LU102676B1 - A Biomarker for Severe Asthma and Application Thereof - Google Patents

Abstract

Disclosed are a biomarker and application for severe asthma. The biomarker is ENSG00000241587 specifically. The present invention detects the expression level of lncRNA in blood samples of normal people and patients with ordinary asthma and severe asthma, and finds for the first time that ENSG00000241587 presents a significant difference in severe asthma, indicating that ENSG00000241587 can be used as a detection target for auxiliary diagnosis of severe asthma.

Description

DESCRIPTION A Biomarker for Severe Asthma and Application Thereof

TECHNICAL FIELD The invention relates to a biomarker for severe asthma and its application, belonging to the field of biomedicine and specifically, the biomarker is ENSG00000241587.

BACKGROUND Bronchial asthma (abbreviated as asthma) is a chronic airway inflammatory disease involving eosinophils, mast cells, neutrophils, airway epithelial cells, etc. The main manifestation is airway hyperresponsiveness, with reversible airflow limitation, repeated episodes of wheezing, shortness of breath, chest tightness, coughing and other symptoms. The vast majority of asthma patients can effectively alleviate the symptoms of asthma after being given anti-inflammatory drugs and bronchodilators. However, about 5% to 10% of asthma patients cannot control their symptoms even if they are given a combination of multiple drugs. This is called severe asthma, which consumes a lot of medical and health resources, resulting in increasing cost of asthma treatment. Although severe bronchial asthma accounts for a small proportion of asthma patients, its pathogenesis and cause are complicated, and its response to glucocorticoids combined with B2 adrenoceptor agonists is poor. In addition, its treatment is more difficult, prone to serious complications, even life-threatening. Correct diagnosis of severe asthma, search for risk factors that induce and aggravate asthma, appropriate treatment measures for each factor, improvement of patient treatment compliance are the key measures to improve the level of asthma control, which should arouse sufficient attention from doctors and patients in clinical practice. At present, the epidemiology, genetic research, pathological characteristics and pathogenesis of severe asthma have not yet been fully clarified, and further research is needed. With the development of human genomics and molecular biology technology, the genetics research of asthma has become an international hot spot. It is generally believed that asthma is a polygenic genetic disease caused by the joint action of immunity, heredity, environment, and other factors. It has an obvious genetic tendency, with a heritability of about 70%-80%, and its occurrence involves disorders of multiple genes. Long non-coding RNA (IncRNA) is a type of transcript of non-coding protein, which are located in the nucleus or cytoplasm and have a similar structure with a length of more than 200 nucleotides. They contain adjacent promoter-related sequences, without open reading frames (ORF). Recently, a large number of studies have confirmed that IncRNA plays an important function, and it has various functions, including chromatin modification, transcription factor regulation and post-transcriptional control. The study of IncRNAs related to severe asthma may provide a new biological indicator for predicting the risk of asthma in the future, so as to realize the early diagnosis and detection of asthma as well as the screening of susceptible individuals.

SUMMARY In order to make up for the shortcomings of the existing technology, the purpose of the present invention is to provide genetic markers related to the occurrence and development of severe asthma. The use of genetic markers to diagnose diseases has timeliness, specificity and sensitivity, so patients can know the risk in the early stage of severe asthma, and corresponding preventive and therapeutic measures can be taken to target the level of risk, thereby improving the life quality of patients.

In order to achieve the above objectives, the present invention adopts the following technical solutions.

The invention provides the application of ENSG00000241587 reagent from detection samples in preparing a product for diagnosing severe asthma.

Further, the product comprises an ENSG00000241587 reagent detected by sequencing technology, nucleic acid hybridization technology and nucleic acid amplification technology.

Furthermore, the reagent comprises a probe that can specifically recognize the ENSGO00000241587 gene; or primers that can specifically amplify the ENSGO00000241587 gene.

Further, the primer sequence that specifically amplifies ENSG00000241587 is shown in SEQ ID NO. 2-3. Further, the sample is selected from human blood.

The present invention provides a product for detecting the expression level of ENSG00000241587 in vitro, which is composed of a preparation, a nucleic acid membrane strip, a chip or a kit.

Further, the chip includes specific primers or oligonucleotide probes for ENSGO00000241587. Further, the kit is a reagent that can detect the expression level of genetic markers by RT- PCR method, qRT-PCR method, biochip detection method, Southern blotting method, and in situ hybridization method.

