Biomarker for evaluating severe capillary bronchitis infected by respiratory syncytial virus of children and application of biomarker

Abstract

The invention discloses a biomarker for evaluating severe capillary bronchitis infected by respiratory syncytial virus of children and application of the biomarker, and belongs to the field of biomarker detection. Through a large number of screening, genes PSMA6, NMI and PIM1 showing differential expression in children respiratory syncytial virus infected patients are found for the first time, one or more of PSMA6, NMI and PIM1 are taken as biomarkers, the expression level of the biomarkers is detected, and the severity of severe capillary bronchitis infected by children respiratory syncytial virus can be accurately judged, so that the clinical application prospect is wide. The specificity and the sensitivity are relatively high. The invention provides a better approach and method for early detection of severe capillary bronchitis infected by children respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the field of biomarker detection, and in particular to biomarkers for evaluating severe bronchiolitis caused by respiratory syncytial virus infection in children and applications thereof. Background Art

[0002] Bronchiolitis is a common acute lower respiratory tract infection in infants and young children, and is also one of the main causes of hospitalization in infants and young children. It is more common in infants under 6 months old. Viruses are the main pathogens causing bronchiolitis, among which respiratory syncytial virus (RSV) is the most common pathogen and the one that is most likely to cause severe illness. An increasing number of studies have shown that severe RSV infection in infancy and childhood is associated with the development of wheezing and bronchial asthma, and severe RSV bronchiolitis in early life can also lead to long-term decreased lung function. The pathogenesis of RSV bronchiolitis is not yet fully understood, especially the factors that affect the severity of the disease.

[0003] Clinically, most studies on bronchiolitis divide the severity of bronchiolitis into three levels: mild, moderate, and severe, based on respiratory rate, lung signs (wheezing and / or moist rales), degree of chest wall inspiratory retraction, pulse oxygen saturation, and general condition according to the Wang score and modified Tal score. However, these disease assessment methods are subjective, and differences in the assessment of the severity of bronchiolitis may also lead to different research results and clinical decisions. However, the application of biomarkers provides a new perspective for the objective assessment of bronchiolitis. Specific biomarkers, such as serum interleukin-8 (IL-8), lactate dehydrogenase (LDH) levels, and viral load in nasal secretions, have been studied to be associated with the severity of bronchiolitis. The application of biomarkers can not only improve the diagnostic accuracy of bronchiolitis, but also provide important support for disease monitoring and treatment effect evaluation, especially in severe cases, and has broad clinical application prospects. Therefore, screening of biological markers can not only accurately predict the severity of the disease, but also help in the formulation of treatment decisions and follow-up plans.

[0004] In recent years, more and more studies have used transcriptome sequencing technology to study the pathogenesis of various infectious diseases and the screening of related drugs. This technology provides a more comprehensive and rapid analytical method for the study of the pathogenesis of RSV bronchiolitis in children, and also provides a new idea for the prediction of the progression of RSV bronchiolitis in children and further treatment plans. RNA sequencing is the most important high-throughput sequencing method in transcriptome sequencing methods. Compared with other transcriptomics technologies, RNA sequencing provides more accurate digital signals, higher detection throughput, and a wider detection range. It can also discover new transcripts and does not require pre-designed probes. It has almost overcome all the limitations of other transcriptomics technologies. So far, it has become the mainstream tool for quantitative gene expression and transcriptome analysis in transcriptomics research, and has been widely used in medical research, clinical research, and drug development. Therefore, using RNA sequencing to study the pathogenesis of RSV bronchiolitis in children is conducive to exploring potential biomarkers of disease severity, developing drug targets, etc., and then guiding clinicians to formulate individualized and precise diagnosis and treatment plans, in order to maximize the treatment effect and minimize side effects. Summary of the invention

[0005] The purpose of the present invention is to provide a biomarker for evaluating severe bronchiolitis caused by respiratory syncytial virus infection in children and its application, so as to solve the problems existing in the above-mentioned prior art. A biomarker for evaluating severe bronchiolitis caused by RSV infection in children is obtained through bioinformatics screening. The biomarker can realize the early evaluation of severe bronchiolitis caused by RSV infection in children and has high specificity, sensitivity and accuracy.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of genes in preparing biomarkers for evaluating severe bronchiolitis caused by respiratory syncytial virus infection in children, wherein the genes include at least one of PSMA6, NMI and PIM1.

