Biomarkers for evaluating severe bronchiolitis due to respiratory syncytial virus infection in children and their applications

Abstract

The present invention discloses biomarkers for evaluating severe bronchiolitis caused by respiratory syncytial virus infection in children and their applications, belonging to the field of biomarker detection. Through a large number of screenings, genes PSMA6, NMI and PIM1 that are differentially expressed in patients with respiratory syncytial virus infection in children are discovered for the first time. Using one or more of PSMA6, NMI and PIM1 as biomarkers and detecting their expression levels can accurately judge the severity of severe bronchiolitis caused by respiratory syncytial virus infection in children, with high specificity and sensitivity. The present invention provides a better way and method for the early detection of severe bronchiolitis caused by respiratory syncytial virus infection in children.

Description

Technical Field

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

[0002] Bronchiolitis is a common acute lower respiratory tract infectious disease in infants and young children, and is also one of the main causes of hospitalization for infants and young children, mostly seen in infants within 6 months. Viruses are the main pathogens causing bronchiolitis, among which respiratory syncytial virus (RSV) is the most common and most likely to cause severe disease. More and more studies have shown that severe RSV infection in infancy is related to the occurrence and development of wheezing and bronchial asthma, and severe RSV bronchiolitis in early life can also lead to long-term reduction of lung function. The pathogenesis of RSV bronchiolitis has not been fully understood, especially the influencing factors for the severity of the disease are not clear.

[0003] Clinically, most studies on bronchiolitis classify the severity of bronchiolitis into three levels: mild, moderate and severe based on the Wang's score and the modified Tal score method, based on respiratory rate, pulmonary signs (wheezing and / or moist rales), degree of inspiratory chest wall retraction, pulse oximetry saturation and general condition. However, these disease assessment methods are all subjective, and the 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, especially in severe cases, but also provide important support for disease monitoring and evaluation of treatment effects, and has broad clinical application prospects. Therefore, screening biological markers can not only accurately predict the severity of the disease, but also contribute to the formulation of treatment decisions and follow-up plans.

[0004] In recent years, more and more studies have utilized transcriptome sequencing technology for research on the pathogenesis of various infectious diseases and the screening of related drugs. This technology provides a more comprehensive and rapid analysis method for studying the pathogenesis of pediatric RSV bronchiolitis, and also offers new ideas for predicting the disease progression and further treatment plans of pediatric RSV bronchiolitis. RNA sequencing is the most important high-throughput sequencing method in transcriptome sequencing. Compared with other transcriptomics technologies, RNA sequencing provides more accurate digital signals, higher detection throughput, a wider detection range, can also discover new transcripts, and does not require pre-designed probes, almost overcoming 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 applied in medical research, clinical research, drug development, etc. Therefore, using RNA sequencing to study the pathogenesis of pediatric RSV bronchiolitis is beneficial for exploring potential biomarkers of disease severity, developing drug targets, etc., and then guiding clinicians to formulate individualized precise diagnosis and treatment plans in order to maximize the therapeutic effect and minimize side effects. Summary of the Invention

[0005] The object of the present invention is to provide biomarkers for evaluating severe bronchiolitis caused by respiratory syncytial virus (RSV) infection in children and their applications, so as to solve the problems existing in the above-mentioned prior art. Biomarkers for evaluating severe bronchiolitis caused by RSV infection in children are obtained through bioinformatics screening. These biomarkers can achieve early evaluation of severe bronchiolitis caused by RSV infection in children and have high specificity, sensitivity, and accuracy.

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

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

[0008] The present invention also provides the application of reagents for detecting gene expression levels in the preparation of products for evaluating the disease severity of severe bronchiolitis caused by respiratory syncytial virus (RSV) infection in children, and the genes include at least one of PSMA6, NMI, and PIM1. In the present invention, the products include reagent kits, chips, or nucleic acid membrane strips.

[0009] The present invention also provides the application of reagents for detecting gene expression levels in the preparation of products for diagnosing severe bronchiolitis caused by respiratory syncytial virus (RSV) infection in children, and the genes include at least one of PSMA6, NMI, and PIM1. In the present invention, the products include reagent kits, chips, or nucleic acid membrane strips.

[0010] The present invention also provides a product for evaluating the severity of severe bronchiolitis caused by respiratory syncytial virus (RSV) infection in children. The product includes reagents for detecting the gene expression level, and the genes include 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.

