Saliva sample-based multiple PCR-NGS screening method for susceptible genes of intrahepatic cholestasis in gestation period

Abstract

The invention belongs to the technical field of clinical gene screening in obstetrics, particularly relates to a multiple PCR-NGS screening method for susceptible genes of intrahepatic cholestasis in pregnancy based on a saliva sample, and aims to solve the problems of high cost and long time consumption of existing ICP gene Panel detection. Specific primers of 40 high-frequency mutation sites of 9 ICP susceptible genes are designed, a sequencing library is constructed through two rounds of PCR amplification and magnetic bead purification, and mutation information is obtained through NGS sequencing and data analysis. The method is non-invasive and adapts to pregnancy requirements, the detection cost and time can be reduced, susceptible gene mutation can be screened out, and support is provided for genetic etiological investigation and clinical intervention of severe and refractory ICP (inductively coupled plasma).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of obstetric clinical gene screening, and particularly relates to a multiple PCR-NGS screening method for intrahepatic cholestasis of pregnancy susceptible genes based on a saliva sample. BACKGROUND

[0002] Intrahepatic cholestasis of pregnancy (ICP) is a common idiopathic complication in the middle and late stages of pregnancy in obstetric clinics, and its onset is closely related to the synergistic effect of genetics, hormones and environmental factors, which poses a significant threat to maternal and infant health. According to literature and clinical data, the global ICP incidence rate fluctuates between <1% and 27.6%, and the ICP prevalence rate of pregnant women in China is as high as 1.2%-6.06%, which is much higher than that in some European and American countries. Severe ICP is defined as serum total bile acid ≥40μmol / L, and the incidence of perinatal adverse outcomes (such as preterm birth, perinatal fetal death, and neonatal respiratory distress syndrome) of such patients is significantly increased. When serum total bile acid is ≥100μmol / L, the treatment and monitoring scheme recommended by the existing clinical guidelines is difficult to effectively reduce the risk of adverse outcomes, and the etiological exploration and precise intervention of severe and refractory ICP have become a clinical problem to be solved.

[0003] From the perspective of pathogenesis, the genetic basis of ICP is complex, but its family aggregation, increased risk of first-degree relatives, and high recurrence rate (60%-70%) confirm that genetic factors play a central role in the onset. Existing studies have found that ICP onset is closely related to mutations in multiple bile acid metabolism-related genes, including the ABCB4 gene encoding the membrane phospholipid translocation protein of the capillary bile duct (MDR3), the ABCB11 gene encoding the bile acid transporter protein, the ATP8B1 and ABCC2 genes regulating bile acid uptake and excretion; In particular, a recent whole-genome study covering nearly 100,000 people suggests that there is a special ICP-related susceptible gene SLC10A1 mutation in East Asian and Southeast Asian populations, which also provides a key target direction for ICP genetic screening in this population. Under normal physiological conditions, bile acids are maintained stable through the "synthesis-secretion-reabsorption-liver uptake" enterohepatic circulation, while mutations in the above genes can cause abnormalities in bile acid metabolism and transport, and under the action of high levels of estrogen during pregnancy, eventually trigger different severity of intrahepatic cholestasis symptoms.

[0004] To explore the genetic cause of ICP, in the early stage of the application, a saliva sample-based ICP gene panel detection technology covering 186 bile acid metabolism-related genes was used to detect 112 ICP patients saliva samples, the results showed that 104 patients (detection rate 92.86%) had gene mutations, and the high-frequency mutations were concentrated in 40 sites of 9 genes ABCB4, ABCB11, ATP8B1, ABCC2, SLC10A1, ATP7B, UGT1A1, MYO5B and TJP2, only 8 patients were not detected for mutations. However, the gene panel detection technology has significant limitations: on the one hand, the detection cost is high (about 1500 yuan), and the detection period is as long as 4 weeks, which is difficult to meet the needs of clinical rapid screening and timely intervention for ICP patients; on the other hand, only 9 of the 186 genes are high-frequency mutation genes (accounting for 4.84%), and the wide range of gene coverage leads to waste of detection resources and increases the complexity of clinical interpretation.

