Analytical method and kit for diagnosing recurrent miscarriage

Abstract

The present invention provides an analytical method comprising the step of measuring the expression level of HtrA4 protein or the expression level of a gene encoding the protein in a sample from a subject, thereby providing information necessary for diagnosing recurrent miscarriage. Furthermore, the present invention provides a kit for diagnosing recurrent miscarriage, comprising a molecule capable of measuring the expression level of HtrA4 protein or the expression level of a gene encoding the protein.

Description

Technical Field

[0001] The present invention relates to an analysis method for providing information required for diagnosing recurrent miscarriage and a kit for diagnosing recurrent miscarriage. Background Art

[0002] Unlike in the past, with the rise in women's social status in modern society, the age of first marriage has increased, leading to a steady rise in the incidence of various infertility-related conditions, including recurrent miscarriage. Furthermore, recurrent miscarriage can lead to psychological distress caused by the pressure to continue the family line and often leads to family breakdown, exacerbating the severity of the problem. Recurrent miscarriage is defined as two or three consecutive miscarriages before 20 weeks of gestation (Kim, MS, Gu, BH, Song, S., Choi, BC, Cha, DH, and Baek, KH, ITI-H4 as a serum biomarker in recurrent pregnancy loss patients. Mol. BioSyst. 2011, 7). Although recurrent miscarriage is the result of the interaction of various factors, such as fetal or parental chromosomal abnormalities, uterine anatomy abnormalities, hormonal abnormalities, and immunological abnormalities, the cause of 40% to 50% of recurrent miscarriage remains unidentified (Li, L.; Choi, BC; Ryoo, JE; Song, SJ; Pei, CZ; Lee, KY; Paek, J.; Baek, KH, H, posing roles of inter-alpha-trypsininhibitor heavy chain 4 in recurrent pregnancy loss. EbioMedicine 2018, 37). Therefore, to date, there are no preventive or diagnostic methods for recurrent miscarriage, and there is no specific treatment.

[0003] In recent years, diagnostic methods for recurrent miscarriage include amniocentesis, cord blood testing, and chorionic villus sampling. However, these methods may cause complications such as miscarriage, leading many patients to refuse these tests and facing significant resistance to their clinical application. Therefore, there is a need in the field to develop biomarkers that can easily diagnose recurrent miscarriage through blood tests and other methods, and to study the role of these biomarkers. Summary of the Invention

[0004] Technical problems to be solved

[0005] The present inventors have conducted various studies on genes whose expression is specifically altered in patients with recurrent miscarriage. As a result, they discovered that HtrA4 protein expression is significantly lower in patients with recurrent miscarriage than in healthy controls, and revealed the function of HtrA4 protein in recurrent miscarriage. Therefore, detection of low-level expression of HtrA4 protein can be useful for diagnosing recurrent miscarriage, and HtrA4 protein or a gene encoding the protein can be used as a biomarker for diagnosing recurrent miscarriage.

[0006] Therefore, an object of the present invention is to provide an analytical method using HtrA4 protein or a gene encoding the protein to provide information required for diagnosing recurrent miscarriage.

[0007] Furthermore, the present invention aims to provide a kit for diagnosing recurrent miscarriage, the kit comprising a molecule capable of measuring the expression level of HtrA4 protein or a gene encoding the protein.

[0008] Technical Solution

[0009] According to one embodiment of the present invention, an analysis method is provided, which includes the step of measuring the expression level of HtrA4 protein or the expression level of the gene encoding the protein in a sample of a subject, so as to provide information required for diagnosing recurrent miscarriage.

[0010] In the analysis method of the present invention, the sample from the subject can be blood or serum. The expression level of the HtrA4 protein can be measured by Western blotting or enzyme-linked immunosorbent assay (ELISA), and the expression level of the gene encoding the HtrA4 protein can be measured by measuring the amount of mRNA, preferably by measuring the amount of mRNA by reverse transcription PCR (RT-PCR) or real-time PCR.

[0011] According to another embodiment of the present invention, a kit is provided, which is a kit for diagnosing recurrent miscarriage, comprising a molecule that can measure the expression level of HtrA4 protein or the expression level of the gene encoding the protein, wherein the molecule is an antibody, substrate, ligand or cofactor that specifically binds to the protein, or a primer having a complementary sequence specific to the gene encoding the protein.

