CN101967511B - Gene chip, detection reagent and kit for detecting GDM susceptible population - Google Patents

Abstract

The invention discloses a gene chip, a detection reagent and a kit for detecting a GDM (gestational diabetes mellitus) susceptible population. A gene chip detection system for screening the polymorphism high-risk population of GDM related genes is constructed by the gene chip, the detection reagent and the kit for detecting the GDM susceptible population; and the gene chip detection system can quickly and accurately detect GDM related SNPs in a clinical sample, has high flux and high sensitivity, has significance for clinical diagnosis of the GDM and early screening and early prevention and intervention of the high-risk population, and can be widely used for efficiently screening the GDM high-risk population in clinic.

Description

Gene chips, detection reagents and kits for detecting GDM susceptible population

technical field

The invention belongs to the technical field of detection, and relates to a gene chip (or called a DNA microarray) and its detection reagent and kit, in particular to a gene chip and its detection reagent for SNP detection and typing of people susceptible to gestational diabetes and detection kits. the

Background technique

Gestational Diabetes Mellitus (GDM) refers to diabetes that occurs or is discovered for the first time during pregnancy, and its incidence is reported to be 1% to 14% in various countries around the world. The incidence of GDM in my country has increased from 2% in 1996 to 4.6% in 2003. Existing evidence shows that Chinese women are one of the most at-risk groups suffering from GDM. The main complications of GDM are macrosomia, polyhydramnios, hypertensive disorders of pregnancy, infection, premature delivery, ketoacidosis and so on. And often due to dystocia, neonatal head hematoma, subgaleal hematoma, brachial plexus injury, clavicle fracture, etc. GDM will not only cause a series of metabolic disorders in pregnant women, but also cause abnormal growth and development of fetuses and newborns, and even have longer-term effects after childhood and adolescence. The most common problem is abnormal glucose metabolism. Babies born to diabetic mothers are prone to hypoglycemia, immature organ development, organ function below the actual gestational age, and immature brain development. Some children of GDM patients have some psychological and behavioral problems, although their intellectual development is within the normal range, such as self-reported various discomforts, learning difficulties, anxiety, depression, etc. to varying degrees. The incidence of type 2 diabetes after postpartum follow-up of GDM patients outside the country is 60% to 70%. The short-term and long-term effects of maternal diabetes and the rising prevalence of GDM make it a problem that will plague Chinese people in the near future. One of the serious problems of health. the

Timely intervention during pregnancy or even earlier can not only reduce or even eliminate the adverse effects of GDM on mother and child, but may become one of the most important medical preventive measures in this century. GDM is a polygenic genetic disease, which is called a complex disease in genetics. It has the following characteristics: more than one gene is involved in the pathogenesis, and each gene has a different effect. The one that plays the main role is called the main effect gene. , those with smaller effects are secondary genes, and there are interactions between different genes; each pathogenic susceptibility gene acts on different links of metabolism. At present, domestic and foreign studies have shown that the following genes may be related to type 2 diabetes, and may also be related to GDM: (1) protein kinase Cξ subtype gene, discovered by single nucleotide polymorphism (singlenucleotide polymorphism, SNP) method Its rs36045(A/G) is related to type 2 diabetes; the correlation between the haplotypes of 15 SNPs located upstream and downstream of rs36045 and type 2 diabetes was analyzed by linkage disequilibrium, and it was found that there were 5 SNPs composed of C-G-T-A-G The ploidy increased significantly in the case group, further confirming that the gene may be a susceptibility gene of the population studied; (2) ALDHIAI-E3/56 gene, in diabetic and non-diabetic populations, genotypes were screened with high-frequency SNPs, This gene was found to be very significant. This gene may be involved in the susceptibility of type 2 diabetes in Chinese Han people; (3) the gene encoding adipocyte differentiation-related protein (ADFP), which is very important for the differentiation and function of adipose tissue, especially the formation of fat and insulin resistance related. The gene mutation may be involved in the formation of insulin resistance and type 2 diabetes; (4) CAPN10 gene, its product is calpain-10 protein, which may be related to intracellular calcium ion-mediated signal transduction. The single nucleotide polymorphism site of this gene is related to the onset of type 2 diabetes. According to statistical data analysis, SNP43 of this gene is related to tissue insulin sensitivity and insulin level in the process of sugar load, and this gene may be a related gene of Chinese type 2 diabetes; Morphological loci are strongly associated with the occurrence of type 2 diabetes. Compared with GC type, CC type has a higher transcription level of IL-6, which increases the level of IL-6 in serum, and IL-6 can reduce some protective factors in the body, such as adiponectin, leptin, etc., so that the individual more prone to diabetes. the