[0005] Therefore, in view of the actual clinical needs, developing a low-cost, short-cycle, target-focused high-frequency mutation site ICP susceptible gene screening technology has become the key direction to solve the pain points of the prior art and promote the clinical popularization of ICP genetic screening. SUMMARY

[0006] In view of the above deficiencies in the prior art, the present application provides a saliva sample-based intrahepatic cholestasis of pregnancy susceptible gene multiplex PCR-NGS screening method to solve the problems in the above background art.

[0007] To solve the above technical problems, the application adopts the following technical solutions: The saliva sample-based intrahepatic cholestasis of pregnancy susceptible gene multiplex PCR-NGS screening method comprises the following steps: A1, saliva sample DNA extraction: collect 1-2ml saliva of pregnant women, extract and purify DNA in the saliva of pregnant women by using a kit, and screen saliva DNA samples by concentration, purity and integrity detection; A2, multiplex PCR primer design and grouping: specific primers are designed for 40 high-frequency mutation sites of 9 intrahepatic cholestasis of pregnancy susceptible genes, and the same gene position is close to the mutation using the same group of specific primers for amplification, and a primer pool is constructed; A3, two-round PCR amplification and library construction: using saliva DNA sample as template, first-round multiplex PCR amplification is carried out with primer pool, and after obtaining amplification product, it is purified by magnetic bead to obtain purified product; the purified product is subjected to second-round PCR amplification to construct sequencing library containing 8bp molecular tag, and the library is purified by magnetic bead; A4, high-throughput sequencing: after quantification of the sequencing library, sequencing is performed on the Illumina sequencing platform to obtain sequencing data; A5, data analysis: quality control, alignment, variant detection and annotation of the sequencing data, and output of genotype information of 40 high-frequency mutation sites of women in the gestational period.

[0008] Further, in step A1, the saliva is collected by a sterile single-pack test tube, and after collection, it is stored at 4°C in a preservation solution; when screening, the concentration and purity of the DNA are preliminarily detected using Nanodrop, and the concentration of the DNA is accurately detected using Qubit 3.0, and the integrity of the DNA is detected by agarose gel electrophoresis.

[0009] Further, in step A2, the 40 high-frequency mutation sites of the 9 intrahepatic cholestasis of pregnancy susceptibility genes are determined by screening the saliva samples of female patients with intrahepatic cholestasis of pregnancy using Panel sequencing technology; the 9 intrahepatic cholestasis of pregnancy susceptibility genes include SLC10A1, ATP7B, UGT1A1, ABCB4, ABCB11, ATP8B1, TJP2, MYO5B, and ABCC2; the 3' end of the specific primer contains at least 2 bases complementary to the target mutation site, and there is no self-complementary or inter-primer complementary sequence.

[0010] Further, in step A3, in the first round of multiplex PCR amplification, the reaction system includes: KAPA2G FastMultiplex Mix 5μL, one round of mixed primers 2μL, saliva DNA sample 1μL, and ddH2O 2μL; the reaction system is subjected to the first amplification by starting the PCR program on a PCR instrument, and the first reaction system product is obtained and stored at 4°C.

[0011] Further, the specific steps of the first amplification are as follows: B1, 95°C pre-denaturation for 5min; B2, annealing and extension: 65°C for 30s in the annealing stage, and then 72°C for 1min in the extension stage; a total of 5 cycles, with the temperature of the annealing stage decreasing by 1°C each cycle; B3, fixed temperature cycle amplification: 94°C denaturation for 30s, 60°C annealing for 30s, and 72°C extension for 1min, a total of 20-30 cycles; B4, final extension and storage: 72°C final extension for 5min, and after the reaction is completed, the first reaction system product is stored at 4°C.