[0012] In the diagnostic kit of the present invention, the molecules may be labeled with a detectable marker. In another embodiment, the kit may be in the form of a microarray in which the primers are immobilized on a substrate.

[0013] Beneficial effects

[0014] The present invention discovered that HtrA4 protein expression is significantly lower in patients with recurrent miscarriage than in healthy individuals. Therefore, the analysis method and kit according to the present invention can be useful for diagnosing recurrent miscarriage. In other words, HtrA4 protein or a gene encoding the protein can be useful as a biomarker for diagnosing recurrent miscarriage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of confirming the expression level of HtrA4 in the serum of patients with recurrent miscarriage and normal controls are shown. (C represents the normal group (control group), and P represents patients with recurrent miscarriage)

[0016] Figure 2 Shown based on Figure 1 The results of statistical analysis of the relative expression rate of HtrA4.

[0017] Figure 3 The results of Western blot analysis showing the expression level of HtrA4 in choriocarcinoma cell lines BeWo, HTR / SVneo, and JEG3 cells are shown.

[0018] Figure 4 The results are shown in which the knockout status of two BeWo cell lines in which the HtrA4 gene was knocked out was confirmed by T7E1 analysis.

[0019] Figure 5 The figures show the results of TA cloning and sequence analysis of the knockout portion of the HtrA4 gene in wild-type and knockout cell lines.

[0020] Figure 6 Shown are the results of confirming the expression level of HtrA4 in wild-type and knockout BeWo cells by Western blot analysis.

[0021] Figure 7 The results of confirming the proliferation ability of wild-type and knockout BeWo cells using CCK-8 are shown.

[0022] Figure 8 The results of confirming the proliferation ability of wild-type and knockout BeWo cells by colony formation assay are shown.

[0023] Figure 9 The results of Western blot analysis showing the expression levels of ERK, p-ERK, p38, and p-p38, which are associated with cell proliferation, in wild-type and knockout BeWo cells are shown.

[0024] Figure 10 is based on Figure 9The results of statistical analysis of the relative protein expression rates of ERK, p-ERK, p38 and p-p38.

[0025] Figure 11 The results of Western blot analysis confirming the expression levels of cyclin E and cyclin A, which are associated with the cell cycle, in wild-type and knockout BeWo cells are shown.

[0026] Figure 12 Shown based on Figure 11 The results are the results of statistical analysis of the relative protein expression rates of cyclin E and cyclin A.

[0027] Figure 13 The figures show the results of confirming the invasion ability of wild-type and knockout BeWo cells by invasion assay.

[0028] Figure 14 The results show that the expression levels of MMP-2 and MMP-9, which are associated with invasion, in wild-type and knockout BeWo cells were confirmed by Western blot analysis.

[0029] Figure 15 Shown based on Figure 14 The results of statistical analysis of the relative protein expression rates of MMP-2 and MMP-9.

[0030] Figure 16 The graph shows the results of confirming the migration ability of wild-type and knockout BeWo cells by scratch wound assay.

[0031] Figure 17 The results show that the expression levels of FAK, which is associated with the migration ability of cells, were confirmed in wild-type and knockout cells by Western blot analysis.

[0032] Figure 18 Shown based on Figure 17 The results of statistical analysis of the relative protein expression rate of FAK.

[0033] Figure 19 Shown are photographs of the morphology of wild-type and knockout cells taken with a microscope at different magnifications.

[0034] Figure 20 The results of Western blot analysis confirming the expression levels of vascular endothelial cadherin (VE-cadherin), which promotes cell-cell junctions, in wild-type and knockout cells are shown.

[0035] Figure 21 Shown based on Figure 20 The results of statistical analysis of the relative protein expression rate of vascular endothelial cadherin. DETAILED DESCRIPTION

[0036] In this specification, "recurrent pregnancy loss" refers to a disease in which a woman suffers 2 to 3 or more consecutive miscarriages before 20 weeks of gestation.

