WO2020007227A1

WO2020007227A1 - Method and kit for detecting chronic rhinosinusitis with nasal polyps subtype and use of alox15 gene as biomarker

Info

Publication number: WO2020007227A1
Application number: PCT/CN2019/093281
Authority: WO
Inventors: 张罗; 王成硕; 闫冰
Original assignee: 首都医科大学附属北京同仁医院; 北京市耳鼻咽喉科研究所
Priority date: 2018-07-03
Filing date: 2019-06-27
Publication date: 2020-01-09

Abstract

Disclosed are a kit for detecting chronic rhinosinusitis with nasal polyps subtype and the use of an ALOX15 gene as a biomarker, and the method for and the use of detecting the expression amount of the ALOX15 gene in cast-off cells in a nasal cavity. The kit comprises specific primers of the ALOX15 gene. The ALOX15 gene, as a biomarker, can be made into a product for detecting chronic rhinosinusitis with nasal polyps subtype. The detection method of the present disclosure comprises the following steps: extracting RNAs from cast-off cells in the nasal cavity, reverse transcribing the total RNA into cDNA, carrying out real-time fluorescence quantitative PCR amplification of the ALOX15 gene and a reference gene of the cDNA respectively with the specific primers of the ALOX15 gene and specific primers of the reference gene by using a quantitative polymerase chain reaction, and calculating the expression amount of the ALOX15 gene based on a detection result of an amplification product.

Description

Method and kit for detecting chronic sinusitis with nasal polyp subtype and application of ALOX15 gene as a biomarker

Cross-reference to related applications

The disclosure requires that the application number of 201810717413.X, which is submitted to the State Intellectual Property Office of the People's Republic of China on July 3, 2018, be named "Kit for detecting chronic sinusitis with nasal polyp subtypes and the application of ALOX15 gene as a biomarker ", And the priority of the Chinese patent application with the application number of 201810720285.4 and the title of" Method and Application for Detecting ALOX15 Gene Expression in Nasal Exfoliated Cells "submitted to the State Intellectual Property Office of China on July 3, 2018. , The entire contents of which are incorporated herein by reference.

Technical field

The present disclosure belongs to the technical field of biomedicine, and particularly relates to a kit for detecting chronic sinusitis with nasal polyp subtype and the application of ALOX15 gene as a biomarker, and a method for detecting the expression level of ALOX15 gene in nasal exfoliated cells. .

Background technique

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammation of the sinus mucosa. Examination revealed nasal or middle nasal polyps. Common symptoms of CRSwNP are nasal congestion, runny nose or runny nose, diminished olfactory feeling, facial bloating or pressure, which lasts more than 12 weeks. The prevalence is about 0.5-4%. CRSwNP is often accompanied by asthma and allergic rhinitis. It has been reported that 7% of asthma patients have CRSwNP and 26-48% of CRSwNP have asthma. The pathogenesis of CRSwNP is still uncertain. The destruction of mucosal epithelial cells, the imbalance of the host immune system, and the invasion of pathogenic microorganisms may be the main causes of its pathogenesis. The main treatments for CRSwNP are surgery and medication. However, research shows that even with standardized medicine or surgery, the recurrence rate of chronic sinusitis with nasal polyps is still as high as 56%, which severely affects the quality of life of patients and brings high medical expenses. However, there is a lack of radical treatment in clinical practice. It has become the focus of research in rhinology.

CRSwNP can be divided into Eosinophilic (CRSwNP, ECRSwNP) and Nonosinophilic (CRSwNP, nonECRSwNP) according to the degree of eosinophil infiltration. The clinical manifestations, medications and prognosis of the two are different. The clinical symptoms of eosinophils are severe, mainly nasal congestion and decreased olfactory symptoms. Most patients have asthma, and the recurrence rate is higher. The degree of eosinophil infiltration in nasal polyp tissue is most closely related to recurrence. When the percentage of cells in the tissue exceeds 27%, the risk of recurrence exceeds 90%. The sensitivity of eosinophilic polyps to glucocorticoids is significantly higher than that of non-eosinophilic polyps. The clinical symptoms of non-eosinophils are generally mild, and there is less chance of asthma, airway inflammation is lighter, and the postoperative recurrence rate is lower than that of eosinophils, and the response to macrolide therapy is good. The western countries are mainly eosinophils, which mainly show TH2 inflammatory response, while the proportion of eosinophils and non-eosinophils in China is about half, and the non-eosinophils mainly show TH1 / TH17 is predominantly inflammatory. In summary, the eosinophilic and non-eosinophilic types are significantly different in immunopathological types, clinical symptoms, drug response, and prognosis. Different chronic sinusitis with nasal polyps have different inflammatory / pathological typing treatment strategies. Therefore, the identification of the pathological classification of chronic sinusitis with nasal polyps is particularly important.