[0011] The present invention also provides a product for diagnosing severe bronchiolitis caused by respiratory syncytial virus (RSV) infection in children. The product includes reagents for detecting the gene expression level, and the genes include 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.

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

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

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

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0016] Figure 1 It is the volcano plot of differential genes for each group; A: mild group vs control group; B: moderate group vs control group; C: severe group vs control group;

[0017] Figure 2 It is the Venn diagram of differential genes for each group;

[0018] Figure 3 It is the evaluation result of the importance of biomarkers for severe RSV bronchiolitis in children;

[0019] Figure 4 It is the real-time quantitative PCR verification result of biomarkers for severe RSV bronchiolitis in children;

[0020] Figure 5 It is the ROC curve for the diagnostic evaluation of biomarkers in severe RSV bronchiolitis in children. Detailed Embodiments

[0021] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0025] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0026] In the present invention, the reagent for detecting the 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. The specific binding agent is, for example, a receptor of a protein, a lectin that binds to a protein, an antibody against a protein, a peptide antibody (peptidebody) against a protein, a bispecific dual binding agent, or a bispecific antibody form. The specific binding agent has an affinity of at least 10 7 mol / L for its corresponding target molecule. The specific binding agent preferably has an affinity of 10 8 mol / L or more preferably 10 9An affinity of mol / L. Examples of specific binding agents are peptides, peptidomimetics, 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 the gene expression level can be detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, and these techniques include, but are not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification techniques, and protein immunological techniques.

[0028] In the present invention, a "chip" is also referred to as an "array", and refers to a solid support containing linked nucleic acid or peptide probes. An array usually contains a variety of different nucleic acid or peptide probes linked to the surface of a substrate at different known positions. These arrays, also known as "microarrays", can generally be produced using mechanical synthesis methods or light-directed synthesis methods, which combine a combination of photolithography methods and solid-phase synthesis methods. The array can contain a flat surface, or can be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array can be packaged in a certain way to allow for the diagnosis or other manipulation of a fully functional device.

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

[0030] A variety of probe arrays have been described in the literature and can be used in the context of the present invention to detect markers associated with the phenotypes described herein. For example, DNA probe array chips or larger DNA probe array wafers (otherwise, individual chips can be obtained by breaking the wafer) are used in one embodiment of the present invention. DNA probe array wafers generally comprise a glass wafer on which an array of high density DNA probes (short DNA fragments) is placed. Each of these wafers can hold, for example, approximately 60 million DNA probes for identifying longer sample DNA sequences (e.g., from an individual or population, e.g., containing the marker of interest). Identification of sample DNA with the DNA probe set on the glass wafer is performed by DNA hybridization. When a DNA sample hybridizes to the DNA probe array, the sample binds to those probes that are complementary to the sample DNA sequence. By evaluating which probes the individual sample DNA hybridizes to more strongly, it is possible to determine whether a known nucleic acid sequence is present in the sample, and thus whether the marker found in the nucleic acid is present. This means can also be used to perform ASH by controlling the hybridization conditions to allow discrimination of single nucleotides, e.g., for SNP identification and genotyping of a sample for one or more SNPs. The array provides a convenient embodiment for simultaneously (or serially) detecting multiple polymorphic markers.

[0031] In the present invention, the nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate can be any substrate suitable for immobilizing oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass slide, silica wafer, microscale magnetic beads, etc.

[0032] In the present invention, the kit can be used to detect the expression levels of the genes or proteins (PSMA6, NMI and PIM1) described in the present invention, and comprises ligands and / or chips of the present invention for gene detection and / or quantification. Optionally, together with the instructions of the kit.

[0033] The kit includes one or more sterile containers, and such containers can be boxes, ampoules, bottles, flasks, tubes, bags, sachets, blister packs, 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 drugs.

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

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

[0036] 1. Research subjects and data collection

[0037] 1) Inclusion criteria for the case group

[0038] ① Aged 1 month to 24 months; ② Clinically diagnosed with bronchiolitis for the first time (meeting the diagnostic criteria of the Expert Consensus on the Diagnosis, Treatment, and Prevention of Bronchiolitis (2014 Edition)); ③ Positive for RSV detected by immunofluorescence in nasopharyngeal secretions; ④ Determined to have no concurrent respiratory bacterial infection based on inflammatory indicators, bacterial culture, and imaging; ⑤ Obtained the informed consent and signature of the legal guardian.