[0037] The present inventors conducted various studies on genes showing differential expression in serum isolated from patients with recurrent miscarriage and normal controls. Their results revealed that expression of a specific protein, HtrA4, was significantly lower in patients with recurrent miscarriage than in normal controls. Furthermore, to investigate the mechanism by which changes in HtrA4 expression contribute to recurrent miscarriage, the present inventors selected a BeWo cell line with high HtrA4 expression levels and constructed cells in which the HtrA4 gene was knocked out. By comparing wild-type and knockout cell lines, they evaluated the effects of HtrA4 on cell proliferation, migration, invasion, and adhesion. Their results revealed that wild-type BeWo cells exhibited more active invasion and adhesion than knockout BeWo cells, but that knockout BeWo cells exhibited more active proliferation and migration than wild-type BeWo cells, and that the cell cycle of HtrA4-knockout BeWo cells was shorter than that of wild-type BeWo cells. These results indicate that HtrA4 protein plays an important role in fertilized egg implantation, placenta formation, development, and function. Therefore, HtrA4 protein and the gene encoding the protein can be useful as a biomarker for predicting recurrent miscarriage and a biomarker for a diagnostic kit.

[0038] The present invention provides an analytical method comprising the step of measuring the expression level of HtrA4 protein or the expression level of a gene encoding the protein in a sample of a subject, so as to provide information required for diagnosing recurrent miscarriage.

[0039] The HtrA4 protein and the gene encoding the protein used as biomarkers in the analytical methods of the present invention are known, and thus, known protein and gene sequences can be used in the analytical methods of the present invention. Specifically, the NCBI accession numbers for the HtrA serine peptidase 4 (HtrA4) protein are NP_710159.1, XP_011542733.1, and XP_011542734.1, and the NCBI accession numbers for the mRNA encoding the protein are NM_153692.4, XM_011544431.2, XM_011544432.1, and BC057765.1.

[0040] In the analysis method of the present invention, the subject sample refers to a sample separated from the human body outside the body, for example, including blood, serum, etc. separated from the human body outside the body.

[0041] The analysis method of the present invention includes the step of measuring the expression level of HtrA4 protein or the expression level of the gene encoding the protein. The amino acid sequence of the HtrA4 protein and the gene sequence encoding the protein can be genes known in GenBank or the like.

[0042] The expression level of the HtrA4 protein or gene can be measured by measuring the level of mRNA of the protein or gene according to a method commonly used in the field of bioengineering.

[0043] The expression level of HtrA4 protein can be measured by Western blotting or enzyme-linked immunosorbent assay. For example, when the expression level of HtrA4 protein is significantly lower than that of a normal person (e.g., at most about 1 / 4), the patient can be determined to be at risk for recurrent miscarriage. Furthermore, the expression level of the gene encoding the proteolysis regulating enzyme can be measured by measuring the amount of mRNA, preferably by measuring the amount of mRNA using RT-PCR or real-time PCR. When the expression level of the HtrA4 gene is significantly lower than that of a normal person, the patient can be determined to be at risk for recurrent miscarriage.

[0044] The present invention also provides a kit for diagnosing recurrent miscarriage, which comprises a molecule capable of measuring the expression level of HtrA4 protein or the expression level of the gene encoding the protein, wherein the molecule is an antibody, substrate, ligand or cofactor that specifically binds to the protein, or a primer having a complementary sequence specific to the gene encoding the protein.

[0045] In the diagnostic kit of the present invention, polyclonal or monoclonal antibodies can be prepared against the HtrA4 protein using methods commonly used in the field of bioengineering, and diagnostic kits containing these antibodies can be prepared. Furthermore, since the functions of the HtrA4 protein have been revealed, the kit of the present invention can also be prepared to contain substrates, ligands, or cofactors specific for the HtrA4 protein. Furthermore, primers having complementary sequences specific for genes encoding proteolysis-regulating enzymes can be prepared using methods commonly used in the field of bioengineering, and diagnostic kits containing these primers can also be prepared.

[0046] In the diagnostic kit of the present invention, the molecule capable of measuring the expression level of the gene encoding the protein can be labeled with a detectable marker (e.g., a chromophore). In addition, the diagnostic kit of the present invention has a microarray format in which the primers are immobilized on a substrate, and thus can also be in the form of a chip such as a DNA chip or a protein chip.

