Primer probe set for noninvasive detection of placenta drug transporter gene polymorphism and application

Abstract

The invention provides a primer probe group for noninvasive detection of gene polymorphism of a placenta drug transporter. The primer probe group comprises a primer probe group for detecting c.80G > A polymorphism of an RFC1 gene or / and c.1236Cgt of an ABCB1 gene, a primer probe set of T polymorphism; the method for noninvasive detection of placenta drug transporter gene polymorphism comprises the following steps: taking peripheral blood free DNA (deoxyribonucleic acid) of a pregnant woman in a gestation period as a template, and performing micro-droplet generation and PCR (polymerase chain reaction) amplification by using a primer probe group to obtain an RFC1 gene c.80Ggt; a or / and an ABCB1 gene c.1236Cgt; and carrying out genotype analysis on copy number information of T mutation sites. The invention further provides application of the primer probe set for noninvasive detection of placenta drug transporter gene polymorphism, and the primer probe set is used for preparing a kit for noninvasive detection of placenta drug transporter gene mutation site genotypes. According to the invention, the peripheral blood DNA of the pregnant woman in the gestation period is detected by using digital PCR, the placenta drug transporter gene mutation information is noninvasively obtained, the transplacental transport of the drug and the possible influence on the fetus are evaluated, and an important reference is provided for the treatment decision of the pregnancy complication / complication and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of placental drug transporter gene polymorphism detection, and particularly relates to a primer probe set for non-invasive detection of placental drug transporter gene polymorphism and application thereof. BACKGROUND

[0002] Pregnancy medication is directly related to maternal and fetal health and birth population quality. The incidence of pregnancy complications in China is as high as 25.04%, the drug use rate during pregnancy is as high as more than 90%, and the proportion of early pregnancy prescription drugs is more than 60%. Among them, antiepileptic drugs, antidepressants and sex hormones can cause adverse pregnancy outcomes. Some pregnancy complications such as fetal growth restriction have no clear treatment drugs, and the above problems need to be studied urgently.

[0003] The efficacy of medication during pregnancy and the safety to the fetus are affected by multiple factors such as the physical and chemical properties of the drug, the medication scheme, the mechanism of drug transplacental barrier, and the gestational age of the characteristic drug. Among them, the proportion of the drug transported across the transplacental barrier to reach the fetus is one of the key factors. The mechanism of drug transplacental barrier transport includes simple diffusion, facilitated diffusion and active transport. Active transport is mainly carried out by drug transporters. Some drugs have specific transporters, and most of these transporters are distributed in syncytial trophoblasts and trophoblasts (genetically belonging to the fetus). There is no literature report on non-invasive acquisition of information about placental drug transporters (i.e. fetal drug transporters) during pregnancy. In addition, pharmacogenomics (PGx) is also a possible reason for the difference in drug treatment effect or safety. PGx aims to study how genetic variations affect individual responses to drugs. Currently, more than 350 drugs approved by the US FDA include PGx information in their product descriptions, and 85%-95% of the population carries potentially intervenable PGx variations that may affect drug dosage or treatment. In clinical practice, it is well known that different patients have different responses to the same drug, and PGx provides a scientific explanation for this individual difference. With the continuous decline in detection costs and the increasing improvement of clinical practice guidelines based on PGx results, the clinical application of PGx has broad prospects. Detection methods include PCR, digital PCR (Digital PCR, dPCR), gene chips, high-throughput sequencing (NGS) and Sanger sequencing, which can detect genotypes.