Based on the fact that these genes may be related to the occurrence of GDM, there is no research involving the use of gene chip probes to predict high-risk groups of diabetes at home and abroad. This patent designs a gene chip system for detecting GDM susceptible populations, and establishes the corresponding relationship between SNP patterns and GDM. The general population can be screened quickly and conveniently through the gene chip, and the high-risk group of GDM can be predicted in the first trimester or even pre-pregnancy examination, so as to intervene as early as possible through diet control and exercise therapy, and reduce or even eliminate the short-term and long-term effects of GDM on mother and child. Influence. the

Contents of the invention

The object of the present invention is to provide a gene chip, a detection method, a testing agent and a kit for detecting GDM susceptible populations in view of the deficiencies in the prior art. the

The purpose of the present invention is achieved by the following technical solutions:

A gene chip for detection and typing of GDM susceptible populations, comprising a solid phase carrier and oligo-glutamic acid probes immobilized thereon, wherein the oligonucleotide probes on the chip are for GDM susceptibility DNA fragments for SNP site detection. the

The above SNP sites include: rs1799884, rs2144908, rs1800629, rs361525, rs266729, rs17300539, rs1801278, rs4994 and rs12255372. Their sequences have the DNA fragments of the nucleotide sequences shown in Seq ID No.1-No.9. the

A detection reagent comprising the above-mentioned gene chip, the detection reagent also comprises PCR primers for amplifying each GDM-related SNPs, and these PCR primers have the nucleotide sequences of Seq ID No.82-No.90. the

A kit for detection and typing of GDM susceptible population, at least including a gene chip, PCR amplification primers, negative control and positive control samples. The kit also includes a hybridization box and a hybridization solution. the

The invention designs specific probes for different GDM susceptibility-related SNP sites to detect, interprets the results of gene chip hybridization, and predicts the disease susceptibility of detection samples. the

The beneficial effect of the present invention is: the present invention is used for detecting the gene chip of GDM susceptibility population, detection reagent and test kit have constructed the gene chip detection system of screening the high-risk population of each relevant gene polymorphism of GDM, can detect rapidly and accurately The GDM-related SNPs in clinical samples have large throughput and high sensitivity, which are of great significance for the clinical diagnosis of GDM, early screening and early preventive intervention of high-risk groups, and can be widely used in clinical and efficient screening of high-risk groups for GDM. the

Description of drawings

Fig. 1 is a general flow chart of making a gene chip; Fig. 2 is a flow chart of PCR amplification. the

Detailed ways

The DNA chip technology with glass matrix as the carrier adopts the DNA microarray technology with micro spotting, and its basic principle is as follows: the DNA probe is an oligonucleotide fragment containing a specific DNA sequence, and its end is labeled with an amino group. The chemically modified glass matrix substrate has an activated aldehyde group, which can be covalently bonded to the modified amino group attached to the DNA probe. Using this method, the DNA probe can be fixed on the surface of the substrate according to a certain arrangement. Its main advantage is that it is simple and easy to operate, and can easily produce chips that meet the purpose according to the requirements of users. On this basis, combined with PCR technology, a large amount of rapid amplification is performed on a small amount of samples to be tested. After amplification, the samples to be tested containing fluorescent markers are specifically hybridized and scanned with the probes on the chip. After that, the strength and effectiveness of the hybridization signal Without analysis, the quantity and nature of the target gene in the sample can be judged. The production process of the gene chip is shown in Figure 1. the

The gene chip of the present invention comprises a substrate and probes fixed on the substrate, and the probes fixed on the substrate are specific probes capable of hybridizing with SNP polymorphic nucleic acids of GDM related genes. the

Each related gene SNP polymorphic nucleic acid of GDM of the present invention is selected as follows:

Table 1: SNP polymorphic nucleic acid gene sequence list of GDM related genes;