[0047] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are merely for illustrating the present invention, and the present invention is not limited to the following examples.

[0048] Example

[0049] 1. Test methods

[0050] (1) Sample preparation

[0051] This study was approved by the Ethics Committee of CHA University of Medicine in Seoul, South Korea. All participants in this study provided written informed consent, and the study of blood samples was approved by the Institutional Review Board (reference number: 08-16). Blood was collected from a group of patients with recurrent miscarriage (60 women) and a normal control group (32 women) who visited the Fertility Center of the CHA General Hospital. The blood was divided into peripheral blood mononuclear cells (PBMCs) and serum. Only serum was used in this study and stored at -80°C. The diagnostic criteria for patients with recurrent miscarriage were women with a history of at least 2-3 spontaneous abortions and consecutive miscarriages before 20 weeks of gestation. The normal group (control group) was composed of women who had undergone at least one delivery without obstetric complications and no history of miscarriage.

[0052] (2) Construction of BeWo cell line with HtrA4 gene knockout

[0053] sgRNA was designed from the homepage (http: / / www.rgenome.net) [forward primer: 5'-CAG CGG CAC AGGTCG AAC ACC GG-3' (SEQ ID NO: 1) and reverse primer: 5'-AAA CCA GAC TTC ACG CTC GGC-3' (SEQ ID NO: 2)] and then cloned into the pSpCas9(BB)-2A-GFP (pX458) vector (Addgene, Plasmid catalog #: 48138) to prepare the sgRNA-pX458 plasmid. The sgRNA-pX458 plasmid was transfected into choriocarcinoma cell line BeWo cells (KCLB: 10098, Korean Cell Bank) using a Lonza 4D-Nucleofector (Lonza, 50829 Clogne, Germany) according to the manufacturer's instructions and electrophoresed. The obtained cells were plated at 1 cell / well in a 96-well plate and cultured for 8 days to reach a confluence of 60-70%. Genomic DNA was extracted using the Accuprep Genomic DNA Extraction Kit (Bioneer Corporation, Daejeon, Korea) according to the manufacturer's instructions, and polymerase chain reaction (PCR) was performed using the forward primer: 5'-GAG GGT TTG CAG GTC CAG AG-3' (SEQ ID NO: 3) and the reverse primer: 5'-ACA TGC TGG GGT AGGTGC-3' (SEQ ID NO: 4). PCR products were analyzed by T7E1 to select only cells with HtrA4 gene knockout, and TA cloning was performed to confirm the base sequence. The expression level of HtrA4 was confirmed by Western blot analysis.

[0054] (3) Colony forming assay

[0055] The wild type and knockout cell lines were seeded in equal numbers and then stained with crystal violet for colony formation analysis. 3Cells / well were seeded into 96-well plates and then cultured for 0 hours, 24 hours, 36 hours, 48 ​​hours, and 60 hours. The cells were treated with Dulbecco's Modified Eagle Medium (DMEM) containing 10 μL of WST-8 solution (Cell Counting Kit-8 Dojindo, Kumamoto, Japan), then cultured for 4 hours, and the OD value was measured at 450 nm. Furthermore, after 14 days, the proliferation ability of the cells was confirmed by comparing the number of colonies formed by wild-type and knockout cells.

[0056] (4) Scratch analysis

[0057] Wild-type and knockout cell lines were seeded into 6-well plates and then, when cells reached 70-80% confluence, scratched with a micropipette tip. Cells were washed with phosphate-buffered saline to remove stripped cells and analyzed using Image-J software at 0, 24, and 48 hours. Digital images were taken using a microscope (Olympus, Tokyo, Japan).

[0058] (5) Invasion analysis

[0059] Wild-type and knockout cell lines were cultured at 1×10 6 Cells were plated at a density of 10 cells / ml in a 24-well upper chamber (80-μm pore membranes; BD Biosciences, Franklin Lakes, NJ, USA) with serum-free medium (DMEM). DMEM medium containing 10% FBS was added to the lower chamber. After incubation at 37°C for 36 hours, non-invasive cells were removed from the upper chamber using cotton swabs, and cells on the lower membrane surface were stained with crystal violet for 10 seconds to allow the attachment of invasive BeWo cells. The invasion chamber was washed twice with phosphate-buffered saline, and the number of invasive BeWo cells was counted under a microscope (Olympus, Tokyo, Japan). Each experiment was repeated three times.