[0004] Fetal pharmacogenomics can predict and analyze drug transport across the placental barrier by analyzing genetic variations of fetal individuals, which can help guide drug selection and dosage determination to promote precise medication decisions during pregnancy, improve the efficacy of pregnancy complications and reduce adverse reactions. Among the many genetic variation types, single nucleotide polymorphism (SNPs) is of high frequency and strong detectability, and is of great concern. Currently, the specimen for detecting fetal PGx can be obtained by amniocentesis or chorionic villus sampling, and some samples come from intrauterine death or post-pregnancy products such as placenta and umbilical cord, umbilical cord blood, and neonatal hair, and obtaining amniotic fluid, umbilical cord blood or chorionic villus tissue during pregnancy is invasive and poses a risk to the mother and baby. The use of gene chips or NGS methods to detect fetal cell-free DNA (cffDNA) in maternal peripheral blood is an important tool for current prenatal screening and diagnosis, i.e. non-invasive DNA testing (NIPT, Non-Invasive Prenatal Testing). This technology realizes early and non-invasive detection of fetal genetic abnormalities by analyzing fetal cell-free DNA in maternal peripheral blood, and has been widely used in screening for fetal chromosomal aneuploidy (such as T21, T18, T13), with high accuracy, but has not been used for SNP detection of placental drug transporters PGx. Theoretically, non-invasive detection of placental drug transporter gene polymorphisms in maternal peripheral blood during pregnancy can be achieved by whole genome sequencing, but the cost is very high and the data analysis is particularly complex, and it has not been applied. There is no literature reported on the application of maternal peripheral blood to directly detect the information of placental drug transporter gene polymorphisms. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a primer probe set for non-invasive detection of placental drug transporter gene polymorphism and its application, which uses digital PCR technology to detect the free DNA in the peripheral blood of pregnant women during pregnancy, and the detection method is safe and reliable, and can non-invasively obtain the information of the mutation site of the placental drug transporter gene, predict the transport of drugs to the fetus during pregnancy, evaluate the effectiveness of drug treatment and the possible adverse effects on the fetus, and provide an important reference for the mechanism research, clinical diagnosis and treatment decision of pregnancy complications.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is: a primer probe set for non-invasive detection of placental drug transporter gene polymorphism, wherein the placental drug transporter gene polymorphism includes RFC1 gene c.80G>A polymorphism or / and ABCB1 gene c.1236C>T polymorphism. The primer and probe set for non-invasive detection of the c.80G>A polymorphism of the RFC1 gene is referred to as primer and probe set a, which includes forward primer RFC1-80-F, reverse primer RFC1-80-R, probe RFC1-80-WP and probe RFC1-80-MP, and the nucleotide sequences are shown in SEQ ID No. 1 to 4 in sequence; The primer and probe set for non-invasive detection of the c.1236C>T polymorphism of the ABCB1 gene is designated as primer and probe set b, which includes forward primer ABCB1-1236-F, reverse primer ABCB1-1236-R, probe ABCB1-1236-WP, and probe ABCB1-1236-MP. The nucleotide sequences of the forward primer ABCB1-1236-F and the reverse primer ABCB1-1236-R are shown in SEQ ID No. 5 to 6, respectively. The nucleotide sequence of the probe ABCB1-1236-WP is: CTTGAAGGG+T+CTGAACC; the nucleotide sequence of the probe ABCB1-1236-MP is: CTTGAAGGG+CCTGAACC, where +T and +C represent that the nucleotide has been modified with a locked nucleotide.

[0007] The method for detecting placental drug transporter genes using the primer and probe set described above for non-invasive detection of placental drug transporter gene polymorphism is as follows: S1. Collect peripheral blood samples from pregnant women, add EDTA anticoagulant, centrifuge, separate plasma and extract cell-free DNA from plasma; S2. Using the cell-free plasma DNA obtained in S1 as a template, microdroplet generation and PCR amplification reactions were performed using primer probe set a and primer probe set b, respectively. Then, microdroplet detection was performed to obtain copy number information of the c.80G>A mutation site of the RFC1 gene and / or the c.1236C>T mutation site of the ABCB1 gene, respectively. S3. Based on the copy number information of the mutation sites obtained in S2, perform genotypic analysis on the c.80G>A mutation site of the RFC1 gene and / or the c.1236C>T mutation site of the ABCB1 gene.

[0008] Preferably, the centrifugation conditions in S1 are: centrifugation at 3000 rpm for 10 min.