[0039] 2) Exclusion criteria for the 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 Concurrent bacterial infection in other systems; ③ Concurrent viral infection in other systems.

[0041] 3) Inclusion criteria for the control group

[0042] ① Aged 1 month to 24 months; ② Healthy children undergoing physical examinations in the child health care department during the same period; ③ No history of bronchiolitis in the past and no history of respiratory tract infection within 1 month.

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

[0044] 2. Sample collection of whole blood specimens

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

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

[0047] Red blood cell lysate was used to lyse red blood cells in whole blood. An appropriate amount of TRIzol was added to lyse cells, and total RNA was extracted according to the operation instructions. 2 μL of the sample was removed from the extracted total RNA, and the integrity of the RNA was detected by agarose gel electrophoresis. Finally, the RIN value was determined using an Agilent 2100 instrument. For single library construction, the total amount of RNA was required to be ≥1 μg, the concentration was ≥35 ng / μL, OD 260 / 280 ≥1.8, OD 260 / 230 ≥1.0.

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

[0049] The qualified RNA samples were used to enrich mRNA with magnetic beads, and the mRNA was fragmented. Random primers and reverse transcriptase were added to synthesize cDNA. Finally, end repair was performed using exonuclease and DNA polymerase, PolyA was added to the ends, and PCR amplification was carried out after adding sequencing adapters to complete the preparation of the cDNA library.

[0050] After the library construction was completed, Qubit 2.0 Fluorometer was first used for preliminary quantification, and then Agilent 2100 bioanalyzer was used to detect the insert size of the library. After the insert size met the expectations, qRT-PCR was used to accurately quantify the effective concentration of the library (the effective concentration of the library was higher than 1.5 nM) to ensure the library quality. After the library quality inspection was qualified, different libraries were pooled according to the requirements of the effective concentration and the target output data volume and sequenced using Illumina NovaSeq 6000 to generate 150 bp paired-end reads.

[0051] 5. Preliminary processing of sequencing data

[0052] Through quality assessment of the original off-machine data, quality control and preprocessing processes such as removing low-quality sequences and adapter contamination, high-quality sequencing data is obtained for downstream analysis.

[0053] 6. Analysis of the expression level of transcriptome sequencing data

[0054] The quality-controlled data, i.e., clean data (reads), is aligned with the human reference genome (GRCh37 version) provided by the GENCODE database to obtain mapped data (reads) for subsequent analysis. At the same time, the alignment results of this sequencing are quality-assessed. Software is used to quantitatively analyze the overall expression level of genes / transcripts respectively, in order to analyze the differential expression of genes / transcripts between different samples subsequently.

[0055] 7. Differential expression analysis

[0056] The children are divided into mild, moderate, and severe groups according to the severity of the disease. Each group is compared with the healthy control group. Using R language and the DESeq2 software package, a significant difference analysis of the expression of all genes in the samples is carried out. Genes with an adjusted P-value less than 0.05 and a log2-fold change value of |log2FoldChange| > 1 between two samples are defined as genes with significant differential expression. Finally, the selected DEGs are used for subsequent analysis, and the final detected DEGs are plotted as a volcano plot using the R package ggplot2 for visualization.

[0057] 8. Results

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

[0059] Furthermore, the differentially expressed genes between the mild, moderate, and severe groups and the control group are further compared and analyzed. The results are as shown in the Figure 2 Venn diagram: The total number of differentially expressed genes in the mild group is 271, in the moderate group is 500, and in the severe group is 829; among them, the number of specifically differentially expressed genes in the mild group is 23, in the moderate group is 143, and in the severe group is 435.

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

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

[0062] Using the R package randomForest, the comparison group is specified as the categorical variable, and the expression values of the differentially expressed genes in this comparison group are used as the feature variables. The importance of each gene is calculated by calling the randomForest function. This importance is measured by the value of MeanDecreaseAccuracy. The larger the value, the more important the role of the gene in distinguishing the two groups. By sorting according to the size of MeanDecreaseAccuracy and selecting the top 3 most important genes as candidate markers.

[0063] 2. Results

[0064] The importance of the differentially expressed genes between children with severe RSV bronchiolitis and the control group is sorted from largest to smallest, as Figure 3 shown. The top three genes with the most important differentially expressed genes are taken as potential candidate markers, namely PSMA6, NMI, and PIM1.