Furthermore, the reagent for detecting expression level of genetic markers by qRT-PCR method includes primers for specific amplification of gene ENSG00000241587, shown as SEQ ID NO.2-3. The invention provides the application of ENSG00000241587 in the construction of a calculation model for predicting severe asthma. Advantages and beneficial effects of the present invention are as follows The present invention discovered for the first time that the expression of ENSG00000241587 gene is associated with severe asthma. By detecting expression level of ENSG00000241587 in subjects, it can be determined whether subjects have severe asthma or whether subjects are at risk of having severe asthma, thereby instructing the clinician to provide subjects with a prevention plan or a treatment plan. A new biomarker for severe asthma-ENSG00000241587 is disclosed in the invention. Compared with traditional detection methods, the use of genetic diagnosis is timelier, more specific and sensitive, and can realize the early diagnosis of severe asthma, thereby reducing the death rate of severe asthma.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing the expression of ENSG00000241587 in asthma patients detected by QPCR. Wherein, *** represents P<0.001 and ns means P>0.05.

DESCRIPTION OF THE INVENTION After extensive and in-depth research, the present invention detects differentially expressed genes in severe asthma patients, ordinary asthma patients and normal people through a high-throughput sequencing method, and explores the relationship between them and the occurrence of severe asthma, thereby providing markers for the early detection of severe asthma.

Through screening, the present invention finds for the first time that ENSG00000241587 is significantly increased in patients with severe asthma, suggesting that ENSG00000241587 can be used as a detection index for clinical diagnosis of severe asthma.

Gene ENSG00000241587 The ENSG00000241587 gene is located on chromosome 3. In the context of the present invention, the “FNSG00000241587 gene” includes the human ENSG00000241587 gene and polynucleotide, functional equivalent of the human ENSG00000241587 gene.

In the specific embodiment of the present invention, the nucleotide sequence of a representative ENSG00000241587 gene is shown in SEQ ID NO 1. Technicians of this field will recognize that the utility of the present invention is not limited to quantify the gene expression of any specific variant of target genes.

If there is nucleotide sequence 1dentity in at least about 60% of nucleoside bases, usually at least about 70%, more generally at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of nucleoside bases when a nucleic acid or a fragment thereof (or complementary chains thereof) 1s optimally aligned with other nucleic acids (that is, with appropriate nucleotide insertion or deletion), the two sequences are “basically homologous” (or similar). The ENSG00000241587 gene of the present invention can be natural or artificially synthesized, or it can be obtained by transfecting cells with a vector that can express the DNA fragment of ENSG00000241587. The vectors include viral vectors and eukaryotic expression vectors.

The viral vector can be any suitable vector, including but not limited to retroviral vector, adenovirus vector, adenovirus-associated virus vector, herpes virus (such as herpes simplex virus, vaccinia virus and Epstein-Barr virus) vector, alpha virus vector.

The eukaryotic expression vector can be any suitable expression vector, including but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEFBos expression vector, pTet expression vector, pTRE expression vector, or vectors modified on the basis of well-known expression vectors, such as pBin438, PCAMBIA1301, etc.

Detection Technology The ENSG00000241587 is detected using a variety of nucleic acid technologies known to ordinary technicians in the field, including but not limited to nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification.

Non-restrictive examples of nucleic acid sequencing technologies include but are not limited to chain terminator (Sanger) sequencing and dye terminator sequencing.

The general technician in the field will recognize that RNA is often reverse transcribed into DNA prior to sequencing because RNA is less stable in cells and more susceptible to nuclease attack in experiments.

Another example of DNA sequencing technology includes next generation sequencing (deep sequencing / high-throughput sequencing). High-throughput sequencing technology is a synthesis and sequencing technology based on single molecular cluster and the proprietary principle of reversible termination of chemical reactions.

When sequencing, random fragments of genomic DNA are attached to the optically transparent glass surface.

After extension and bridge amplification, these DNA fragments form billions of clusters on the glass surface. Each cluster is a single molecule cluster with thousands of identical templates. Then, the template DNA is sequenced by reversible synthesis and sequencing technology using four kinds of special deoxyribonucleotides with fluorescent groups.

