Application of mRNA marker in preparation of Stevens-Johnson syndrome / toxic epidermal necrolysis syndrome detection kit

Abstract

The invention provides an application of an mRNA (messenger Ribonucleic Acid) marker in preparation of a Stevens-Johnson syndrome / toxic epidermal necrosis debonding disease detection kit, and the mRNA marker can be used for preparing the Stevens-Johnson syndrome / toxic epidermal necrosis debonding disease detection kit and can be used for detecting the Stevens-Johnson syndrome / toxic epidermal necrosis debonding disease. Differential mRNA expression profiles in PBMC and skin tissues of SJS / TEN patients and healthy individuals can be analyzed, and the clinical value of the differential mRNA expression profiles can be analyzed, so that the differential mRNA expression profiles can be used for SJS / TEN diagnosis or severity prediction.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of mRNA markers in the preparation of a Stevens-Johnson syndrome / toxic epidermal necrolysis detection kit. Background Art

[0002] Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are important types of severe skin and mucosal adverse reactions, characterized by epidermal necrosis and high mortality. When the area of ​​epidermal lysis is <10% of the body surface area (BSA), it is defined as SJS, when the area of ​​epidermal lysis is 10%-30% BSA, it is defined as SJS-TEN coexistence, and when the area of ​​epidermal lysis is ≥30% BSA, it is defined as TEN. The larger the area of ​​epidermal lysis, the worse the prognosis. The skin manifestations of SJS / TEN are painful erythema, bullae and erosions on the face, trunk and limbs. The Nikolsky sign is usually positive, often accompanied by liver and kidney damage and respiratory complications, secondary severe infection, so the mortality rate is very high. At the same time, almost all SJS / TEN patients will have eye, oral and genital mucosal involvement. Even after recovery, patients may suffer from sequelae such as dry eyes, decreased vision, conjunctivitis, corneal erosion, trichiasis, and even serious blindness. Therefore, a biomarker for early diagnosis and severity prediction is needed so that SJS / TEN patients can be accurately diagnosed and start reasonable treatment as early as possible, which is of great significance for the treatment and prognosis of SJS / TEN patients.

[0003] Regarding the pathogenesis of SJS / TEN, it is traditionally considered to be a T cell-mediated disease, in which drugs bind to antigen presenting cells and activate T cell receptors, resulting in a strong immune response. There are currently three hypotheses about T cell activation: (1) hapten / prehapten model, (2) pharmacological interaction theory, and (3) altered peptide model. These all involve human leukocyte antigen presenting drugs and / or metabolite-altered peptides to T cell receptors. In recent years, researchers have made significant progress in the pathogenesis of SJS / TEN. However, the factors that lead to keratinocyte necrosis remain to be determined. Epidermal damage in the skin lesions of SJS / TEN patients is considered to be the origin of apoptosis. In the early stage of the disease, cytotoxic CD8+ T cells mainly infiltrate the blister fluid and epidermis, and CD4+ T cells mainly infiltrate the dermis. In the later stage, lymphocytes decrease and the number of monocytes increases. Therefore, peripheral blood mononuclear cells (PBMCs) are involved in the occurrence and development of SJS / TEN and are closely related to keratinocyte necrosis. Cytotoxic CD8+T cells induce apoptosis through the Fas-Fas ligand pathway or the perforin / granzyme pathway. Monocytes play an important role in epidermal damage by enhancing the cytotoxicity of CD8+T cells. Therefore, the development of new biomarkers for SJS / TEN is a technical problem that needs to be solved by those skilled in the art. Summary of the invention

[0004] The object of the present invention is to provide an application of an mRNA marker in the preparation of a Stevens-Johnson syndrome / toxic epidermal necrolysis detection kit for SJS / TEN diagnosis or severity prediction.

[0005] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0006] The present invention provides the use of an mRNA marker in preparing a Stevens-Johnson syndrome / toxic epidermal necrolysis detection kit, wherein the nucleotide sequence of the mRNA marker is shown in SEQ ID NO:1.

