute. Remove the supernatant. Add 200 μL of 70°C preheated Wash Buffer 2, vortex mix for 5 seconds, centrifuge, and incubate at 70°C for 5 minutes on a ThermoMixer. After a brief centrifugation, place the centrifuge tube on a magnetic stand for 2 minutes, remove the supernatant, and repeat the wash 5 times, for a total of 6 times. Finally, completely remove Wash Buffer 2 for the last time. Add 38 μL of Nuclease-free water to the centrifuge tube, remove the centrifuge tube from the magnetic stand, and pipette 6 times to resuspend the magnetic beads.

9. The method for sequencing a gene panel for intrahepatic cholestasis of pregnancy according to claim 1, wherein the PCR reaction system is configured to perform a post-PCR reaction and purification to obtain supernatant C, characterized in that: The steps include: Prepare the PCR reaction system in a 0.2 mL centrifuge tube, including 37.5 μL of bead-bound target-enriched DNA sample, 10 μL of 5× Herculase II Reaction Buffer, 1 μL of Herculase II Fusion DNA Polymerase, 0.5 μL of 100 mM dNTP Mix, and 1 μL of SureSelect Post-Capture Primer Mix; Mix the prepared PCR reaction system by pipetting and pumping 6 times, and then place it on the PCR instrument; Run the PCR instrument to cycle the reaction system; After the PCR instrument finishes running, vortex the PCR product to mix it evenly, centrifuge it at a light speed, add 70 μL of Inlighten magnetic beads to the PCR product, and pipette it up and down 6 times to mix it evenly; Incubate in the greenhouse for 5 min, then place the PCR tube on a magnetic stand for 3 min; The supernatant was removed, and the PCR tube was placed on the magnetic stand again. 200 μL of 80% anhydrous ethanol was added and allowed to stand for 30 seconds. Remove the supernatant and add 200 μL of 80% anhydrous ethanol to the PCR tube. Let it stand for 30 seconds and then completely remove the supernatant. Place in the greenhouse for 5 minutes to allow the residual ethanol to evaporate; Add 25 μL of Nuclease-free water, remove the PCR tube from the magnetic stand, pipette to mix and resuspend the magnetic beads, and place in the room temperature for 2 minutes; Place the PCR tube on a magnetic stand for 2 minutes; Use a pipette to draw 24.5 μL of the supernatant into a 1.5 mL centrifuge tube to prepare supernatant C.

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