[0012] Further, in step A3, the second round of PCR amplification, the reaction system comprises: KOD enzyme solution 20 μL, second round index primer 1 μL, primer 1.0 1 μL and first reaction system product 10 μL; the second round of amplification is started by a PCR instrument, and a second reaction system product is obtained, which is stored at 4 DEG C.

[0013] Further, the specific steps of the second round of amplification are as follows: C1, pre-denaturation: 94 DEG C pre-denaturation for 2 min; C2, cycle amplification: 94 DEG C denaturation for 15 s, 62 DEG C annealing for 30 s, 72 DEG C extension for 30 s, and 7 cycles of amplification are performed; C3, final extension and storage: 72 DEG C final extension for 10 min, and after the reaction is completed, the product is stored at 4 DEG C.

[0014] Further, in step A3, the magnetic beads are Yinglai shield magnetic beads; after the first round of multiplex PCR amplification, 16 μL Yinglai shield magnetic beads are added for purification in the first reaction system product; after the second round of PCR amplification, 32 μL Yinglai shield magnetic beads are added for purification in the second reaction system product.

[0015] Further, the step of magnetic bead purification comprises the following steps: incubation at room temperature for 5 min, clarification by being placed in a magnetic stand for 3 min, removal of supernatant, washing of the surface of the magnetic beads with 200 μL 80% ethanol solution twice, room temperature standing for 5 min to volatilize residual ethanol, addition of nuclease-free water to resuspend the magnetic beads and room temperature standing for 5 min, and finally, suction of supernatant.

[0016] Compared with the prior art, the present application has the following beneficial effects: 1. In view of the high cost and long time of the early gene Panel detection technology, 40 high-frequency mutation sites of 9 susceptible genes verified by ICP patient saliva samples are focused on, and the multiplex PCR-NGS technology is used to realize targeted detection, which significantly reduces the cost of money and time, and is more suitable for large-scale, multi-center ICP susceptible gene screening work in clinical.

[0017] 2. Saliva is selected as the DNA collection sample, and no puncture operation is needed, which can effectively reduce the physiological discomfort and psychological anxiety of pregnant women, and the sample collection compliance is significantly higher than that of invasive methods such as venous blood; at the same time, through optimization of the saliva preservation and extraction process, the DNA quality is guaranteed, the subsequent detection requirements are met, and the special needs during pregnancy are adapted.

[0018] 3. Ensure the accuracy of detection by triple precision design: first, design specific primers for 40 high-frequency mutation sites, 3' end contains at least 2 bases complementary to the target mutation, to avoid non-specific binding; second, introduce 8bp UMI molecular tags in two rounds of PCR amplification, effectively eliminate PCR repetitive sequences caused by saliva DNA degradation, improve mutation detection accuracy; third, the magnetic bead purification adopts targeted proportion (1.6x in the first round, 1x in the second round), which is suitable for the low concentration characteristics of saliva amplification product; finally, the ultra-high depth coverage of 40 sites can be realized, which provides support for clinical diagnosis of severe and refractory ICP susceptible genes. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The flow chart of the present application based on saliva sample for intrahepatic cholestasis of pregnancy susceptible gene multiplex PCR-NGS screening method. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the present application, the technical solutions of the present application are further described below in combination with the drawings and examples.

[0021] Among them, the drawings are only used for illustrative explanation, and the representation is only a schematic diagram, not a physical diagram, and cannot be understood as a limitation on the present application; in order to better illustrate the embodiments of the present application, some components of the drawings will be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some known structures and their descriptions in the drawings can be omitted.

[0022] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components; in the description of the present application, it should be understood that if the terms "upper", "lower", "left", "right", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, therefore the terms describing the positional relationship in the drawings are only used for illustrative explanation, and cannot be understood as a limitation on the present application, for those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0023] In the description of the present application, unless otherwise explicitly specified and limited, if the term "connection" and the like appear to indicate the connection relationship between components, the term should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or it can be integrated; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the internal communication of two components or the interaction relationship between two components. For those skilled in the art, the specific meaning of the above-mentioned terms in the present application can be understood according to the specific circumstances.