[0060] (6) Western blotting

[0061] Wild-type and knockout cells were lysed in lysis buffer on ice for 20 minutes and then centrifuged at 13,000 rpm for 20 minutes. The samples were boiled with 2X SDS protein loading buffer for 10 minutes, then loaded onto SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) microporous membranes (Millipore, Billerica, Massachusetts (MA), USA). The primary antibody was reacted with the membrane overnight at 4°C, followed by washing, adding the secondary antibody, and reacting at room temperature for 1 hour. The blots were detected using ECL reagent solution (YoungIn Frontier, Seoul, South Korea).

[0062] (7) Antibodies

[0063] HtrA4 antibody was purchased from Proteintech (Proteintech, Rosemont, Illinois (IL), USA); ERK1 / 2 antibody, p-ERK (Thr202 / Tyr204) antibody, p38 antibody, p-p38 antibody, cyclin A antibody, cyclin E antibody, and vascular endothelial cadherin antibody were purchased from Cell Signaling Technology (Cell Signaling Technology Inc., Danvers, Massachusetts (MA), USA); MMP-2 antibody, MMP-9 antibody, p-FAK (Try397) antibody, and β-actin antibody were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, California (CA), USA).

[0064] (9) Confirmation and analysis of the results

[0065] Densitometric analysis was performed using Image J (National Institutes of Health, Bethesda, MD, USA), and t-tests were performed using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA). ANOVA was performed using one-way analysis to show significant differences.

[0066] 2. Test results

[0067] The blood of patients with recurrent miscarriage and normal controls was separated into serum and PBMC, and the expression level of HtrA4 in serum was confirmed by Western blot analysis. Figure 1 and Figure 2 shown. Figure 1 The results showed that the expression level of HtrA4 was confirmed in the serum of patients with recurrent miscarriage and normal controls. Figure 2 is based on Figure 1 The results of statistical analysis of the relative protein expression rate of HtrA4. Figure 1 and Figure 2 The results confirmed that HtrA4 was expressed at a significantly low level in patients with recurrent miscarriage.

[0068] Figure 3 The results of confirming the expression level of HtrA4 in choriocarcinoma cell lines BeWo, HTR / SVneo, and JEG3 cells are shown, and it was confirmed that HtrA4 was most highly expressed in BeWo cells. Figure 4 Results from T7E1 analysis of two HtrA4 knockout BeWo cell lines and a wild-type BeWo cell line confirming knockout. Both KO1 and KO2 cell lines had HtrA4 knockout, while both alleles were knocked out in KO2. Figure 5 The results of TA cloning and sequence analysis of two bands in the KO2 cell line confirmed the deletion of 95 bases in allele 1 and 35 bases in allele 2. Subsequent experiments were conducted using the KO2 cell line.

[0069] Figure 6 The results of Western blot analysis confirming the expression levels of HtrA4 in wild-type and knockout BeWo cells showed that HtrA4 expression was significantly low in the knockout cells.

[0070] Figure 7 The results of confirming the proliferation capacity of wild-type and knockout BeWo cells using CCK-8 confirmed that the proliferation capacity of knockout cells showed statistical significance after 24 hours and proliferated faster with the passage of time. Figure 8 The results of confirming the proliferation ability of wild-type and knockout BeWo cells by colony formation analysis confirmed that the proliferation ability of knockout cells was stronger as expected.

[0071] Figure 9 The results are the results of confirming the expression levels of ERK, p-ERK, p38, and p-p38, which are related to cell proliferation, in wild-type and knockout BeWo cells. Figure 10 is based on Figure 9The results of statistical analysis of the relative protein expression rates of ERK, p-ERK, p38 and p-p38. Figure 9 and Figure 10 The results showed that ERK, p-ERK, p38, and p-p38, which promote cell proliferation, were expressed more in knockout cells with stronger cell proliferation ability.