The invention can amplify nucleic acid before detection or at the same time with detection. Exemplary non-restrictive embodiments of nucleic acid amplification techniques include but are not limited to polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). The average technologist in the field will recognize that some amplification techniques (for example, PCR) require reverse transcription of RNA into DNA prior to amplification (for example, RT-PCR), while other amplification techniques can directly amplify RNA (such as, TMA and NASBA). Generally, PCR uses denaturation, annealing of primer pair and opposite strand, and multiple cycles of primer extension to increase the copy number of target nucleic acid sequence exponentially. In RT-PCR, reverse transcriptase (RT) is used to prepare complementary DNA (cDNA) from mRNA, and then the cDNA is amplified by PCR to produce multiple copies of DNA. TMA catalyzes the synthesis of multiple copies of target nucleic acid sequence under the condition of basically constant temperature, ion strength and pH. Wherein, multiple RNA copies of target sequence can generate other copies autocatalytically. TMA optionally uses blocking, termination and other modification parts to improve the sensitivity and accuracy of TMA process. LCR uses two sets of complementary DNA oligonucleotides hybridized with adjacent regions of the target nucleic acid. DNA oligonucleotides are covalently linked by DNA ligase in repeated cycles of thermal denaturation, hybridization and ligation to produce detectable double stranded oligonucleotide products. SDA uses multiple loops of the following steps. Th opposite chain of primer sequence pair and target sequence are annealed, and the primer extension is carried out in the presence of dNTPaS to produce the double stranded hemi-phosphorothioated primer extension product. Endonuclease mediated cleavage is performed at the semi-modified restriction enzyme recognition site. Polymerase mediated primer extension is performed at the 3’ end of the incision to replace the existing chain and generate the chain for the next round of primer annealing, cutting and chain replacement, which leads to the geometric amplification of the product.

The nucleic acid hybridization technology in the invention includes but is not limited to in situ hybridization (ISH), microarray and Southern or Northern blotting. In ISH, labelled complementary DNA or RNA strands are used as probes to locate specific DNA or RNA sequences in a part or section of tissue (in situ) or the whole tissue (whole tissue embedded ISH) if the tissue is small enough. DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNA and other transcripts (e.g., ncRNA) in tissue sections or whole embedded tissue. Sample cells and tissues are usually treated to immobilize target transcripts in situ and increase probe entry. The probe is hybridized with the target sequence at high temperature, and then the surplus probe is washed away. Base is labelled with radiation, fluorescence, or antigen. Then radioautography, fluorescence microscopy or immunohistochemistry are used to localize and quantify base-labelled probes. ISH can also use two or more probes labelled with radioactive or other non-radioactive markers to detect two or more transcripts simultaneously.

Southern blotting and Northern blotting are used to detect specific DNA or RNA sequences, respectively.

Break the DNA or RNA extracted from a sample, and then separate them by gel electrophoresis on the matrix gel and transfer them to the membrane filter.

The DNA or RNA that the filter binds is hybridized with labelled probe with complementary sequence.

The hybridization probe bound to the filter is detected.

A variation of this procedure is reverse Northern blotting, in which the substrate nucleic acid fixed to the membrane is a collection of separated DNA fragments, while the probe is RNA extracted from tissue and labelled.

The nucleic acids not amplified or amplified in the invention can be detected by any conventional means.

Nucleic acid membrane strip, chip and kit In the invention, the nucleic acid membrane strip comprises a substrate and an oligonucleotide probe fixed on the substrate for specifically recognizing ENSG00000241587. The substrate can be any substrate suitable for fixing the oligonucleotide probe, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica wafer, miniature magnetic bead, etc.

In the invention, the chip comprises an oligonucleotide probe orderly fixed on the solid carrier.

The oligonucleotide probe specifically corresponds to part or all of the sequences shown in ENSG00000241587. The solid phase carrier comprises an inorganic carrier and an organic carrier.

The inorganic carrier includes but is not limited to silicon carrier, glass carrier, ceramic carrier, etc.

The organic carrier comprises polypropylene membrane and nylon membrane, etc.

A “probe” is a molecule that can bind to a specific sequence or subsequence or other part of another molecule.

Unless otherwise noted, the term “probe” generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a “target polynucleotide”) by complementary base pairing.

According to the rigor of hybridization conditions, the probe can bind to the target polynucleotide which lacks complete sequence complementarity.

Probes can be used for direct or indirect labelling, including primers.