[0007] Beneficial effects:

[0008] The research results of the present invention show that in PBMC, ZMAT4, DNASE1L3, SH2D1B, and GNLY in the SJS / TEN patient group were significantly downregulated compared with the normal control group, and TNFAIP6, BMX, and IFI27 were significantly upregulated; in skin tissue, SH2D1B, FCGR3B, GNLY, TNFAIP6, BMX, and IFI27 in the SJS / TEN patient group were significantly upregulated compared with the normal control group. When IFI27 was used to distinguish SJS / TEN patients from healthy people in PBMC and skin tissue, the ROC curve area was ≥0.90, and the specificity was 1, which had good diagnostic value. The key gene (IFI27) was verified to be significantly highly expressed in PBMC and skin tissue of SJS / TEN patients. In addition, it was found that the expression level of IFI27 in PBMC and skin tissue was significantly correlated with the area of ​​epidermolysis. Further analysis showed that in PBMC and skin tissue, IFI27 was significantly upregulated in the SJS / TEN patient group compared with the erythema multiforme (EM), maculopapular drug eruption (MPE), drug hypersensitivity syndrome (DRESS), autoimmune blistering disease (AIBD), and burns (Brun) patient groups. The ROC curve area of ​​IFI27 in PBMC and skin tissue was ≥0.78 when used to distinguish SJS / TEN from EM, MPE, DRESS, AIBD, and Brun patients, which has good diagnostic value.

[0009] Therefore, the present invention proposes an mRNA marker associated with Stevens-Johnson syndrome / toxic epidermal necrolysis, which can be used to prepare a Stevens-Johnson syndrome / toxic epidermal necrolysis detection kit, and can analyze the differential mRNA expression in PBMC and skin tissues of SJS / TEN patients and healthy individuals and analyze its clinical value, so as to be used for SJS / TEN diagnosis or severity prediction. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The figure shows the difference analysis of 8 mRNAs in PBMCs of SJS / TEN group and normal control group;

[0011] Figure 2 The figure shows the difference analysis of 8 mRNAs in the skin tissues of SJS / TEN group and normal control group;

[0012] Figure 3 ROC curve analysis of IFI27 expression levels in PBMC and skin tissues of SJS / TEN patients;

[0013] Figure 4This is a correlation analysis chart between the IFI27 expression level and epidermal lysis area in PBMC and skin tissues of SJS / TEN patients.

[0014] Figure 5 This is a differential analysis diagram of IFI27 in PBMCs of SJS / TEN and EM, MPE, DRESS, AIBD, Brun and normal control groups;

[0015] Figure 6 This is a differential analysis of IFI27 in the skin tissues of SJS / TEN and EM, MPE, DRESS, AIBD, Brun and normal control groups. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0017] 1. Materials and Methods

[0018] 1.1 Sample collection

[0019] Methods: Twenty-three patients with SJS / TEN who were hospitalized in the Department of Dermatology, the First Affiliated Hospital of Nanchang University from January 2023 to December 2023 were collected. PBMC and skin tissues of patients with clinical diagnosis or pathological confirmation of SJS / TEN, erythema multiforme (EM), maculopapular drug eruption (MPE), drug hypersensitivity syndrome (DRESS), autoimmune blistering disease (AIBD), and burns (Brun) were selected, and the clinical data of the patients were recorded. The epidermolysis area of ​​toxic epidermal necrolysis was evaluated. At the same time, 20 healthy volunteers whose age and gender matched those of the experimental group were recruited as the normal control group. All subjects signed informed consent, and the study was subject to ethical approval from the First Affiliated Hospital of Nanchang University.

[0020] The ratio of epidermolysis area to body surface area (BSA) at admission is an important indicator for assessing the severity and prognosis of SJS / TEN. The area of ​​epidermolysis is assessed only for skin that has been exfoliated, necrotic, or has a positive Nikolsky sign, and does not include unseparated and inseparable erythema or purpura. The body surface area can be assessed using the nine-point principle (Table 1): the adult body is divided into 13 parts, with a BSA value of 9% for the head and neck; 18% for the upper limbs on both sides; 27% for the trunk; 45% for the lower limbs on both sides; and 1% for the groin. In addition, the palm method can be used, using the patient's palm as a reference value for BSA1% for overall assessment.

[0021] Table 1

[0022]

[0023] 1.2 Human peripheral blood mononuclear cells (PBMC) isolation method

[0024] (1) Pour 10 ml of whole blood into a 50 ml centrifuge tube, then add 10 ml of PBS solution to dilute and mix gently.

[0025] (2) Next, prepare two 15 ml centrifuge tubes and add 5 ml of Ficoll solution to each tube. Then, gently add the diluted blood to the top layer of Ficoll in each centrifuge tube. Be gentle to avoid mixing the two solutions. Add 10 ml of diluted blood to each centrifuge tube.