[0008] The present invention also provides the use of a reagent for detecting gene expression levels in the preparation of a product for assessing the severity of severe bronchiolar disease caused by respiratory syncytial virus infection in children, wherein the gene includes at least one of PSMA6, NMI and PIM1. In the present invention, the product includes a kit, a chip or a nucleic acid membrane strip.

[0009] The present invention also provides the use of a reagent for detecting gene expression levels in the preparation of a product for diagnosing severe bronchiolar infection in children with respiratory syncytial virus infection, wherein the gene includes at least one of PSMA6, NMI and PIM1. In the present invention, the product includes a kit, a chip or a nucleic acid membrane strip.

[0010] The present invention also provides a product for evaluating the severity of severe bronchiolar disease caused by respiratory syncytial virus infection in children, the product comprising a reagent for detecting gene expression levels, the gene comprising at least one of PSMA6, NMI and PIM1. In the present invention, the product comprises a kit, a chip or a nucleic acid membrane strip.

[0011] The present invention also provides a product for diagnosing severe bronchiolar infection in children with respiratory syncytial virus, the product comprising a reagent for detecting gene expression levels, the gene comprising at least one of PSMA6, NMI and PIM1. In the present invention, the product comprises a kit, a chip or a nucleic acid membrane strip.

[0012] The present invention discloses the following technical effects:

[0013] The present invention discovered for the first time that PSMA6, NMI and PIM1 are associated with severe bronchiolitis caused by RSV infection in children. By detecting the expression levels of the above genes in the peripheral blood of subjects, accurate assessment of the severity of bronchiolitis caused by RSV infection in children can be achieved.

[0014] The present invention discovered for the first time that a combination of one or more of PSMA6, NMI, and PIM1 can be used to diagnose severe RSV bronchiolitis in children and determine the severity of the disease with high specificity, sensitivity, and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The volcano plots of differentially expressed genes in each group; A: mild group vs control group; B: moderate group vs control group; C: severe group vs control group;

[0017] Figure 2 The Venn diagrams of differentially expressed genes in each group;

[0018] Figure 3 To provide the results of the significance assessment of biomarkers for severe RSV bronchiolitis in children;

[0019] Figure 4 To validate the results of real-time quantitative PCR for biomarkers of severe RSV bronchiolitis in children;

[0020] Figure 5 ROC curves for the evaluation of biomarker diagnostics in children with severe RSV bronchiolitis. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] In the present invention, the reagent for detecting gene expression level can be selected from a probe that specifically recognizes the gene; or a primer that specifically amplifies the gene; or a specific binding agent that specifically binds to the protein encoded by the gene, and the specific binding agent is, for example, a protein receptor, a lectin that binds to the protein, an antibody against the protein, a peptide body against the protein, a bispecific dual binding agent or a bispecific antibody. The specific binding agent has at least 10 7 mol / L affinity. A specific binding agent preferably has a 10 8 mol / L or preferably 10 9mol / L affinity. Examples of specific binding agents are peptides, peptide mimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies, and monovalent antibody fragments. In a specific embodiment, the specific binding agent is an antibody or a monovalent antibody fragment, preferably a monovalent fragment derived from a monoclonal antibody. Monovalent antibody fragments include, but are not limited to, Fab, Fab'-SH, single domain antibodies, Fv, and scFv fragments.

[0027] In the present invention, the method for detecting gene expression levels can be detected using a variety of nucleic acid and protein technologies known to ordinary technicians in the field, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology, and protein immunoassay technology.

[0028] In the present invention, "chip" is also referred to as "array", which refers to a solid support comprising connected nucleic acid or peptide probes. The array generally comprises a plurality of different nucleic acid or peptide probes connected to the surface of a substrate according to different known positions. These arrays, also referred to as "microarrays", can generally be produced using mechanical synthesis methods or light-guided synthesis methods, which incorporate a combination of photolithography and solid phase synthesis methods. The array may comprise a flat surface, or may be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass or any other suitable substrate. The array may be packaged in a manner to allow for diagnosis of a fully functional device or manipulation of other methods.