[0024] The present application takes saliva as the DNA collection object, has non-invasiveness and high compliance, compared with the invasive sampling mode such as venous blood, saliva collection does not need puncture operation, can effectively reduce the psychological burden and physiological discomfort of women during pregnancy, is more easily accepted by the examinee, is especially suitable for this special physiological stage of pregnancy, greatly improves the convenience and coverage rate of sample collection. Secondly, saliva is rich in oral epithelial cells, which can release a sufficient amount of stable genomic DNA, and the DNA concentration, purity and integrity can meet the requirements of multiple PCR amplification and high-throughput sequencing (NGS) on templates, and can stably support accurate detection. In addition, the storage and transportation conditions of saliva samples are loose, short-term storage can be realized at 4°C low temperature, without complex cold chain support, reducing the sample management cost, facilitating the clinical development of large-scale, multi-center pregnancy intrahepatic cholestasis (ICP) susceptible gene screening work, providing feasibility guarantee for the wide application of technology.

[0025] Secondly, it should be noted that when sampling the pregnant women, the objects infected by hepatitis virus, EB virus, cytomegalovirus and the like should be excluded, and it is recommended to routinely perform ultrasonic examination to exclude the hepatobiliary diseases of the pregnant women during pregnancy, and the detection of common cholestasis gene high-frequency mutation sites can further determine the susceptible gene mutation or exclude genetic metabolic cholestasis and the like diseases.

[0026] The present application first designs an intrahepatic cholestasis of pregnancy (ICP) gene Panel detection technology based on saliva detection, covering 186 bile acid metabolism related genes, 112 ICP patient saliva samples are subjected to cholestasis gene Panel detection, 40 site gene mutations containing 9 genes are detected in 104 patients, 8 patients are negative, as shown in Table 1.

[0027] Table 1 40 high-frequency mutation sites of 9 intrahepatic cholestasis of pregnancy susceptible genes Note: *Inheritance: AD, autosomal dominant inheritance; AR, autosomal recessive inheritance. Test results: homozygous mutation: a pair of alleles on the chromosome both have mutations; heterozygous mutation: only one gene of a pair of alleles on the chromosome has a mutation.

[0028] In view of the characteristics of high cost and long time of cholestasis gene panel detection technology, and the high frequency mutation gene only accounts for 4.84%, the present application is based on the 40 high frequency mutation sites of the 9 intrahepatic cholestasis of pregnancy susceptible genes screened out by the previous intrahepatic cholestasis of pregnancy gene panel sequencing technology, and a saliva sample based intrahepatic cholestasis of pregnancy susceptible gene multiplex PCR-NGS screening method is invented, aiming to solve the problems of high cost and long time of the existing cholestasis gene panel detection technology, realize the rapid and accurate screening of intrahepatic cholestasis of pregnancy susceptible gene mutations in pregnant women, and provide technical support for the genetic etiology elucidation of severe and refractory intrahepatic cholestasis of pregnancy.

[0029] As Figure 1 , the saliva sample based intrahepatic cholestasis of pregnancy susceptible gene multiplex PCR-NGS screening method of the present application comprises the following steps: A1, saliva sample DNA extraction: collect 1-2ml of saliva of a pregnant woman, extract and purify the DNA in the saliva of the pregnant woman by using a kit, and screen the saliva DNA sample by concentration, purity and integrity detection; A2, multiplex PCR primer design and grouping: specific primers are designed for 40 high frequency mutation sites of 9 intrahepatic cholestasis of pregnancy susceptible genes, and the same group of specific primers is used for amplification of mutations close to the same gene position, and a primer pool is constructed; A3, two rounds of PCR amplification and library construction: using saliva DNA sample as template, first round of multiplex PCR amplification is carried out with primer pool, and after obtaining amplification product, it is purified by magnetic beads to obtain purified product; the purified product is subjected to second round of PCR amplification to construct sequencing library containing 8bp molecular tag (UMI), and the library is purified by magnetic beads; A4, high-throughput sequencing: after quantifying the sequencing library, sequencing is carried out on Illumina sequencing platform to obtain sequencing data; A5, data analysis: quality control, alignment, variation detection and annotation are carried out on the sequencing data, and genotype information of 40 high frequency mutation sites of the pregnant woman is output.