[0026] (3) During the centrifugation process, the centrifuge should be centrifuged at a speed of 2000 rpm for 20 minutes. When decelerating, the centrifuge can be set to horizontal inertia braking, or only 1-2 braking.

[0027] (4) After centrifugation, the tube will be divided into four layers, from top to bottom: plasma layer, PBMC layer, Ficoll solution layer and red blood cell layer.

[0028] (5) Transfer the PBMC cell layer to another 15 ml centrifuge tube. Then add an appropriate amount of PBS and centrifuge at 1500 rpm for 10 minutes, then remove the supernatant. The PBMC cell layer appears white.

[0029] (6) Add 1 ml of TransZolUp to a 1.5 ml centrifuge tube to allow TransZolUp to be evenly distributed on the cell surface and lyse the cells. Then use a pipette to blow the cells to make them fall off, and transfer the cell lysate to the centrifuge tube.

[0030] (7) Can be frozen at -80℃ before RNA extraction.

[0031] 1.3 High-throughput sequencing library construction

[0032] Total RNA from PBMCs of three SJS / TEN patients and three normal subjects was extracted for whole transcriptome sequencing and database was established. The first step was to extract total RNA from the samples, and then remove ribosomal RNA using a ribo-zero kit. Subsequently, RNA was sheared into shorter fragments and single-stranded cDNA was synthesized by random primers. Next, the second-strand cDNA was prepared by a two-strand synthesis reaction system, using dUTP instead of dTTP. After connecting the adapter, the cDNA chain containing dUTP was digested by UNG enzyme method, and only the cDNA chain connected to different adapters was retained. After purification, repair, A-tailing and connecting sequencing adapters, fragment size selection and PCR amplification were performed. Finally, the constructed library was qualified by Agilent 2100 bioanalyzer, and then 2×150bp read length double-end sequencing was performed by Illumina sequencer. The obtained raw reads (Raw Data) were then filtered and processed using trimmomatic software to filter low-quality bases, N bases or low-quality reads, thereby obtaining high-quality sequencing data (Clean Data). In order to carry out subsequent bioinformatics analysis of mRNAs.

[0033] 1.4 Screening of differentially expressed mRNA

[0034] In the sequencing data, the expression level of each mRNA in different samples was evaluated by the number of sequencing fragments (Fragments Per Kilobase of exon model per Million mapped reads, FPKM), and the differentially expressed genes (DEGs) in the data set were screened using the limma package, and DEGs were selected to meet FC>2 or <0.5 (|log2FC|>1) and p value<0.05. Volcano maps and heat maps of different mRNAs were generated using R software to show the differential distribution and differential expression of sample DEGs.

[0035] 1.5 RNA extraction

[0036] Adjust the low-temperature centrifuge to 4°C before operation, and dilute Wash Buffer 9 5 times by adding anhydrous ethanol before use.

[0037] 1.5.1 Homogenization

[0038] a. Isolation of PBMC cells

[0039] Take out the frozen PBMC cells with transZol Up from the -80℃ freezer, thaw naturally (this step can be omitted if they are not frozen and directly extracted), add 0.2ml RNA Extraction Agent, pipette repeatedly until there is no precipitation, and then shake at room temperature for 5 minutes.

[0040] b. Skin tissue

[0041] After ultra-low temperature freezing, the tissue should be quickly transferred to a pre-cooled liquid nitrogen mortar and ground into powder with a pestle. Liquid nitrogen can be added during the process. Then transfer the powdered sample to a centrifuge tube, add TransZol Up and RNAExtraction Agent, and mix using an electric homogenizer. Finally, shake at room temperature for 5 minutes.

[0042] 1.5.2. Centrifuge the mixture at 10,000g and 4°C for 15 minutes. It will separate into a colorless aqueous phase, a middle phase, and a pink organic phase. RNA is mainly present in the aqueous phase.

[0043] 1.5.3. Then transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol, and gently invert to mix.

[0044] 1.5.4. The subsequent steps can be completed at room temperature. First, add the solution and precipitate to the centrifuge column, centrifuge at 12,000 g for 30 seconds at room temperature, and discard the effluent.

[0045] 1.5.5. Then, add 500ul of Clean Buffer 9, centrifuge at 12,000g for 30 seconds at room temperature, and discard the flow-through. Repeat this step once.