[0065] Example 3: Verifying the differential expression of candidate genes by real-time fluorescence quantitative PCR

[0066] 1. Sample collection of whole blood specimens for real-time fluorescence quantitative PCR verification

[0067] Collect the blood of 30 children with severe bronchiolitis positive for RSV infection according to the sample collection method in Example 1; additionally, collect the peripheral blood of 30 healthy children.

[0068] 2. Total cellular RNA extraction

[0069] Extract total RNA and detect the concentration and purity of RNA according to the operation instructions in Example 1. Qualified specimens are those with a purity OD 260 / OD280 of 1.9 - 2.1 and a concentration greater than 200 ng / μL at the same time. Detect the integrity of RNA by agarose gel electrophoresis and measure the RIN value with an Agilent 2100.

[0070] 3. Reverse transcription to synthesize cDNA

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

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

[0073] (3)The reverse transcription step is carried out according to the instruction manual. The specific steps are as follows: Add 4 μL of oligonucleotide primer, i.e., Oligo(dT), to each system treated in the above steps. After gently mixing, incubate at 70°C for 10 min, immediately take it out and place it on ice, and incubate 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, 1 μL of reverse transcriptase (M-MLV). Gently mix the reaction system, incubate at 42°C for 60 min, 70°C for 10 min, and finally cool to 4°C.

[0075] 4. Design of primers for real-time fluorescence quantitative PCR amplification

[0076] Search for the coding sequence of the gene in the Ensembl database and design the QPCR amplification primer sequences on the Primer3 website. The sequence range is approximately around 100 - 150 bp. All primer sequences are synthesized by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The specific primer sequences are shown in Table 1.

[0077] Table 1 Primer sequences for real-time quantitative PCR

[0078]

[0079] 5. Verify the expression of candidate genes by real-time fluorescence quantitative PCR amplification

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

[0081] (2)Operate according to the instructions of the TAKARA kit to construct a 25 μL PCR reaction system. The volumes of each component are detailed as follows: 10 μL of SYBR Premix Ex Taq II (2×), 0.5 μL of forward primer and reverse primer, 0.5 μL of ROX Reference Dye II (50×), 2.5 μL of cDNA template, and finally make up to 25 μL with sterile distilled water.

[0082] (3)Perform real-time fluorescence quantitative PCR reaction in an ABI 7300 PCR instrument. The reaction conditions are as follows: pre-denature at 95 °C for 30 seconds, and then perform 40 amplification cycles (one cycle is 15 seconds of denaturation at 95 °C followed by 35 seconds of annealing and extension at 60 °C). Each sample's PCR should be repeated at least three times.

[0083] (4)After the fluorescence quantitative PCR reaction, an amplification curve is generated, and the lowest cycle number Ct (Threshold cycle) value is analyzed according to the melting curve. In addition, each cDNA is repeated three times, and the relative expression calculation method (2 -△△Ct method) is used to calculate the relative quantification value. Statistical analysis is performed using GraphPad Prism 7.0 software, and the t-test is used to compare the differences in gene expression levels among groups. When P < 0.05, the difference is statistically significant.

[0084] 6. Results

[0085] The results are as Figure 4 shown. Compared with normal children, the expressions of the three biomarker genes PSMA6, NMI, and PIM1 were significantly up-regulated in the blood of children with severe bronchiolitis caused by RSV infection, and the differences were all 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 plotting and analysis

[0088] For each group, different combinations of important differentially expressed genes are selected, and the ROC curve is plotted and analyzed by the "pROC" R software package. Calculate the area under the curve (AUC) and its 95% confidence interval to evaluate the diagnostic ability of gene markers for disease severity. According to different AUC values, it 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 is considered statistically significant.

[0089] 2. Results

[0090] The results are as Figure 5 shown. The area under the curve for the combined detection of biomarker genes in the severe disease group for evaluating severe bronchiolitis caused by RSV infection in children ranges from 0.907 to 0.996, indicating that the combined application of one or more of PSMA6, NMI, and PIM1 has high accuracy in evaluating severe bronchiolitis caused by RSV infection in children.

[0091] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a gene in the preparation of a product for diagnosing 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 level in the preparation of a product for diagnosing severe bronchiolitis caused by respiratory syncytial virus infection in children, characterized in that, The gene includes at least one of PSMA6, NMI, and PIM1.

3. The application according to claim 2, wherein The product includes a kit, a chip, or a nucleic acid membrane strip.

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