Seq ID Gene dbSNP SNP Sequence No.1 Glucokinase rs1799884 CATGACAACCACAGGCCCTCTCAGGA[A/G]CACAGT AAGCCCTGGCAGGAGAATC No.2 HNF rs2144908 TATGTCATCAATTGCTGAGGACAGAG[A/G]GCCAGG GAATGACAGAATCAGGGCC No.3 TNFa gene polymorphism rs1800629 GGAGGCAATAGGTTTTGAGGGGCATG[A/G]GGACGG GGTTCAGCCTCCAGGGTCC No.4 TNFa gene polymorphism rs361525 TGGCCCAGAAGACCCCCCTCGGAATC[A/G]GAGCAG GGAGGATGGGGAGTGTGAG No.5 Adiponectin gene polymorphism rs266729 CTTGCAAAGAACCGGCTCAGATCCTGC[C/G]CTTCAA AAACAAAACATGAGCGTGC No.6 adiponectin gene rs17300539 ATCAGAATGTGTGGCTTGCAAGAACC[A/G]GCTCAG

the Polymorphism the ATCCTGCCCTTCAAAAACA No.7 Polymorphisms of Insulin Receptor and Receptor Substrates rs1801278 TGGGCAGACTGGGCCCTGCACCTCCC[A/G]GGGCTG CTAGCATTTGCAGGCCTAC No.8 β3 adrenergic receptor gene rs4994 ACCTGCTGGTCATCGTGGCCATCGCC[C/T]GGACTCC GAGACTCCAGACCATGAC No.9 Transcription factor 7 rs12255372 CTGCCCAGGAATATCCAGGCAAGAAT[G/T]ACCATA TTCTGATAATTACTCAGGC

The specific probe sequences for each related gene polymorphism of GDM are as follows:

Table 2. Design of SNP detection probes based on SNP sites and flanking sequences (Seq ID No.10-No.81 for each SNP site, a total of 8 probes are designed for the positive and negative 2 DNA strands):

SNP(rs_) probe_strand 1 probe_strand 2 4994 GTGGCCATCGCCAGGACTCCGAGAC GTCTCGGAGTCCAGGCGATGGCCAC 4994 GTGGCCATCGCCCGGACTCCGAGAC GTCTCGGAGTCCCGGCGATGGCCAC 4994 GTGGCCATCGCCGGGACTCCGAGAC GTCTCGGAGTCCGGGCGATGGCCAC 4994 GTGGCCATCGCCTGGACTCCGAGAC GTCTCGGAGTCCTGGCGATGGCCAC 361525 TTGAGGGGCATGAGGACGGGGTTCA TGAACCCCGTCCACATGCCCCTCAA 361525 TTGAGGGGCATGCGGACGGGGTTCA TGAACCCCGTCCCCATGCCCCTCAA 361525 TTGAGGGGCATGGGGACGGGGTTCA TGAACCCCGTCCGCATGCCCCTCAA 361525 TTGAGGGGCATGTGGACGGGGTTCA TGAACCCCGTCCTCATGCCCCTCAA 1801278 CCTGCACCTCCCAGGGCTGCTAGCA TGCTAGCAGCCCAGGGAGGTGCAGG 1801278 CCTGCACCTCCCCGGGCTGCTAGCA TGCTAGCAGCCCCGGGAGGTGCAGG 1801278 CCTGCACCTCCCGGGGCTGCTAGCA TGCTAGCAGCCGGGGAGGTGCAGG 1801278 CCTGCACCTCCCTGGGCTGCTAGCA TGCTAGCAGCCCTGGGAGGTGCAGG 1799884 GCCCTCTCAGGAACACAGTAAGCCC GGGCTTACTGTGATCCTGAGAGGGC 1799884 GCCCTCTCAGGACCACAGTAAGCCC GGGCTTACTGTGCTCCTGAGAGGGC 1799884 GCCCTCTCAGGAGCACAGTAAGCCC GGGCTTACTGTGGTCCTGAGAGGGC 1799884 GCCCTCTCAGGATCACAGTAAGCCC GGGCTTACTGTGTTCCTGAGAGGGC 1800629 TTGAGGGGCATGAGGACGGGGTTCA TGAACCCCGTCCACATGCCCCTCAA 1800629 TTGAGGGGCATGCGGACGGGGTTCA TGAACCCCGTCCCCATGCCCCTCAA 1800629 TTGAGGGGCATGGGGACGGGGTTCA TGAACCCCGTCCGCATGCCCCTCAA 1800629 TTGAGGGGCATGTGGACGGGGTTCA TGAACCCCGTCCTCATGCCCCTCAA 12255372 TCCAGGCAAGAAAGACCATATTCTG CAGAATATGGTCATTCTTGCCTGGA 12255372 TCCAGGCAAGAACGACCATATTCTG CAGAATATGGTCCTTCTTGCCTGGA 12255372 TCCAGGCAAGAAGGACCATATTCTG CAGAATATGGTCGTTCTTGCCTGGA 12255372 TCCAGGCAAGAATGACCATATTCTG CAGAATATGGTCTTTCTTGCCTGGA 266729 CTCAGATCCTGCACTTCAAAAACAA TTGTTTTTGAAGAGCAGGATCTGAG 266729 CTCAGATCCTGCCCTTCAAAAACAA TTGTTTTTGAAGCGCAGGATCTGAG 266729 CTCAGATCCTGCGCTTCAAAAACAA TTGTTTTTGAAGGGCAGGATCTGAG 266729 CTCAGATCCTGCTCTTCAAAAACAA TTGTTTTTGAAGTGCAGGATCTGAG 17300539 CTTGCAAGAACCAGCTCCAGATCCTG CAGGATCTGAGCAGGTTCTTGCAAG