[0046] 1.5.6. Then, add 500ul of Wash Buffer 9 containing anhydrous ethanol, centrifuge at 12,000g for 30 seconds at room temperature, and discard the flow-through. Repeat this step once.

[0047] 1.5.7. Then, centrifuge at 12000g for 2 minutes at room temperature to completely remove the residual ethanol. Then gently pull out the centrifuge column and place it in the RNase-free Tube, add 50ml of RNase-free water to the central filter of the centrifuge column, and let it stand for 1 minute.

[0048] 1.5.8. Finally, centrifuge at 12,000 g for 1 minute at room temperature to elute the RNA.

[0049] 1.5.9. Use nanodrop spectrophotometer to measure the concentration of extracted RNA and record the results.

[0050] 1.5.10. Store RNA at -80°C.

[0051] 1.6 cDNA synthesis and gDNA removal

[0052] First-strand cDNA synthesis and gDNA removal should be performed on ice.

[0053] 1.6.1. In an RNase-free centrifuge tube, add reagents according to Table 2:

[0054] Table 2

[0055] Reagents dose Total RNA / mRNA 0.1ng-5μg / 10pg-500ng Anchored Oligo(dT)Primer(0.5ug / ul) lμl 2×ES Reaction Mix 10μl <![CDATA[EasyScripr 2 RT / RI Enzyme Mix]]> 1μl gDNA Remover 1μl RNase-free Water Variable Total volume 20μl

[0056] 1.6.2. Mix by shaking.

[0057] 1.6.3. Incubate at 42°C for 15 min in a PCR amplifier and heat at 85°C for 5 s.

[0058] 1.7 Real-time qPCR

[0059] 1.7.1 Primer design

[0060] The primer sequences used in this experiment are as follows:

[0061] The qRT-PCR primers for IFI27 gene are:

[0062] Upstream primer sequence: 5′-CCTCATCAGCAGTGACCAGTGTG-3′, as shown in SEQ ID NO:1.

[0063] Downstream primer sequence: 5'-TCGCAATGACAGCCGCAATGG-3', as shown in SEQ ID NO:2.

[0064] The qRT-PCR primers for the BMX gene are:

[0065] Upstream primer sequence: 5′-AGCCGAAGTCAGTGGTTGAAAGC-3′, as shown in SEQ ID NO:3.

[0066] Downstream primer sequence: 5′-TGTCTGGGAAGGTGGGAACTCTG-3′, as shown in SEQ ID NO:4.

[0067] The qRT-PCR primers for TNFAIP6 gene are:

[0068] Upstream primer sequence: 5′-GTCTGTGCTGCTGGATGGATGG-3′, as shown in SEQ ID NO:5.

[0069] Downstream primer sequence: 5′-CGTACTCATTTGGGAAGCCTGGAG-3′, as shown in SEQ ID NO:6.

[0070] The qRT-PCR primers for the GNLY gene are:

[0071] Upstream primer sequence: 5′-AAGGTCTGGTCTTCTCTCGTCTGAG-3′, as shown in SEQ ID NO:7.

[0072] Downstream primer sequence: 5′-ACACTTCTCTGGGTGGGCTTATCC-3′, as shown in SEQ ID NO:8.

[0073] The qRT-PCR primers for the FCGR3B gene are:

[0074] Upstream primer sequence: 5′-GTGGTGTTCCTGGAGCCTCAA-3′, as shown in SEQ ID NO:9.

[0075] Downstream primer sequence: 5′-GGAGCAACAGCCAGCCGATATG-3′, as shown in SEQ ID NO:10.

[0076] The qRT-PCR primers for the SH2D1B gene are:

[0077] Upstream primer sequence: 5′-AGTCCTGTGCCTCTGTGTCTCG-3′, as shown in SEQ ID NO:11.

[0078] Downstream primer sequence: 5′-GCTGGGGCTGGTTCTCTTTATTGG-3′, as shown in SEQ ID NO:12.

[0079] The qRT-PCR primers for DNASE1L3 gene are:

[0080] Upstream primer sequence: 5′-TGCTGCTTCTCCTCCTCTCCATC-3′, as shown in SEQ ID NO:13.

[0081] Downstream primer sequence: 5′-CACATCTGCGTCTCCATCCTGATAG-3′, as shown in SEQ ID NO:14.