[0029] "Microarray" is an orderly arrangement of hybridization array elements such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies) on a substrate. The substrate can be a solid substrate, for example, a glass or silica slide, a bead, a fiber optic binder, or a semi-solid substrate, such as a nitrocellulose membrane. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.

[0030] Various probe arrays have been described in the literature and can be used in the context of the present invention to detect markers associated with phenotypes described herein. For example, a DNA probe array chip or a larger DNA probe array wafer (otherwise, each individual chip can be obtained by breaking the wafer) is used for an embodiment of the present invention. The DNA probe array wafer generally comprises a glass wafer, on which a high-density DNA probe (short DNA fragment) array is placed. These wafers can each hold, for example, about 60 million DNA probes for identifying a longer sample DNA sequence (for example, from an individual or a population, for example, comprising a marker of interest). The DNA probe group on the glass wafer is used to identify the sample DNA by DNA hybridization. When the DNA sample is hybridized with the DNA probe array, the sample is bound to those probes complementary to the sample DNA sequence. By evaluating that the individual sample DNA is hybridized more firmly with those probes, it is possible to determine whether a known nucleic acid sequence is present in the sample, thereby determining whether the marker found in the nucleic acid is present. This means can also be used to control the hybridization conditions to allow the distinction of single nucleotides, for example, for SNP identification and sample genotyping of one or more SNPs to carry out ASH. The array provides a convenient embodiment for detecting multiple polymorphic markers simultaneously (or in series).

[0031] In the present invention, the nucleic acid membrane strip comprises a substrate and an oligonucleotide probe fixed on the substrate; the substrate can be any substrate suitable for fixing the oligonucleotide probe, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica gel wafer, micro-magnetic beads, etc.

[0032] In the present invention, the kit can be used to detect the expression level of the gene or protein (PSMA6, NMI and PIM1) of the present invention, comprising the ligand and / or chip of the present invention for gene detection and / or quantification, optionally together with the instructions of the kit.

[0033] Test kit includes one or more sterile containers, such containers can be box, ampoule, bottle, vial, tube, bag, pouch, blister pack or other suitable container forms known in the art. Such containers can be made of plastics, glass, laminated paper, metal foil or other materials suitable for container medicine.

[0034] After research and extensive screening, the present invention has discovered for the first time genes that are differentially expressed in children with respiratory syncytial virus infection, and has sought better ways and methods for early assessment of the severity of bronchiolitis caused by respiratory syncytial virus infection in children. The biomarkers screened by the present invention, as a non-limiting example, are nucleotide sequences of a representative human PSMA6, NMI, and PIM1 genes as shown in NC_000014.9 (located at positions 35278558 to 35317493 of the nucleotide sequence), NC_000002.12 (located at positions 151270470 to 151289668 of the nucleotide sequence, complementary chain), and NC_000006.12 (located at positions 37170152 to 37175428 of the nucleotide sequence) in the current international public nucleic acid database GeneBank. The following is an explanation with specific examples.

[0035] Example 1: Screening of biological markers for evaluating severe RSV bronchiolitis in children based on RNA sequencing

[0036] 1. Research subjects and data collection

[0037] 1) Inclusion criteria of case group

[0038] ①1 month to 24 months of age; ②First clinical diagnosis of bronchiolitis (in accordance with the diagnostic criteria of the expert consensus on diagnosis, treatment and prevention of bronchiolitis (2014 edition)); ③RSV positive in nasopharyngeal secretion immunofluorescence test; ④No concurrent respiratory bacterial infection determined based on inflammatory indicators, bacterial culture and imaging; ⑤Informed consent and signature obtained from the legal guardian.

[0039] 2) Exclusion criteria for case group

[0040] ① Those with the following underlying diseases: premature birth, chronic lung disease, congenital heart disease, immunodeficiency disease, neuromuscular disease and other chronic diseases; ② Combined with other systemic bacterial infections; ③ Combined with other viral infections.

[0041] 3) Inclusion criteria for the control group

[0042] ①1-24 months old; ②Healthy children who underwent physical examination in the pediatric health department during the same period; ③No history of bronchiolitis or respiratory tract infection within 1 month.