At present, the judgment of the two subtypes is mainly based on the staining of histopathological specimens after nasal mucosal biopsy, and the lack of non-invasive biological markers for differential diagnosis. After obtaining polyp specimens under nasal endoscope, the patients were routinely treated with pathological specimens such as paraffin fixation, and then stained with hematoxylin and eosin, and then the tissue-infiltrated inflammatory cells were observed with a high-power microscope (mainly inflammatory cells include eosinophil , Neutrophils, lymphocytes, plasma cells) infiltration number for cell typing. The disadvantages of nasal mucosal biopsy are as follows: 1. It is an invasive test: it increases the risk of infection of the patient and is not suitable for people with low immunity, such as children and the elderly; nasal bleeding when collecting materials often causes patients to fear and worry. 2. It is difficult to obtain real-time dynamic changes of the disease: pathological biopsy of the mucosa in the healing period cannot be performed after surgery. However, clinical data indicate that the pathological classification of chronic sinusitis with nasal polyps will change with drug treatment and surgical treatment, and the pathological biopsy results before treatment cannot represent the characteristics of all disease stages. 3. It takes time and increases the cost of medical treatment. It usually takes 3-4 working days from obtaining tissue samples to obtaining pathological results. Due to the inability to obtain results on the same day or the next day, additional transportation, accommodation, and registration fees were incurred for medical treatment of patients from other places, which increased the cost of medical treatment. 4. There is a certain human error. The number of inflammatory cells counted by different pathologists may be different, which affects the judgment of polyp typing. 5. Histopathological sections are more limited and can only reflect the inflammatory status of the specimen at the location of the section. They cannot reflect the overall picture of the tissue and may cause misdiagnosis. 6. Each film requires manual counting by a pathologist, which is difficult to operate in batches.

It can be seen that providing a method that does not rely on nasal mucosal pathological biopsy and can quickly and accurately detect chronic sinusitis with nasal polyp subtypes has become a technical problem to be solved urgently by those skilled in the art.

The rise of real-time PCR in recent years makes up for the shortcomings of the above techniques. In this method, a fluorescent group is added to the PCR reaction system, and the entire PCR process is monitored in real time by using the accumulation of fluorescent signals. Finally, the measured DNA sample is quantitatively analyzed based on the fluorescent signals. The method is simple, highly sensitive, repeatable, and The results are accurate. However, the prior art has not disclosed a method for effectively detecting the expression level of ALOX15 gene in nasal exfoliated cells to better obtain the ALOX15 gene content in nasal exfoliated cells.

Summary of the invention

The present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtype, wherein the kit includes a specific primer for the ALOX15 gene.

The present disclosure also provides an application of ALOX15 gene as a biomarker in the preparation of a product for detecting chronic sinusitis with nasal polyp subtypes.

The present disclosure also provides the use of the ALOX15 gene as a biomarker for detecting chronic sinusitis with nasal polyp subtypes.

The present disclosure also provides the use of the kit according to the present disclosure for detecting chronic sinusitis with nasal polyp subtypes.

The present disclosure also provides the use of a reagent for detecting ALOX15 for detecting chronic sinusitis with nasal polyp subtypes.

The present disclosure also provides a method for detecting the expression level of ALOX15 gene in nasal cavity exfoliated cells, including the following steps: extracting RNA from nasal cavity exfoliated cells, reverse transcription of total RNA into cDNA, and using quantitative polymerase chain reaction to convert ALOX15 in cDNA Genes and internal reference genes were amplified by real-time fluorescent quantitative PCR using specific primers of the ALOX15 gene and specific primers of the internal reference gene, and the expression of ALOX15 gene was calculated based on the detection results of the amplified products.

The present disclosure also provides an application of the above method for detecting the expression level of ALOX15 gene in nasal exfoliated cells in preparing a kit for detecting a chronic sinusitis subtype with nasal polyps.

The present disclosure also provides a method for detecting a chronic sinusitis subtype with nasal polyps, which comprises detecting the ALOX15 gene expression amount in nasal exfoliated cells by the method described in the present disclosure.