[0009] Preferably, the PCR amplification reaction system in S2 is as follows: 7.5 μL of 4× premix A, 0.18 μL of forward primer, 0.18 μL of reverse primer, 0.09 μL / probe, 10 μL of cell-free plasma DNA, and DEPC water to a final volume of 30 μL; the PCR amplification reaction program is as follows: 95℃ for 10 min; 94℃ for 30 s, 57℃ for 60 s, for 40 cycles.

[0010] This invention also provides the application of the above-mentioned primer and probe set for non-invasive detection of placental drug transporter gene polymorphism, wherein the primer and probe set for non-invasive detection of placental drug transporter gene polymorphism is used to prepare a kit for non-invasive detection of genotypes of mutation sites in placental drug transporter genes.

[0011] Compared with the prior art, the present invention has the following advantages: 1. This invention utilizes cell-free DNA from peripheral blood in early pregnancy to non-invasively obtain information on mutation sites of placental drug transporter genes. Compared with techniques such as amniocentesis and cordocentesis, the risks of placental and maternal infection are reduced to zero.

[0012] 2. This invention utilizes digital PCR for detection, which is safe and reliable, and can achieve absolute quantification of gene copy number, accurately quantifying the genotype copy number of placental drug transporter genes.

[0013] 3. This invention utilizes primer and probe combinations to sensitively detect placental drug transporter genes in maternal peripheral blood during pregnancy, predict drug transport to the fetus during pregnancy, assess drug efficacy and potential effects on the fetus, and provide important reference for mechanism research, clinical diagnosis and treatment decisions of pregnancy complications.

[0014] The present invention will be further described in detail below with reference to the embodiments. Detailed Implementation

[0015] Example 1 This embodiment describes the application of a primer-probe set for non-invasive detection of placental drug transporter gene polymorphism in detecting the genotype of the c.80G>A mutation site in the RFC1 gene. The primer-probe set for non-invasive detection of the c.80G>A polymorphism in the RFC1 gene is referred to as primer-probe set a, which includes the forward primer RFC1-80-F, the reverse primer RFC1-80-R, the probe RFC1-80-WP, and the probe RFC1-80-MP, with nucleotide sequences as shown in SEQ ID No. 1 to 4.

[0016] The non-invasive method for detecting placental drug transporter gene polymorphisms in this embodiment is as follows: S1. With informed consent, peripheral blood and amniotic fluid samples were collected from 7 pregnant women in accordance with the sample collection specifications. EDTA anticoagulant was added, and the samples were centrifuged at 3000 rpm for 10 min to separate the plasma. Free DNA was extracted from the plasma using a free DNA nucleic acid extraction kit, with a final elution volume of 60 μL. Fetal amniotic fluid genomic DNA was extracted from the amniotic fluid samples using a cell DNA nucleic acid extraction kit.

[0017] S2. Using cell-free plasma DNA obtained in S1 as a template and amniotic fluid cell DNA as a template, respectively, as experimental groups, DEPC water as a template instead of cell-free plasma DNA as a negative control group, and plasmid DNA (artificially synthesized plasmid DNA containing the target sequence of the RFC1 gene) as a template as a positive control group, microdroplet generation and PCR amplification reaction were performed using the primer and probe group a, followed by microdroplet detection to obtain the copy number information of the c.80G>A mutation site of the RFC1 gene; The PCR amplification reaction system consisted of: 7.5 μL of 4× premix A (Xinyi Manufacturing Technology Beijing Co., Ltd.), 0.18 μL (100 μM) of forward primer RFC1-80-F, 0.18 μL (100 μM) of reverse primer RFC1-80-R, 0.09 μL (100 μM) of probe RFC1-80-W-P, 0.09 μL (100 μM) of probe RFC1-80-MP, 10 μL of template, and DEPC water to a final volume of 30 μL. The PCR amplification reaction program was: 95℃ for 10 min; 94℃ for 30 s; 57℃ for 60 s; 40 cycles. S3. Based on the copy number information of the c.80G>A mutation site of the RFC1 gene obtained in S2, and combined with the proportion of fetal cell-free DNA measured by NGS (NGS FF%), the genotype of the c.80G>A mutation site of the fetal RFC1 gene was interpreted. The results are shown in Table 1. The copy number of each site in the negative control group should be 0, and the copy number of each site in the positive control group should be no less than 1000 copies. The copy number of different genotypes at the same site should be close to 1:1. The results of the control experiment meet the requirements of data analysis. The results of plasma cell-free DNA detection are consistent with the results of fetal amniotic fluid cell detection, indicating that the accuracy of using digital PCR to detect placental drug transporter gene polymorphism in cell-free DNA of peripheral blood of pregnant women is reliable.