17300539 CTTGCAAGAACCCGCTCCAGATCCTG CAGGATCTGAGCCGGTTCTTGCAAG 17300539 CTTGCAAGAACCGGCTCAGATCCTG CAGGATCTGAGCGGGTTCTTGCAAG 17300539 CTTGCAAGAACCTGCTCCAGATCCTG CAGGATCTGAGCTGGTTCTTGCAAG 2144908 CTGAGGACAGAGAGCCAGGGAATGA TCATTCCCTGGCACTCTGTCCTCAG 2144908 CTGAGGACAGAGCGCCAGGGAATGA TCATTCCCTGGCCCTCTGTCCCTCAG 2144908 CTGAGGACAGAGGGCCAGGGAATGA TCATTCCCTGGCGCTCTGTCCTCAG 2144908 CTGAGGACAGAGTGCCAGGGAATGA TCATTCCCTGGCTCTCTGTCCCTCAG

In the above sequences, two sequences, positive and negative, were designed for each GDM-related SNP. the

On the basis of the above-mentioned designed GDM-related SNPs, those skilled in the art can carry out probe synthesis by conventional methods in the field, and carry out poly(T) and amino modification at the 5' end. the

In one embodiment of the present invention, the chip preparation is carried out by a chip spotter, the probes are dissolved in the spotting solution, and the spotting is carried out according to the set spotting program, and the gene chip after spotting is hydrated and hydroborated After sodium reduction, it can be used for detection. the

The detection reagent used to detect the GDM-related SNPs in the sample at least includes a pair of PCR primers used to amplify the SNPs in the sample and the above-mentioned gene chip of the present invention. According to the characteristics of each related SNPs of GDM, the PCR primers of the present invention utilize their respective specific flanking sequences to design primers, and the specific SNP sequences can be broadly amplified through 9 PCR reactions. In a preferred embodiment of the present invention, designed as SEQ ID NO.82-90 primer sequence is as follows:

Table 3. Upstream and downstream primers (Seq ID No.82-No.99) for PCR amplification designed according to the more distal sequences at both ends of the SNP site:

SNP(rs_) primer_f Seq primer_r Seq length 4994 4994_f1 CCAATACCGCCAACA CCA 4994_r1 FAM-CCAGCGAAGTCACGAACAC 174bp 361525 361525_ f1 GAAGGAAACAGACCA CAGACC 361525_ r1 FAM-GGACACACAAGCATCAAGG A 185bp 1801278 1801278 _f1 GGAGTACATGAAGAT GGACCTG 1801278 _r1 FAM-TGCTGAGGATGAGGAGGC 301bp 1799884 1799884 _f1 GCTCTTTGCCACCAG TCC 1799884 _r1 FAM-TCCTCTCCCTTCTGTGACC T 271bp 1800629 1800629 _f1 GAAGGAAACAGACCA CAGACC 1800629 _r1 FAM-GGACACACAAGCATCAAGG A 185bp 12255372 1225537 2_f1 GAAACTAAGGCGTGA GGG 1225537 2_r1 FAM-AATGGAGGCTGAATCTGG 219bp 266729 266729_ f1 TCTGTGTGGACTGTG GAGATG 266729_ r1 FAM-ACCTTGGACTTTCTTGGCA C 220bp 17300539 1730053 9_f1 TCTGTGTGGACTGTG GAGATG 1730053 9_r1 FAM-ACCTTGGACTTTCTTGGCA C 220bp 2144908 2144908 _f1 CGTGCCTCTTGCTTT CTTTC 2144908 _r1 FAM-TTTCTTCAGCCTCTGCCAT T 276bp