[0072] Figure 11 The results of confirming the expression levels of cyclin A and cyclin E, proteins that regulate the cell cycle, by Western blot analysis, Figure 12 is based on Figure 11 The results of the statistical analysis of the relative protein expression rates of cyclin A and cyclin E. Figure 11 and Figure 12 The results confirmed that the expression level of cyclin E, which is associated with the transition from G1 phase to S phase, was higher in wild-type BeWo cells, but the expression level of cyclin A, which is associated with the transition from S phase to G2 phase, was higher in knockout cells.

[0073] Figure 13 The results of confirming the invasive ability of wild-type and knockout BeWo cells by invasion analysis showed that the invasive ability of knockout cells was weaker than that of wild-type cells. Figure 14 Western blot analysis was used to confirm the expression levels of MMP-2 and MMP-9, which are associated with invasion, in wild-type and knockout BeWo cells. Figure 15 is based on Figure 14 The results of statistical analysis of the relative protein expression rates of MMP-2 and MMP-9. Figure 14 and Figure 15 The results showed that the expression levels of MMP-2 and MMP-9, which promote cell invasion, were low in the knockout cells.

[0074] Figure 16 The results of the scratch assay confirming the migration ability of wild-type and knockout BeWo cells indicate that the knockout cells have a stronger migration ability than the wild-type cells. Figure 17 The results confirm the expression level of FAK in wild-type and knockout cells, which is related to the migration ability of cells. Figure 18 is based on Figure 17 The results of statistical analysis of the relative protein expression rate of FAK. Figure 17 and Figure 18 The results showed that FAK, which promotes cell migration, is more expressed in knockout cells with strong migration ability than in wild-type cells.

[0075] Figure 19 The morphology of wild-type and knockout cells was photographed using a microscope at different magnifications. Figure 19 It can be confirmed that wild-type cells grow in close contact with each other, but knockout cells grow without close cell-to-cell contact. Figure 20 Western blot analysis confirmed the expression levels of vascular endothelial cadherin, which promotes cell-cell junctions, in wild-type and knockout cells. Figure 21 is based on Figure 20 The results of statistical analysis of the relative protein expression of vascular endothelial cadherin (VE-cadherin) confirmed that, as expected, VE-cadherin, which promotes cell-cell junctions, was expressed less in knockout cells compared to wild-type cells.

[0076] 3. Discussion

[0077] While much research has been conducted on the causes of recurrent miscarriage, the role of proteases in the pathogenesis of recurrent miscarriage remains insufficient. The present inventors have discovered that the expression of the HtrA4 gene in the serum of patients with recurrent miscarriage is lower than that in normal controls. This demonstrates that HtrA4 is a biomarker for recurrent miscarriage and can be used to develop a kit that can predict and diagnose recurrent miscarriage using a small amount of blood.

[0078] Based on these results, the function of the HtrA4 gene in recurrent miscarriage was investigated. To this end, a BeWo cell line with HtrA4 knockout was constructed and compared with wild-type BeWo cells. The results showed that HtrA4 promoted the invasion and adhesion of placental chorionic cells and inhibited their proliferation and migration. Therefore, it was found that low HtrA4 expression in patients with recurrent miscarriage affects the function of placental chorionic cells and leads to abnormalities in placental formation, development, and function, thereby causing miscarriage. <110> Che Medical Science University Industry-Academic Cooperation Group <120> Analytical method and kit for diagnosing recurrent miscarriage <130> KHP222111304.6 <150> KR 10-2020-0026269 <151> 2020-03-03 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 1 cagcggcaca ggtcgaacac cgg 23 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 2 aaaccagact tcacgctcgg c 21 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 3 gagggtttgc aggtccagag 20 <210> 4 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 acatgctggg gtaggtgc 18

Claims

1. Use of a molecule capable of measuring the expression level of human HtrA4 protein or the expression level of a gene encoding said protein in the preparation of a kit for diagnosing recurrent miscarriage in humans, wherein said molecule is an antibody, substrate, ligand or cofactor that specifically binds to said protein, or a primer having a complementary sequence specific to the gene encoding said protein.

2. The use according to claim 1, characterized in that The molecule is labeled with a detectable label.

3. The use according to claim 1, characterized in that The kit is in the form of a microarray in which the primers are fixed on a substrate.

Images