Hybridization methods include but are not limited to solution phase, solid phase, mixed phase or in situ hybridization.

The oligonucleotide probe for ENSG00000241587 gene in the invention can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.

The length of probe is not limited, as long as the specific hybridization and specific binding with the target nucleotide sequence can be completed.

The length of the probe can be as short as 25, 20, 15, 13 or 10 bases.

Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the whole gene.

Because different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally not more than 30 base pairs.

The best length complementary to the target nucleotide sequence is 15-25 base pairs.

The self-complementary sequence of the probe should preferably be less than 4 base pairs, so as not to affect the hybridization efficiency.

The ENSG00000241587 chip of the invention can be prepared by the conventional manufacturing method of biochip known in the field.

For example, if the solid carrier is a modified glass or silicon wafer, and the 5°’ end of the probe contains amino modified poly dT string, the oligonucleotide probe can be prepared into a solution, and then the oligonucleotide probe can be spotted on the modified glass or silicon wafer by a spotter to form a predetermined sequence or array, and then the oligonucleotide probe can be fixed by placing overnight to obtain the IncRNA chip of the invention.

Exemplary probes in the present invention include PCR primers and gene specific DNA oligonucleotide probes, such as microarray probes fixed on microarray substrates, probes for quantitative nuclease protection test, probes connected with molecular barcodes, and probes fixed on beads.

The invention provides a kit which can be used to detect the expression level of ENSG00000241587 gene, comprising primers and/or chips of the invention for detection and/or quantification of ENSG00000241587. Preferably, it is placed together with the instruction manual of the kit The kit includes one or more sterile containers, which may be boxes, ampoules, bottles, tube bottles, tubes, bags, small bags, blister packaging, or other suitable container forms known in the art.

Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for the drug in the container.

The kit or chip in the invention can be used to detect the expression level of multiple genes including ENSG00000241587 gene (for example, multiple genes related to severe asthma), and simultaneously detect multiple markers of severe asthma, which can greatly improve the accuracy of severe asthma diagnosis.

The present invention provides a computational model for predicting severe asthma, which can implement and realize the steps of associating marker level with certain possibility or risk in different ways. Preferably, by mathematically combining the determination levels of ENSG00000241587 and one or more other markers as well as associating the combined values with the actual diagnostic problems, the marker values can be combined with the determination levels of ENSG00000241587 by any suitable prior art bioinformatics method.

Preferably, the method applied in the combination of markers is a logarithmic function. Besides, the result of this method is a single value. Based on the actual diagnostic problems, such values can be easily associated with intended diagnostic purposes that can help assess a patient with asthma, for example, an individual’s risk of severe asthma. In a preferred way, such logarithmic functions are obtained as follows. a) Classify individuals into groups, such as normal people, individuals at risk of asthma, asthma patients, etc. b) Univariate analysis is used to identify markers that differed significantly among these groups. c) Logarithmic regression analysis is used to evaluate markers which can be used to assess the independent differences between these different groups, and d) logarithmic function is constructed to combine independent differences.

In such analysis, markers are no longer independent, but represent a combination of markers. In the present invention, when screening and analysing differentially expressed genes, the original gene expression level and alternative splicing data can be improved by applying an algorithm designed to normalize and/or improve the reliability of the data. In some embodiments of the invention, due to the large number of single data points processed, data analysis requires a computer or other device, machine or device to apply the various algorithms described herein. "Machine learning algorithm" refers to a computer-based prediction method for characterizing gene expression profile, which is also called "classifier" by ordinary technicians in the art.

Signals corresponding to a specific level of expression (which are obtained by, for example, microarray-based hybridization analysis) are usually algorithmically processed to classify expression profiles.

Supervised learning usually includes train of classifiers to recognize the differences between classes, and accuracy test of classifiers on independent test sets.

For a new unknown sample, a classifier can be used to predict the class to which the sample belongs.

In the present invention, the up-regulation or increase of expression level refers to that the gene expression level in disease sample is higher than that in reference sample.

Reference samples are biological samples provided by reference groups of self-evident healthy individuals for in vitro evaluation.

As used in the present invention, “reference level” refers to the value established in the reference group of self-evident healthy individuals.

Technicians in the field know that when comparing the measured concentration with the reference concentration, the reference concentration can be determined by using negative reference sample, positive reference sample, or mixed reference sample including one or more control types.