During the primer synthesis process, the 5' end of one of the paired primers was fluorescently labeled in advance,

When in use, extract the genomic DNA of the sample to be detected, design upstream and downstream primers for each SNP site to be detected and perform PCR amplification, and introduce fluorescent markers during the amplification process. Fluorescently labeled nucleic acid target molecules hybridize with the probes on the chip through the principle of complementary base pairing, and the fluorescent signal of each site is obtained by scanning the chip. the

The above-mentioned sample processing reagents, PCR amplification reagents, and hybridization reagents can all use various reagents that are well known to those skilled in the art and need to be used in these operations. These reagents can be included in the kit if necessary, or their formula Listed in the instructions of the kit, users should follow the instructions in the instructions to prepare by themselves. the

The kit of the present invention also includes corresponding negative control and/or positive control samples. the

According to yet another aspect of the present invention, a method is provided for evaluating each GDM-related SNPs in a sample to be tested, thereby efficiently screening GDM high-risk groups. The samples from the individual to be tested are detected by the detection reagent of the present invention, and the possibility of the individual developing GDM or even developing type 2 diabetes in the future is evaluated by the expression of each GDM-related SNPs in the sample. the

Below, with reference to the accompanying drawings, the present invention will be described in detail but not limited by describing the preferred embodiments of the present invention. the

Embodiment 1: Preparation of each related SNPs detection chip of GDM

1. Manufacturers and specifications of raw materials

name manufacturers Specification Product name Aldehyde substrate Capitalbio 75.5×25.2×1.0 Polymer three-dimensional substrate D Gene Chip Spotting Solution Capitalbio 5ml the

The probe sequence design is shown in Seq ID No.10-No.81, and the 5'-terminal amino-labeled probe was synthesized according to methods well known to those skilled in the art. the

2. Chip preparation process:

(1) Dissolve the synthesized probe in the gene chip spotting solution, and spot it on a 75×25 mm chip substrate modified with aldehyde groups with a SmartArray ^TM (CapitalBioCorp.) spotting instrument, and store it after drying.

(2) The chip needs to undergo the following pre-processing before use:

1) Place the chip in a closed, 100% humidity humid box and process overnight at 37°C. the

2) Put the chip in 0.25% NaBH4 solution, and reduce it on a horizontal shaker (80 rpm) for 5 minutes. the

3) Wash in distilled water on a horizontal shaker (80 rpm) for 5 minutes. the

4) Repeat 3) once. the

5) Place the chip in a 50ml special centrifugal drying tube and centrifuge at 2000rpm for 1min. as

The processed chips are ready for hybridization. the

Example 2: GDM related SNPs detection kit sample detection

The clinical verification results show that the detection system of the present invention has large throughput and high sensitivity, and the introduction of the gene chip technology makes the method have a sensitivity comparable to that of fluorescent quantitative PCR. The specific reagents and detection methods are as follows:

1. Kit composition

Composition quantity Storage Conditions Composition quantity Storage Conditions Lysate 50 servings 4°C POD mother liquor 1 tube (100ul) 4°C PCR Mix1-9 50 tubes -20℃ TMB (2mg/ml) 1 bottle (20ul) 4°C PC 1 tube (100ul) -20℃ Sterile pure water 1 tube -20℃

Wherein, PCR Mix1-9 contains the primers of SEQ ID NO.82-90. the

2. The instruments and reagents to be equipped are as follows:

DNA electrophoresis instrument, PCR instrument, chip hybridization instrument, shaker, 0.25% NaBH4 solution. the

Other required reagents were prepared according to the following formula:

20×SSC NaCl 175.3g Sodium citrate 88.2g Add H ₂ O to dissolve to 1000ml, adjust the pH to 7.0 with a pH meter, autoclave 10% SDS, pH7.0 Dissolve 20gSDS with H ₂ O 180ml, adjust the pH to 7.0 with HCl, and finally adjust the volume to 200ml Solution A (2×SSC, 0.1% SDS, pH 7.4) 20×SSC 100ml 10% SDS 10ml Add H ₂ O to dissolve to 1000ml, adjust pH to 7.4 Solution B (0.5×SSC, 0.1% SDS, pH7.4) 20×SSC 25ml 10% SDS 10ml Add H ₂ O to dissolve to 1000ml, adjust the pH to 7.4 Solution C (0.1M Sodium Citrate, pH5.0) Sodium Citrate 14.7g Dissolve in 450ml of water, adjust the pH to 5.0 with concentrated HCl, and finally set the volume to 500ml the

3. Operation steps

(1) Sample collection and storage

The screening samples of GDM susceptible population are mainly taken from peripheral venous blood samples for detection. The collected samples should be stored at room temperature for no more than 2 hours, stored at 4°C for no more than 24 hours, and stored at -20°C for no more than 3 months. Samples should avoid repeated freezing and thawing. the

(2) Sample processing

Transfer the blood sample to a 1.5ml microcentrifuge tube, centrifuge at 13000rpm for 10 minutes, discard the supernatant, and keep the cell mass at the bottom of the tube. the

Add 50ul of lysate to suspend the precipitate, heat at 100°C for 10 minutes, centrifuge at 13000rpm for 10 minutes, and save the supernatant for later use. the

(3) PCR amplification

Take the PCR reaction tube, mark it on the tube wall, add 5 μl of the extracted DNA of the sample to be tested, and the total reaction system is 25 μl. For each experiment, a negative control must be set, that is, 5 μl of sterile pure water is used as a template. the

Amplify according to the flow chart in Figure 2:

(4) hybridization

Take 2 μl of each PCR amplification product of the same sample to be tested and mix, after purification by the PCR product purification kit, add 15 μl of hybridization solution, denature at 95°C for 3 minutes, place on ice for more than 2 minutes, and then load the sample on the chip. the

(5) Chip cleaning

Take out the chip, move it to a 50ml tube filled with preheated solution B, and shake and wash at 42°C for 5 minutes. the

(6) scan

Place the chip in the scanner (LuxScan 10K) and scan according to the established procedures. the

(7) Result judgment

As a control hybridization point, a green fluorescent signal will appear at the PC position, indicating that the hybridization is successful; at the same time, other sites should not develop color, and the interpretation of the result at this time is considered valid. the

The results are shown in the table below. the

Arrangement order of probes on membrane strips

Gene 1SNP1 Gene Gene 1SNP3 Gene

the 1SNP2 the 1SNP4 Gene 2SNP1 Gene 2SNP2 Gene 2SNP3 Gene 2SNP4 Gene 3SNP1 Gene 3SNP2 Gene 3SNP3 Gene 3SNP4 Gene 4SNP1 Gene 4SNP2 Gene 4SNP3 Gene 4SNP4 Gene 5SNP1 Gene 5SNP2 Gene 5SNP3 Gene 5SNP4 Control PC the the the

Whether the patient carries the relevant SNPs of GDM can be judged by the positions displayed by the signal points. the

Claims (3)

1. a gene chip that is used to detect GDM Susceptible population comprises solid phase carrier and is fixed on the oligonucleotide probe on this carrier, it is characterized in that the probe on this chip can be hybridized with GDM related SNP s respectively; Said solid phase carrier is the surface chemistry group modified glass matrix of process, and 5 ' end of said probe carries out amino labeled; The nucleotide sequence of the dna fragmentation of said GDM related SNP s is shown in Seq ID No.1-No.9; The nucleotide sequence of said probe is shown in Seq ID No.10-No.81.

2. a detection reagent that comprises the said gene chip of claim 1 is characterized in that, this detection reagent also comprises the PCR primer of each GDM related SNP s that is used to increase, and the nucleotide sequence of these PCR primers is shown in Seq ID No.82-No.99.

3. a test kit that comprises the said detection reagent of claim 2 is characterized in that, this test kit also comprises a negative control sample and a positive control.

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