[0043] According to the inclusion and exclusion criteria, children with bronchiolitis caused by RSV infection were recruited as the case group, and divided into mild, moderate and severe groups according to the severity of bronchiolitis. At the same time, healthy children who met the inclusion criteria in the child health department were recruited as the control group.

[0044] 2. Sample collection of whole blood specimens

[0045] 86 children with bronchiolitis who were positive for RSV infection were recruited, including 28 mild cases (32.56%), 30 moderate cases (34.88%), and 28 severe cases (32.56%). 2 mL of peripheral venous blood was collected within 48 hours of admission. 2 mL of peripheral venous blood was collected from 30 healthy children during the visit, and placed in EDTA anticoagulation tubes. All were stored in a -80°C refrigerator for whole blood leukocyte separation and RNA extraction. The collection of the above samples was approved by the legal guardian with informed consent and signature, and approved by the hospital ethics committee (approval number: LCKY2019-148).

[0046] 3. Preparation and quality analysis of RNA samples

[0047] The red blood cells in the whole blood were lysed with red blood cell lysis buffer, and an appropriate amount of TRIzol was added to lyse the cells. Total RNA was extracted according to the operating instructions. 2 μL of sample was removed from the extracted total RNA, and the RNA integrity was tested by agarose gel electrophoresis. Finally, the RIN value was determined using an Agilent 2100 instrument. A single library construction requires a total RNA amount of ≥1 μg, a concentration of ≥35 ng / μL, and an OD 260 / 280 ≥1.8, OD 260 / 230 ≥1.0.

[0048] 4. Library construction, quality inspection, and sequencing

[0049] Qualified RNA samples were enriched with magnetic beads for mRNA, and the mRNA was fragmented. Random primers and reverse transcriptase were added to synthesize cDNA. Finally, exonucleases and nucleic acid polymerases were used for end repair, PolyA was added to the ends, and sequencing adapters were added for PCR amplification to complete the preparation of cDNA library.

[0050] After the library construction is completed, the Qubit2.0 Fluorometer is used for preliminary quantification, and then the insert size of the library is detected using the Agilent2100 bioanalyzer. After the insert size meets expectations, the effective concentration of the library is accurately quantified by qRT-PCR (the effective concentration of the library is higher than 1.5nM) to ensure the quality of the library. After the library quality inspection is qualified, different libraries are pooled according to the effective concentration and the target data volume requirements, and then sequenced using the Illumina NovaSeq 6000, and 150 bp paired-end reads are generated.

[0051] 5. Preliminary processing of sequencing data

[0052] By performing quality control and preprocessing on the original data, removing low-quality sequences and adapter contamination, etc., high-quality sequencing data is obtained for downstream analysis.

[0053] 6. Analysis of expression levels of transcriptome sequencing data

[0054] The quality-controlled data, i.e., clean data (reads), were aligned with the human reference genome (GRCh37 version) provided by the GENCODE database to obtain mapped data (reads) for subsequent analysis, and the quality of the alignment results of this sequencing was evaluated. The software was used to quantitatively analyze the overall expression levels of genes / transcripts, respectively, so as to subsequently analyze the differential expression of genes / transcripts between different samples.

[0055] 7. Differential Expression Analysis

[0056] The children were divided into mild, moderate and severe groups according to the severity of the disease. Each group was compared with the healthy control group. The expression of all genes in the samples was analyzed for significant differences using the R language and the DESeq2 software package. The genes with a corrected P value less than 0.05 and the logarithm of the difference fold value between the two samples, i.e., |log2FoldChange|>1, were defined as genes with significant expression differences. The selected DEGs were finally used for subsequent analysis, and the R package ggplot2 was used to plot the final detected DEGs into a volcano map for visualization.

[0057] 8. Results

[0058] The mild, moderate and severe groups were compared with the control group to screen for differentially expressed genes and the number of up-regulated and down-regulated genes. Figure 1 The volcano plot shown: 216 genes (79.7%) were upregulated and 55 genes (20.30%) were downregulated in the mild group; 415 genes (83.0%) were upregulated and 85 genes (17.0%) were downregulated in the moderate group; 610 genes (73.58%) were upregulated and 219 genes (26.42%) were downregulated in the severe group. As the severity of the disease increases, the number of differentially expressed genes increases, and the number of differentially expressed genes is considered to be related to the severity of RSV disease.