The expression “comparing the measured concentration with the reference concentration” is only used to further illustrate what is obvious to skilled technicians.

The reference concentration 1s established in the internal or external control sample.

In one embodiment, an internal control sample is used, that is, to assess the marker level in the test sample and one or more other samples collected from the same subject to determine whether there is any change in the level of the marker.

In another embodiment, an external control sample is used.

For external control samples, the presence or amount of markers in samples derived from individuals is compared with that in samples derived from individuals known to have a given condition or risk of a given condition or individuals known to have no given condition (i.e. normal individuals). Statistical method In the invention, the experiment is repeated for at least three times, and the result data is expressed in the form of mean + standard deviation. The statistical analysis is carried out by using SPSS18.0 statistical software. The difference between the two is analysed by t test and it is statistically significant when p < 0.05. Embodiment The invention is further described in detail below in combination with the attached drawings and embodiments. The following embodiments are used only to illustrate the invention and not to limit the scope of the invention. The test method without specific conditions in the embodiment is usually in accordance with the normal conditions or the conditions recommended by the manufacturer. Embodiment 1 Screening of genetic markers associated with asthma

1. Sample collection 3ml of peripheral venous blood was collected from 3 normal people, 3 common adult asthma patients and 3 severe adult asthma patients. All patients had informed consent. All the above samples were obtained through the consent of the ethics committee. Inclusion criteria: (1) The general asthma patients should meet the diagnostic criteria of bronchial asthma in the 2016 prevention and treatment guidelines of bronchial asthma. (2) Severe asthma should conform to the Chinese expert consensus on diagnosis and management of severe asthma. (3) No medication is taken in the short term. (4) Age > 18. Exclusion criteria: Patients with infection, pulmonary embolism, chronic bronchitis, tuberculosis and hematological diseases; patients with abnormal liver function.

2. Preparation and quality analysis of RNA samples Use the RNA extraction kit of Promega company to extract total RNA. See instructions for specific steps. The concentration and purity of RNA were detected by Nanodrop2000. The integrity of RNA was detected by agarose gel electrophoresis, and the RIN value was measured by Agilent2100. The concentration was >200ng/ul and OD260/280 is between

1.8 and 2.2.

3. Construction of cDNA library The cDNA library was constructed by using Truseq RNA sample Prep Kit of Illumina. Ribosomal rRNA was removed by the kit, and fragmentation buffer was added to the reaction system to break the RNA into short fragments. Then using mRNA as template, the first cDNA strand was synthesized by random hexamers, and then the second cDNA strand was synthesized by adding buffer, ANTPs, RNase H and DNA polymerase I. After being purified by QiaQuickPCR kit and eluted with EB buffer, terminal repair, addition of base A, joining of sequencing adapters were carried out. Then agarose gel electrophoresis was used to select fragment size, and UNG enzyme was used to digest the second cDNA strand. Finally, PCR amplification was carried out, and the target fragments were recovered by agarose gel electrophoresis. The constructed library was used for computer sequencing.

4. Computer sequencing Illumina Hiseq X-ten/Miseq sequencing platform was used for 2*100bp/300bp sequencing. The specific operation was carried out according to the instructions.

5. Analysis of high-throughput transcriptome sequencing data TopHat was used to compare high-quality sequencing sequence obtained after quality control with designated reference genome. The reference genome is from Ensembl V84. The number of RNA-seq fragments was standardized, and the relative abundance of transcripts was calculated. The differential expression of genes was analysed by R- package limma package, and the screening criteria was p < 0.01.

6. Result RNA-seq results showed that the expression level of ENSG00000241587 gene in the blood of patients with severe asthma was significantly higher than that of normal people and normal asthma patients, suggesting that FNSG00000241587 can be used as a possible personalized index in the diagnosis of severe asthma. Embodiment 2 QPCR sequencing to verify the differential expression of ENSG00000241587 gene

1. Based on the results of high-throughput sequencing, ENSG00000241587 gene was selected for large sample QPCR verification. According to the method in Embodiment 1, blood samples were collected from 45 patients with common asthma, 38 patients with severe asthma and 40 normal people.

2. RNA extraction The total RNA was extracted by the RNA extraction kit of Promega company. Please refer to the instructions for the specific steps.

3. Reverse transcription TIANGEN’s FastQuant cDNA first strand synthesis kit was used for reverse transcription. The specific operation is detailed in the manual.