[0082] The qRT-PCR primers for ZMAT4 gene are:

[0083] Upstream primer sequence: 5′-GATCTCCGAATCGCAGCGTGTG-3′, as shown in SEQ ID NO:15.

[0084] Downstream primer sequence: 5′-GCGTGGATTTTGCCTTGATAATGGG-3′, as shown in SEQ ID NO:16.

[0085] 1.7.2. In the eight-tube PCR tube, add the corresponding reagents according to Table 3 to prepare the PCR reaction solution (20 μl system).

[0086] Table 3

[0087] Reagents dose cDNA 1μl Forward Primer (10μM) 0.4μll Reverse Primer (10μM) 0.4μll 2×PerfectStart Green qPCR SuperMix 10μll Nuclease-free Water Variable Total volume 20μl

[0088] 1.7.3. Set up the program and perform qRT-PCR reaction on the machine according to the following conditions (Roche and Bio-Rad are both acceptable)

[0089]

[0090] Dissociation Stage.

[0091] 1.7.4. When comparing the expression levels of different target genes using GAPDH as the internal reference, use 2 -ΔΔCt The relative expression level was calculated.

[0092] 1.8 Statistical analysis

[0093] Data processing and analysis were completed using SPSS26.0 and Graphpad Prism9.5 software. Enumeration data are usually described statistically using the number of cases (n) and percentage (%). For measurement data that follow a normal distribution, mean ± standard deviation ( ) for statistical description; for measurement data that do not conform to normal distribution, the median and interquartile range M (P25, P75) are used to describe the data distribution. When comparing the experimental group and the control group, the t test is often used for measurement data with normal distribution, the Wilcoxon rank sum test is used for measurement data with non-normal distribution variables, and the chi-square test or Fisher's exact test is used for counting data. P < 0.05 indicates a significant difference between the groups. When analyzing the correlation between mRNA expression levels and clinical severity, Pearson correlation analysis is used to evaluate the correlation between normally distributed variables, while Spearman correlation analysis is used for non-normally distributed variables. When the P value is less than 0.05, it indicates that there is a correlation between the groups. In addition, the receiver operating characteristic curve (ROC curve) was used to evaluate the diagnostic value of candidate mRNA for SJS / TEN. All test methods were performed under the bilateral assumption.

[0094] 2. Results

[0095] 2.1 Verification of key genes using qRT-PCR

[0096] Taking the high-throughput RNA sequencing results into consideration, the key genes with large expression fold changes and high expression abundance (pValue<0.05&|log2FC|>1) among the differentially expressed mRNAs and pathways enriched by GO and KEGG analysis that may be related to the pathogenesis of SJS / TEN were selected. Finally, 8 differential mRNAs were selected (Table 4), namely: ZMAT4: related to the p53-mediated intrinsic apoptosis signaling pathway; DNASE1L3: related to neutrophil activation, neutrophil-mediated cytotoxicity, acute inflammatory response and programmed cell death; SH2D1B: related to the regulation of innate immune response, adaptive immune response and natural killer cell-mediated immune response; FCGR3B: related to natural killer cell-mediated cytotoxicity; GNLY: related to humoral response and defense response against bacteria, fungi and other microorganisms; TNFAIP6: related to various biological processes such as inflammatory response, cell adhesion, signal transduction, cell migration and neutrophil degranulation; BMX: related to NKT cell differentiation and cell apoptosis; IFI27: involved in the cell apoptosis process and related to the type I interferon signaling pathway and apoptosis signaling pathway.

[0097] Table 4

[0098]

[0099]

[0100] Finally, the qRT-PCR method was used to verify the expression levels of key genes in PBMC and skin tissues in 20 SJS / TEN patients and 20 normal controls to clarify whether the key genes screened by bioinformatics methods were consistent with the actual experimental detection results.

[0101] The results showed that in PBMC, compared with the normal control group, the SJS / TEN group had significantly downregulated ZMAT4, DNASE1L3, SH2D1B, and GNLY, and significantly upregulated TNFAIP6, BMX, and IFI27. There was no statistical difference in FCGR3B (P=0.5744), but compared with the control group, there was a downward trend. Figure 1In the skin tissue, compared with the normal control group, SH2D1B, FCGR3B, GNLY, TNFAIP6, BMX, and IFI27 in the SJS / TEN patient group were significantly upregulated, while ZMAT4 (P=0.5744) and DNASE1L3 (P=0.2543) showed no statistical difference. Figure 2 shown.