[0030] Specifically, in the step of saliva sample DNA extraction in A1, 1-2 ml of saliva of a female in the gestation period is collected in a sterile single-pack test tube according to the standard, and after collection, the saliva is stored at 4°C in a storage solution and is transported to the laboratory as soon as possible to avoid degradation of DNA due to long-time storage. DNA is extracted and purified using a commercial kit, the concentration and purity of the DNA are preliminarily detected using a Nanodrop, and the concentration of the DNA is accurately detected using a Qubit 3.0, and the integrity of the DNA is detected by agarose gel electrophoresis. The qualified saliva DNA sample enters the next step of operation.

[0031] In the step of multiplex PCR primer design and grouping in A2, 9 ICP susceptible genes including SLC10A1, ATP7B, UGT1A1, ABCB4, ABCB11, ATP8B1, TJP2, MYO5B and ABCC2, 40 high-frequency mutation sites of the 9 genes are designed with specific primers, the length of the primers is controlled to be 20-25 bp, and the GC content is maintained at 40%-60%, which can ensure stable combination of the primers and the template and high amplification efficiency in the PCR amplification process. The core of the primer design is that the 3' end needs to contain at least 2 bases complementary to the target mutation site, for example, for the SLC10A1 gene c.800C>T mutation, the 3' end of the downstream primer is designed as "TTC", and the last two bases "TC" are complementary to the mutation site, which can effectively avoid non-specific binding of the primer and the wild-type sequence. As shown in Table 2, the specific primers for 40 high-frequency mutation sites of 9 gestational intrahepatic cholestasis susceptible genes are designed. In this step, the same specific primers are used to amplify the mutations close to the same gene position, and finally, a primer pool directly used for the first round of multiplex PCR is constructed.

[0032] Table 2 Specific primer information table of high-frequency mutation sites of gestational intrahepatic cholestasis susceptible genes In A3, the steps of two-round PCR amplification and library construction are the key to achieve the target site amplification and sequencing adaptation. Before the first round of multiplex PCR amplification with primer pool, KAPA2G FastMultiplex Mix, a round of mixed primers and other reagents stored in the kit at -20°C are taken out, dissolved on the ice box, dissolved and gently inverted for mixing 3 times, then kept on ice for standby, to avoid the influence of repeated freezing and thawing on the activity of the reagents. In the PCR tube, prepare a reaction system with a total volume of 10 μL, which includes KAPA2G FastMultiplex Mix 5 μL, a round of mixed primers 2 μL, saliva DNA sample 1 μL and ddH2O 2 μL. The preparation process is carried out on the ice box to prevent premature activation of enzyme activity. Use a pipette to gently pipette 6 times to fully mix the system, then centrifuge briefly; then place the PCR tube on the PCR instrument, start the PCR program: first pre-denature at 95°C for 5 minutes, which can completely unwind the DNA double strands and activate the heat-stable DNA polymerase in KAPA2G FastMultiplex Mix; then 5 cycles of annealing and extension, each cycle has an annealing stage of 65°C for 30 seconds, followed by an extension stage of 72°C for 1 minute, and the annealing temperature of each cycle is 1°C lower than the previous cycle, i.e. the annealing temperatures of the 5 cycles are 65°C, 64°C, 63°C, 62°C and 61°C respectively. Through the drop PCR, the specific binding efficiency of multiple primers and target sites can be improved; then 20-30 cycles of fixed temperature amplification, each cycle includes 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, and 72°C extension for 1 minute, the fixed temperature can realize the exponential amplification of the target fragment; finally, 72°C terminal extension for 5 minutes to ensure the complete synthesis of all amplified fragments. After the reaction is completed, the first reaction system product is stored in a 4°C environment, and the used reagents should be returned to the original reagent box and stored at -20°C to avoid reagent failure.