[0059] The differentially expressed genes between the mild, moderate, severe groups and the control group were further compared and analyzed. Figure 2 The Venn diagram shown: there are a total of 271 differentially expressed genes in the mild group, a total of 500 differentially expressed genes in the moderate group, and a total of 829 differentially expressed genes in the severe group; among them, there are 23 differentially expressed genes specific to the mild group, 143 differentially expressed genes specific to the moderate group, and 435 differentially expressed genes specific to the severe group.

[0060] Example 2: Evaluating the importance of genes using the random forest method

[0061] 1. Use random forest to screen important differentially expressed genes in children with severe RSV bronchiolitis

[0062] The R package randomForest was used to specify the comparison group as the classification variable, and the expression value of the differentially expressed gene in the comparison group was used as the characteristic variable. The importance of each gene was calculated by calling the randomForest function. The importance was measured by the value of MeanDecreaseAccuracy. The larger the value, the more important the gene was in distinguishing the two groups. The genes were sorted according to the size of MeanDecreaseAccuracy, and the top three most important genes were selected as candidate markers.

[0063] 2. Results

[0064] Rank the differentially expressed genes between children with severe RSV bronchiolitis and the control group from most to least important, such as Figure 3 As shown, the top three most important differentially expressed genes were taken as potential candidate markers, namely PSMA6, NMI, and PIM1.

[0065] Example 3: Real-time fluorescence quantitative PCR to verify differential expression of candidate genes

[0066] 1. Whole blood specimen collection for real-time fluorescence quantitative PCR verification

[0067] Blood samples were collected from 30 children with severe bronchiolitis who were positive for RSV infection according to the sample collection method in Example 1; and peripheral blood samples were collected from 30 healthy children.

[0068] 2. Extraction of total RNA from cells

[0069] According to the instructions in Example 1, total RNA was extracted and the concentration and purity of RNA were tested. The qualified samples were: purity OD 260 / OD280 The RNA integrity was tested by agarose gel electrophoresis and the RIN value was determined by Agilent2100.

[0070] 3. Reverse transcription to synthesize cDNA

[0071] (1) DNase treatment: Incubate with RNase-free DNase at 37°C for 30 min to remove DNA contamination in the sample. Add 2 μg of total RNA, 2 μL of DNase, and 2 μL of 10× DNase buffer to an RNase-free PCR tube. Finally, add DEPC-treated water to 20 μL, and then incubate the entire reaction system at 37°C for 30 min.

[0072] (2) Add 2 μL of stop solution to each reaction system after the above treatment and incubate at 65°C for 10 min to terminate the remaining DNase in the above step.

[0073] (3) The reverse transcription step was carried out according to the instructions. The specific steps are as follows: Add 4 μL of oligonucleotide primer, i.e. Oligo(dT), to each system treated in the above steps, mix gently and incubate at 70°C for 10 min. Immediately remove and place on ice for 3 min.

[0074] (4) Add 24 μL of premixed reverse transcription reaction solution to each reaction system after ice bath. The premixed reverse transcription system is as follows: 13 μL of DEPC-treated water, 8 μL of 5×RT buffer, 1.25 μL of dNTP (10 mM), 0.75 μL of RNase inhibitor, and 1 μL of reverse transcriptase (M-MLV). Gently mix the reaction system, incubate at 42°C for 60 min, 70 min for 10 min, and finally cool to 4°C.

[0075] 4. Real-time fluorescence quantitative PCR amplification primer design

[0076] The coding sequence of the gene was searched according to the Ensembl database, and the QPCR amplification primer sequence was designed in the Primer3 website. The sequence range was approximately 100-150 bp. All primer sequences were synthesized by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The specific primer sequences are shown in Table 1.

[0077] Table 1 Real-time quantitative PCR primer sequences

[0078]

[0079] 5. Real-time fluorescence quantitative PCR amplification to verify candidate gene expression

[0080] (1) Dilute the cDNA obtained by reverse transcription 4 times with sterile distilled water.