4. QPCR verification

4.1 Primer design QPCR primers were designed according to the sequence of ENSG00000241587 gene and GAPDH gene, and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific primer sequence is as follows. ENSG00000241587: Forward primer: 5’-CTAAGTTCAGTATCAGTGT-3’(SEQ ID NO.2). Reverse primer: 5’-TTTGACCTACTCTGTTTC-3’(SEQ ID NO.3). GAPDH: Forward primer: 5’-CTCTGGTAAAGTGGATATTGT-3’ (SEQ ID NO.4). Reverse primer: 5”-GGTGGAATCATATTGGAACA-3’ (SEQ ID NO.5).

4.2 QPCR amplification test SuperReal PreMix Plus (SYBR Green) from TIANGEN company was used for amplification. The dissolution curve was analysed at 60-95 °C, and then the target band was determined by melting curve analysis and electrophoresis, and the relative quantification was carried out by 2-AACt method. The experimental operation was detailed in the manual.

5. Result

Results as shown in Figure 1, compared with normal people and normal asthma, the expression level of ENSG00000241587 in severe asthma patients was significantly up- regulated, and the difference was statistically significant (P < 0.001), while compared with normal people, the expression level of ENSG00000241587 in normal asthma had no significant change (P > 0.05). These results suggest that ENSG00000241587 can be used as a personalized index in the auxiliary diagnosis of severe asthma.

The above description of the embodiment is only for understanding the method and the core idea of the invention. It should be pointed out that for ordinary technicians in the art, on the premise of not departing from the principle of the invention, a number of improvements and modifications can be made to the invention, and these improvements and modifications will also fall into the protection scope of the claims of the invention.

SEQUENCE LISTING LU102676 <110> The Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University <120> A Biomarker for Severe Asthma and Application Thereof <130> PT187@LU <160> 5 <170> BiSSAP 1.3.6 <210> 1 <211> 298 <212> DNA <213> Homo sapiens <220> <223> Seql <400> 1 actgggtgtg gtggtgtgcg ccagttgtcc cagctacttg ggaggctgag acaggaggat 60 ctgttgagcc caggaggtct gggctgtaga gcagtatgcc aattgggtgt ccacactaag 120 ttcagtatca gtgtgatgac ctcctggaag caaggaccac catgttgcct aaggaggggt 180 gaactggccc aggttggaaa cagagtaggt caaaactccc atgctgatca gtagtgggat 240 agtgcctgtg aatagccgtt acactccagc ctgggcaatg aaaaaaaagt gtgtcttt 298 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Seq2 <400> 2 ctaagttcag tatcagtgt 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220>

<223> Seq3 LU102676 <400> 3 tttgacctac tctgtttc 18 <210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Seq4

<400> 4 ctctggtaaa gtggatattg t 21 <210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Seq5

<400> 5 ggtggaatca tattggaaca 20

Claims (10)

CLAIMS:

1. An application of the detection reagent for ENSG00000241587 in preparing a product for diagnosing severe asthma.

2. The application as stated in Claim 1, wherein the product comprises a reagent for detecting ENSG00000241587 level that can be used in sequencing technology, nucleic acid hybridization technology and nucleic acid amplification technology.

3. The application as stated in Claim 2, wherein the reagent comprises a probe that can specifically recognize the ENSG00000241587 gene; or primers that can specifically amplify the ENSG00000241587 gene.

4. The application as stated in Claim 3, wherein the primer sequence that specifically amplifies ENSG00000241587 1s shown in SEQ ID NO. 2-3.

5. The application as stated in Claims 1-4, wherein the sample is selected from human blood.

6. A product for detecting the expression level of ENSG00000241587 in vitro, which is composed of a preparation, a nucleic acid membrane strip, a chip or a kit.

7. The product as stated in Claim 6, wherein the chip includes specific primers or oligonucleotide probes for ENSG00000241587.

8. The product as stated in Claim 6, wherein the kit is a reagent that can detect the expression level of genetic markers by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, and in situ hybridization method.

9. The product as stated in Claim 8, wherein the reagent for detecting expression level of genetic markers by qRT-PCR method includes primers for specific amplification of gene ENSG00000241587, shown as SEQ ID NO.2-3.

10. An application of ENSG00000241 587 in the construction of a calculation model for predicting severe asthma.