[0033] After the first round of multiplex PCR amplification, magnetic bead purification is required to remove impurities. Add 16 μL of Yinglai magnetic beads to the reaction system tube, which corresponds to a 1.6x ratio, which can efficiently bind the low concentration of amplification products in the saliva DNA sample. Mix gently with a pipette for 10 times to avoid the formation of bubbles that can cause uneven distribution of magnetic beads. Incubate at room temperature for 5 minutes to allow the DNA to fully adsorb to the surface of the magnetic beads. Place the PCR tube on the magnetic stand for 3 minutes until the liquid in the tube is completely clear to ensure that the magnetic beads are fully adsorbed to the tube wall. Remove the supernatant slowly with a pipette, taking care not to touch the magnetic beads. Leave the PCR tube on the magnetic stand and add 200 μL of 80% ethanol solution to the tube. Gently blow the magnetic bead surface with a pipette for 5 times to remove residual dNTPs and primer dimers. Then remove the supernatant. Add another 200 μL of 80% ethanol solution and repeat the washing step to completely remove the supernatant. Use a 10 μL pipette to carefully remove the residual ethanol solution at the bottom of the tube to prevent the residual ethanol from affecting the subsequent PCR. Incubate at room temperature for 5 minutes to allow the residual ethanol on the surface of the magnetic beads to evaporate completely. Add 10 μL of Nuclease-free water to the tube, remove the PCR tube from the magnetic stand, and resuspend the magnetic beads by gently pipetting 10 times. Incubate at room temperature for 5 minutes to allow the DNA to fully elute from the magnetic beads. Place the PCR tube on the magnetic stand again for 2 minutes, and use a pipette to collect 10 μL of supernatant and transfer it to a new PCR tube. Label the sample number on the new tube, and the supernatant is the purified product of the first round of PCR.

[0034] The core of the second round of PCR amplification is to construct a sequencing library containing UMI. Before amplification, take out the KOD enzyme solution, second round index primer, Primer1.0, and other reagents from the -20°C stored kit, dissolve and mix on ice, and keep on ice for standby. Prepare a reaction system with a total volume of 32 μL in a PCR tube, containing KOD enzyme solution 20 μL, second round index primer 1 μL, Primer1.0 1 μL, and purified product from the first round of PCR 10 μL. The preparation process is carried out on the ice box to ensure enzyme activity. Mix the system gently for 6 times, and then centrifuge briefly. Place the PCR tube in the PCR instrument, and start the PCR program: first, pre-denature at 94°C for 2 minutes to activate the high-fidelity amplification activity of KOD enzyme; then perform 7 cycles of amplification, each cycle including 94°C denaturation for 15 seconds, 62°C annealing for 30 seconds, and 72°C extension for 30 seconds. During this process, the 8bp UMI carried by the second round index primer will be integrated into the amplified fragments, which will be used to distinguish different original DNA molecules in the subsequent amplification products and reduce the interference of repeated sequences. Finally, extend at 72°C for 10 minutes to ensure complete synthesis of all fragments. After the reaction is completed, the second reaction system product is stored at 4°C. The used reagents are returned to the original kit and stored at -20°C.