[0102] 2.2 Using ROC curve to evaluate the diagnostic value of differentially expressed genes

[0103] The diagnostic value of genes (TNFAIP6, BMX, IFI27) that have been verified to have significant differences and consistent expression in PBMC and skin tissue of SJS / TEN patients was evaluated to determine the effectiveness of differentially expressed genes in predicting the occurrence or development of SJS / TEN. The ROC curve was drawn using graphpad prism for intuitive description, and then the optimal cutoff value and the corresponding sensitivity and specificity were calculated and analyzed, which were plotted in Tables 5 and 6, respectively. In PBMC, IFI27 has the best diagnostic value; in skin tissue, IFI27 has the best diagnostic value; overall, when IFI27 is used to distinguish SJS / TEN patients from healthy people in PBMC and skin tissue, the ROC curve area is ≥0.90, and the specificity is 1, which has good diagnostic value, such as Figure 3 shown.

[0104] Table 5 ROC results of differentially expressed genes in PBMC

[0105]

[0106] Table 6 ROC results of differentially expressed genes in skin tissue

[0107]

[0108] 2.3 Correlation analysis between key gene expression levels and clinical severity

[0109] The above experiments have verified that the key gene (IFI27) is significantly overexpressed in PBMC and skin tissues of SJS / TEN patients. To determine whether it can be used as a biomarker for severity prediction, a correlation analysis of clinical severity is required. Therefore, the epidermal lysis area was selected as an indicator of clinical severity and the expression level of the IFI27 gene in PBMC and skin tissue was analyzed for correlation. Finally, it was found that the expression level of IFI27 in PBMC and skin tissue was significantly correlated with the epidermal lysis area, such as Figure 4 shown.

[0110] 2.4 Analysis of the diagnostic value of IFI27 gene in patients with SJS / TEN, EM, MPE, DRESS, AIBD, Brun and normal control group

[0111] The specific differential expression of IFI27 in SJS / TEN patients and skin diseases with similar pathogenesis or phenotype was analyzed. The qRT-PCR method was used to verify the specific expression level of IFI27 in PBMC and skin tissue in 20 SJS / TEN groups, 12 EM groups, 12 MPE groups, 8 DRESS groups, 10 AIBD groups, 5 Brun groups and 20 normal control groups. The results showed that in PBMC, compared with the normal control group, SJS / TEN was significantly upregulated (P<0.0001), EM and DRESS were significantly upregulated (P<0.05), and there was no statistical difference in MPE, AIBD, and Brun; compared with the EM and DRESS groups, the SJS / TEN group was significantly upregulated (P<0.01). Figure 5 In skin tissue, compared with the normal control group, SJS / TEN was significantly upregulated (P<0.0001), EM and DRESS were significantly upregulated (P<0.05), and MPE, AIBD, and Brun had no statistical difference; compared with the EM and DRESS groups, SJS / TEN was significantly upregulated in the SJS / TEN group (P<0.01). Figure 6 As shown. At the same time, the diagnostic value of IFI27 was evaluated between SJS / TEN patients and skin diseases with similar pathogenesis or phenotype. The ROC curve was drawn using graphpad prism to calculate and analyze the optimal cutoff value and the corresponding sensitivity and specificity, which were plotted in Table 7 and Table 8, respectively. The results showed that the ROC curve area of ​​IFI27 in PBMC and skin tissue was ≥0.78 when it was used to distinguish SJS / TEN from EM, MPE, DRESS, AIBD, and Brun patients, which had good diagnostic value.

[0112] Table 7 ROC results of IFI27 in PBMC

[0113]

[0114]

[0115] Table 8 ROC results of IFI27 in skin tissue

[0116]

[0117] In summary, the present invention proposes an mRNA marker associated with Stevens-Johnson syndrome / toxic epidermal necrolysis, which can be used to prepare a Stevens-Johnson syndrome / toxic epidermal necrolysis detection kit, and can analyze the differential mRNA expression in PBMC and skin tissues of SJS / TEN patients and healthy individuals and analyze its clinical value, so as to be used for SJS / TEN diagnosis or severity prediction.

[0118] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of mRNA markers in the preparation of a Stevens-Johnson syndrome / toxic epidermal necrolysis detection kit, characterized in that: The nucleotide sequence of the mRNA marker is shown in SEQ ID NO:1.

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