[0018] Table 1. Detection results of the c.80G>A site in the RFC1 gene. Note: For the c.80G>A site of the RFC1 gene, genotype "GG" represents wild type, genotype "GA" represents heterozygous type, and genotype "AA" represents mutant type.

[0019] Example 2 This embodiment describes the application of a primer-probe set for non-invasive detection of placental drug transporter genes in detecting the genotype of the c.1236C>T mutation site in the ABCB1 gene. The primer-probe set for non-invasive detection of the c.1236C>T polymorphism of the ABCB1 gene is denoted as primer-probe set b, which includes forward primer ABCB1-1236-F, reverse primer ABCB1-1236-R, probe ABCB1-1236-WP, and probe ABCB1-1236-MP. The nucleotide sequences of the forward primer ABCB1-1236-F and the reverse primer ABCB1-1236-R are shown in SEQ ID No. 5 to 6, respectively. The nucleotide sequence of the probe ABCB1-1236-WP is: CTTGAAGGG+T+CTGAACC; the nucleotide sequence of the probe ABCB1-1236-MP is: CTTGAAGGG+CCTGAACC, where +T and +C represent that the nucleotide has been modified with a locked nucleotide.

[0020] The non-invasive method for detecting placental drug transporter gene polymorphisms in this embodiment is as follows: S1. With informed consent, peripheral blood and amniotic fluid samples were collected from 7 pregnant women in accordance with the sample collection specifications. EDTA anticoagulant was added, and the samples were centrifuged at 3000 rpm for 10 min to separate the plasma. Free DNA was extracted from the plasma using a free DNA nucleic acid extraction kit, with a final elution volume of 60 μL. Fetal amniotic fluid genomic DNA was extracted from the amniotic fluid samples using a cell DNA nucleic acid extraction kit.

[0021] S2. Using cell-free plasma DNA obtained in S1 as a template and amniotic fluid cell DNA as a template, respectively, as experimental groups, DEPC water as a template instead of cell-free plasma DNA as a negative control group, and plasmid DNA (artificially synthesized plasmid DNA containing the ABCB1 target sequence) as a template as a positive control group, microdroplet generation and PCR amplification reaction were performed using the primer and probe group b, followed by microdroplet detection to obtain the copy number information of the ABCB1 gene c.1236C>T mutation site; The PCR amplification reaction system consisted of: 7.5 μL of 4× premix A (Xinyi Manufacturing Technology Beijing Co., Ltd.), 0.18 μL (100 μM) of forward primer ABCB1-1236-F, 0.18 μL (100 μM) of reverse primer ABCB1-1236-R, 0.09 μL (100 μM) of probe ABCB1-1236-WP, 0.09 μL (100 μM) of probe ABCB1-1236-MP, 10 μL of template, and DEPC water to a final volume of 30 μL. The PCR amplification reaction program was: 95℃ for 10 min; 94℃ for 30 s; 57℃ for 60 s; 40 cycles. S3. Based on the copy number information of the c.1236C>T mutation site of the ABCB1 gene obtained in S2, and combined with the proportion of fetal cell-free DNA measured by NGS (NGS FF%), the genotype of the c.1236C>T mutation site of the fetal ABCB1 gene was interpreted. The results are shown in Table 2. The copy number of each site in the negative control group should be 0, and the copy number of each site in the positive control group should be no less than 1000 copies. The copy number of different genotypes at the same site should be close to 1:1. The results of the control experiment meet the requirements of data analysis. The results of plasma cell-free DNA detection are consistent with the results of fetal amniotic fluid cell detection, indicating that the detection of placental drug transporter genes in peripheral blood cell-free DNA of pregnant women by digital PCR is accurate and reliable.