[0081] (2) According to the instructions of the TAKARA kit, a 25 μL PCR reaction system was constructed. The volumes of the components were as follows: 10 μL SYBR Premix Ex TaqⅡ (2×), 0.5 μL forward primer and reverse primer, 0.5 μL ROX Reference Dye II (50×), 2.5 μL cDNA template, and finally, sterile distilled water was added to make up to 25 μL.

[0082] (3) Real-time fluorescence quantitative PCR reaction was performed in an ABI 7300 PCR instrument. The reaction conditions were as follows: pre-denaturation at 95°C for 30 seconds, followed by 40 amplification cycles (denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 35 seconds per cycle). PCR for each sample was repeated at least three times.

[0083] (4) After the fluorescence quantitative PCR reaction is completed, an amplification curve is generated, and the minimum cycle number Ct (Threshold cycle) value is analyzed based on the melting curve. In addition, each cDNA is repeated three times and the relative expression level is calculated using the method (2 -△△Ct The relative quantitative values ​​were calculated using the GraphPad Prism 7.0 software, and the differences in gene expression among the groups were compared using the t-test. When P < 0.05, the differences were considered statistically significant.

[0084] 6. Results

[0085] The results are as follows Figure 4 As shown, compared with normal children, the expression of three biomarker genes, PSMA6, NMI, and PIM1, in the blood of children with severe bronchiolitis caused by RSV infection was significantly upregulated, and the differences were statistically significant (P<0.05), which was consistent with the RNA sequencing results.

[0086] Example 4: Receiver Operating Characteristic (ROC) Curve Analysis of Differentially Expressed Genes

[0087] 1. ROC curve drawing and analysis

[0088] Different combinations of important differentially expressed genes were screened for each group, and the ROC curve was drawn and analyzed using the "pROC" R software package. The area under the curve (AUC) and its 95% confidence interval were calculated to evaluate the diagnostic ability of gene markers for disease severity. According to the different AUC values, they can be divided into no predictive performance (AUC≤0.5), low predictive performance (0.5<AUC≤0.7), moderate predictive performance (0.7<AUC≤0.9) and high predictive performance (0.9<AUC≤1). P<0.05 was considered statistically significant.

[0089] 2. Results

[0090] The results are as follows Figure 5 As shown, the area under the curve of the combined detection of marker genes in the severe group for evaluating severe bronchiolitis caused by RSV infection in children ranged from 0.907 to 0.996, suggesting that one or several combinations of PSMA6, NMI, and PIM1 have high accuracy in evaluating severe bronchiolitis caused by RSV infection in children.

[0091] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of a gene in the preparation of a biomarker for evaluating severe bronchiolitis caused by respiratory syncytial virus infection in children, characterized in that: The gene includes at least one of PSMA6, NMI and PIM1.

2. Use of a reagent for detecting gene expression levels in the preparation of a product for assessing the severity of severe bronchiolar infection in children with respiratory syncytial virus infection, characterized in that: The gene includes at least one of PSMA6, NMI and PIM1.

3. The use according to claim 2, characterized in that The product includes a test kit, a chip or a nucleic acid membrane strip.

4. Use of a reagent for detecting gene expression levels in the preparation of a product for diagnosing severe bronchiolar infection in children with respiratory syncytial virus, characterized in that: The gene includes at least one of PSMA6, NMI and PIM1.

5. The use according to claim 4, characterized in that The product includes a test kit, a chip or a nucleic acid membrane strip.

6. A product for assessing the severity of severe bronchiolar disease caused by respiratory syncytial virus infection in children, characterized in that: The product includes a reagent for detecting gene expression levels, and the gene includes at least one of PSMA6, NMI and PIM1.

7. The product according to claim 6, characterized in that The product includes a test kit, a chip or a nucleic acid membrane strip.

8. A product for diagnosing severe bronchiolar infection of respiratory syncytial virus in children, characterized in that: The product includes a reagent for detecting gene expression levels, and the gene includes at least one of PSMA6, NMI and PIM1.

9. The product according to claim 8, characterized in that The product includes a test kit, a chip or a nucleic acid membrane strip.

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