[0035] After the second round of PCR amplification, magnetic bead purification is performed. 32 μL of Yinglai magnetic beads is added to the product of the second reaction system, which corresponds to a 1x proportion and meets the purification requirements of the sequencing library. The mixture is mixed several times with a pipette to avoid air bubbles. After incubation at room temperature for 5 minutes, the PCR tube is placed on a magnetic stand for 3 minutes until the liquid is clear. The supernatant is removed, and the PCR tube is left on the magnetic stand. 200 μL of 80% ethanol solution is added to wash the surface of the magnetic beads, and then the supernatant is removed. The washing step is repeated once and the residual ethanol is completely removed. The tube is incubated at room temperature for 5 minutes to allow the ethanol to evaporate completely. 40 μL of nuclease-free water is added, and the magnetic beads are resuspended by gently pipetting. The tube is incubated at room temperature for 5 minutes. The PCR tube is placed back on the magnetic stand for 2 minutes, and 20 μL of supernatant is transferred to a new PCR tube using a pipette and placed in an ice box. The sample number is labeled, and the purified sequencing library is obtained. 1 μL of the sequencing library is quantified using the Qubit dsDNA HS Assay Kit, and the library concentration is recorded and labeled on the tube side wall. If immediate sample mixing and sequencing are not performed, the library can be stored at -20°C for no more than 1 month.

[0036] In A4, high-throughput sequencing step, the operation is performed according to the required sequencing data volume of the sample. The sequencing data volume of each sample needs to meet the ultra-high depth coverage of 40 high-frequency mutation sites (the coverage depth of each site is not less than 500x). The library quantification result is used to calculate the mixing volume. For example, if the library concentration is 10 ng / μL and the required on-machine volume is 1 μg, 100 μL is taken. The sequencing libraries of multiple samples are mixed in proportion to the calculated volume to ensure that the sequencing data volume of each sample after mixing is balanced. The mixed library is sent to the Illumina sequencing platform for sequencing.

[0037] In A5, data analysis step, the sequencing data is processed using conventional bioinformatics procedures. First, quality control is performed using FastQC software to filter out low-quality reads and adapter contamination sequences, and effective reads are retained. Then, BWA software is used to align the effective reads with the human reference genome (such as GRCh38) to generate a SAM format file. Samtools software is used to convert the SAM file to BAM format and sort it. Based on the aligned BAM file, GATK software is used for variant detection to identify single nucleotide variants and insertion / deletion variants in the target region. Finally, Annovar software is used to annotate the detected variant sites, including mutation type (such as missense mutation, nonsense mutation), gene and exon location, corresponding amino acid change, and related disease phenotype information. The genotype information of the 40 high-frequency mutation sites of the pregnant woman is finally output, providing genetic evidence for clinical judgment of ICP susceptibility genes and elucidation of the reasons for poor treatment effect of severe ICP.

[0038] The above is only an embodiment of the present application, which relates to the circuit and electronic components and modules are prior art, those skilled in the art can be fully realized, without further ado, the content of the present application does not involve the improvement of software and method. The specific structure and characteristics of the scheme known in the art are not described in detail, and the ordinary skilled in the art knows all the ordinary technical knowledge in the art before the filing date or the priority date, can know all the prior art in the field, and has the ability to apply the conventional experimental means before the date, the ordinary skilled in the art can perfect and implement the scheme under the inspiration of the present application combined with their own ability, some typical known structure or known method should not be an obstacle for the ordinary skilled in the art to implement the present application. It should be pointed out that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can also be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent.

Claims

1. A multiplex PCR-NGS screening method for susceptibility genes to intrahepatic cholestasis of pregnancy based on saliva samples, characterized in that, Includes the following steps: A1. Saliva DNA Extraction: Collect 1-2 ml of saliva from pregnant women, extract DNA from the saliva using a kit and purify it, and screen saliva DNA samples by detecting concentration, purity and integrity. A2, Multiplex PCR primer design and grouping: Specific primers were designed for 40 high-frequency mutation sites of 9 genes susceptible to intrahepatic cholestasis of pregnancy, and mutations in close proximity of the same gene location were amplified using the same set of specific primers to construct a primer pool. A3, Two-round PCR amplification and library construction: Using saliva DNA samples as templates, the first round of multiplex PCR amplification was performed using a primer pool. The amplified products were then purified by magnetic beads to obtain the purified products. The purified product was then subjected to a second round of PCR amplification to construct a sequencing library containing an 8bp molecular tag. The library was purified by magnetic beads. A4, High-throughput sequencing: After quantifying the sequencing library, sequencing is performed on the Illumina sequencing platform to obtain sequencing data; A5, Data Analysis: Perform quality control, alignment, variant detection and annotation on sequencing data, and output genotype information for 40 high-frequency mutation sites in pregnant women.

2. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 1, characterized in that: In step A1, the saliva was collected using sterile, individually packaged test tubes and stored in a preservation solution at 4°C after collection. During screening, Nanodrop was used to initially detect the concentration and purity of the DNA, Qubit 3.0 was used to accurately detect the concentration of the DNA, and agarose gel electrophoresis was used to detect the integrity of the DNA.

3. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 1, characterized in that: In step A2, 40 high-frequency mutation sites of the nine susceptibility genes for intrahepatic cholestasis of pregnancy are identified by screening and sequencing saliva samples from female patients with intrahepatic cholestasis of pregnancy using panel sequencing technology. The nine susceptibility genes for intrahepatic cholestasis of pregnancy include SLC10A1, ATP7B, UGT1A1, ABCB4, ABCB11, ATP8B1, TJP2, MYO5B, and ABCC2. The 3' end of the specific primer contains at least two bases complementary to the target mutation site and has no self-complementary or inter-primer complementary sequences.

4. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 1, characterized in that: In step A3, the reaction system in the first round of multiplex PCR amplification includes: 5 μL of KAPA2G FastMultiplex Mix, 2 μL of first-round mixed primers, 1 μL of saliva DNA sample, and 2 μL of ddH2O. The reaction system is used to start the PCR program of the PCR instrument for the first round of amplification to obtain the first reaction system product, which is stored at 4°C.

5. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 4, characterized in that: The specific steps of the first round of amplification are as follows: B1, pre-denaturation at 95℃ for 5 min; B2, Annealing and Extension: Annealing at 65°C for 30 seconds, followed by extension at 72°C for 1 minute; 5 cycles in total, with the annealing temperature decreasing by 1°C in each cycle. B3, fixed temperature cyclic amplification: denaturation at 94℃ for 30s, annealing at 60℃ for 30s, extension at 72℃ for 1min, for a total of 20-30 cycles; B4, final extension and storage: final extension at 72℃ for 5 min, after which the product of the first reaction system is stored at 4℃.

6. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 5, characterized in that: In step A3, the reaction system for the second round of PCR amplification includes: 20 μL of KOD enzyme solution, 1 μL of second-round index primers, 1 μL of Primer 1.0, and 10 μL of the product from the first reaction system. The second round of amplification is performed by starting the PCR program using a PCR instrument to obtain the product from the second reaction system, which is then stored at 4°C.

7. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 6, characterized in that: The specific steps of the second round of amplification are as follows: C1, pre-denaturation: pre-denaturation at 94℃ for 2 min; C2, Cyclic amplification: denaturation at 94℃ for 15s, annealing at 62℃ for 30s, extension at 72℃ for 30s, for 7 cycles of amplification; C3, final extension and storage: final extension at 72℃ for 10 min, after which the product is stored at 4℃.

8. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 7, characterized in that: In step A3, the magnetic beads are Intracere magnetic beads; after the first round of multiplex PCR amplification, 16 μL of Intracere magnetic beads are added to the product of the first reaction system for purification; after the second round of PCR amplification, 32 μL of Intracere magnetic beads are added to the product of the second reaction system for purification.

9. The method for screening susceptibility genes for intrahepatic cholestasis of pregnancy based on saliva samples as described in claim 8, characterized in that: The purification steps of the magnetic beads include: incubating at room temperature for 5 min, placing on a magnetic rack for 3 min until clear, removing the supernatant, washing the surface of the magnetic beads twice with 200 μL of 80% ethanol solution, letting stand at room temperature for 5 min to evaporate residual ethanol, adding Nuclease-free water to resuspend the magnetic beads and letting stand at room temperature for 5 min, and finally aspirating the supernatant.

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