[0022] Table 2. Detection results of the c.1236C>T site in the ABCB1 gene. Note: For the c.1236C>T site of the ABCB1 gene, genotype "CC" represents wild type, genotype "CT" represents heterozygous type, and genotype "TT" represents mutant type.

[0023] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A primer probe set for non-invasive detection of genetic polymorphism of placental drug transporters, characterized in that, The placental drug transporter gene polymorphism comprises an RFC1 gene c.80G>A polymorphism and / or an ABCB1 gene c.1236C>T polymorphism. The primer probe set for non-invasive detection of the RFC1 gene c.80G>A polymorphism is referred to as primer probe set a, and comprises a forward primer RFC1-80-F, a reverse primer RFC1-80-R, a probe RFC1-80-W-P, and a probe RFC1-80-M-P, the nucleotide sequences of which are shown in SEQ ID No. 1-4 in sequence. The primer probe set for non-invasive detection of the ABCB1 gene c.1236C>T polymorphism is referred to as primer probe set b, and comprises a forward primer ABCB1-1236-F, a reverse primer ABCB1-1236-R, a probe ABCB1-1236-W-P, and a probe ABCB1-1236-M-P, the nucleotide sequences of the forward primer ABCB1-1236-F and the reverse primer ABCB1-1236-R being shown in SEQ ID No. 5-6 in sequence. The nucleotide sequence of the probe ABCB1-1236-W-P is CTTGAAGGG+T+CTGAACC, and the nucleotide sequence of the probe ABCB1-1236-M-P is CTTGAAGGG+CCTGAACC, wherein +T and +C represent that the nucleotides are modified by locked nucleotides.

2. A method for detecting placental drug transporter gene using the primer probe set for non-invasive detection of placental drug transporter gene polymorphism according to claim 1, wherein the method comprises the steps of: (a) amplifying the target gene by PCR using the primer probe set; (b) detecting the amplified target gene by hybridization using the primer probe set; and (c) determining the genotype of the target gene. The method comprises: S1, collecting a peripheral blood sample of a pregnant woman during pregnancy, adding an EDTA anticoagulant, centrifuging, separating plasma, and extracting plasma free DNA; S2, using the plasma free DNA obtained in S1 as a template, using the primer probe set a and the primer probe set b respectively for microdroplet generation and PCR amplification reaction, and then performing microdroplet detection to obtain the copy number information of the RFC1 gene c.80G>A or / and the ABCB1 gene c.1236C>T mutation site; S3, performing genotype analysis of the RFC1 gene c.80G>A mutation site or / and the ABCB1 gene c.1236C>T mutation site according to the copy number information of the mutation site obtained in S2.

3. The method for detecting placental drug transporter genes according to claim 2, characterized in that, The centrifugation condition in S1 is 3000 rpm for 10 min.

4. The method for detecting placental drug transporter genes according to claim 2, characterized in that, The reaction system of the PCR amplification in S2 is 4×pre-mixed solution A 7.5 μL, forward primer 0.18 μL, reverse primer 0.18 μL, probe 0.09 μL per strip, plasma free DNA 10 μL, and DEPC water to 30 μL; the reaction program of the PCR amplification is 95℃ for 10 min; 94℃ for 30 s, 57℃ for 60 s, 40 cycles.

5. The use of the primer probe set for non-invasive detection of genetic polymorphism of placental drug transporters according to claim 1, characterized in that, The primer probe set for non-invasive detection of the placental drug transporter gene polymorphism is used for preparing a kit for non-invasive detection of the genotype of the mutation site of the placental drug transporter gene.

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