The present disclosure also provides a method for detecting a chronic sinusitis subtype with nasal polyps, comprising detecting and detecting the expression level of ALOX15 gene in nasal exfoliated cells, preferably using the kit of the present disclosure to detect Expression of ALOX15 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene as part of an experimental example of the present disclosure;

2 is a real-time quantitative PCR amplification curve diagram of ALOX15 gene in another part of the experimental example of the present disclosure;

3 is a real-time quantitative PCR amplification curve diagram of ALOX15 gene in the experimental example of the present disclosure;

FIG. 4 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene in another part of the experimental example of the present disclosure;

FIG. 5 is a melting curve diagram of ALOX15 gene real-time quantitative PCR;

FIG. 6 is a melting curve diagram of ALOX15 gene real-time quantitative PCR in another part of the experimental example of the present disclosure;

FIG. 7 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR in the experimental example of the present disclosure;

FIG. 8 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR, which is part of the experimental example of the present disclosure;

FIG. 9 is an optional ROC curve for detecting chronic sinusitis with nasal polyp typing in Experimental Example 1 of the present disclosure.

FIG. 10 is a real-time fluorescent quantitative PCR amplification curve of ALOX15 gene in the second part of the experimental example of the present disclosure; FIG.

FIG. 11 is a real-time quantitative PCR amplification curve diagram of ALOX15 gene in another part of Experimental Example 2 of the present disclosure; FIG.

FIG. 12 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene in a second part of Experimental Example 2 of the present disclosure; FIG.

13 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR in the second part of the experimental example of the present disclosure;

14 is a melting curve diagram of ALOX15 gene real-time quantitative PCR in another part of Experimental Example 2 of the present disclosure;

FIG. 15 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR in a second part of the experimental example of the present disclosure;

16 is a real-time fluorescent quantitative PCR amplification curve of ALOX15 gene in Experimental Example 3 of the present disclosure;

FIG. 17 is a melting curve diagram of real-time fluorescent quantitative PCR of ALOX15 gene in Experimental Example 3 of the present disclosure; FIG.

18 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene in Experimental Example 4 of the present disclosure;

FIG. 19 is a melting curve diagram of ALOX15 gene real-time quantitative PCR in Experimental Example 4 of the present disclosure.

detailed description

The technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, but not all of the embodiments. Based on the implementations in the present disclosure, all other implementations obtained by a person of ordinary skill in the art without creative labor shall fall within the protection scope of the present disclosure.

The term "primer", "amplification primer" or "oligonucleotide primer" as used herein refers to an oligonucleotide that is capable of specifically annealing to an RNA or DNA region site adjacent to a target sequence, and as appropriate under conditions Primer for DNA synthesis. It is usually single-stranded, which can be naturally occurring or synthetic. A "primer" or "oligonucleotide primer" typically comprises a sequence between about 5 to about 50 nucleotides, more preferably about 10 to about 30 nucleotides, or more preferably about 15 to 25 cores Glycylic acid.

Relative to the region of the nucleic acid to be amplified, the term "upstream primer" as used herein generally binds to a region closer to the 5 ' end of the nucleic acid molecule. On the other hand, the term "downstream primer" as used herein generally binds to a region closer to the 3 'end of the nucleic acid molecule relative to a region on the nucleic acid to be amplified. As used herein, the term "internal reference gene" generally refers to a gene that is expressed relatively constant in various tissues and cells, such as GAPDH gene in nasal polyp tissue or cells exfoliated from nasal mucosa, and is mainly used as a reference when detecting gene expression levels.

As used herein, "polymerase chain reaction" or PCR is the amplification of a nucleic acid consisting of: the initial denaturation step of separating the strands of a double-stranded nucleic acid sample, followed by repeating (i) allowing the amplification primers to be specific to the flanking position of the target sequence Annealing step of thermal annealing; (ii) an extending step of extending primers in the 5 'to 3' direction to form an amplicon polynucleotide complementary to the target sequence, and (iii) denaturation causing separation of the amplicon from the target sequence step. The above steps can be performed at different temperatures, preferably using an automatic thermal cycler.

As used herein, "Real-time quantitative PCR", also known as quantitative PCR, or QPCR for short, refers to adding a fluorescent group to a PCR reaction system, and using fluorescence signal accumulation to monitor the entire PCR process in real time. Method for quantitative analysis of unknown template by standard curve.

As used herein, the term "Ct" (cycle threshold) or "Ct value" generally refers to the number of cycles that a fluorescent signal undergoes when a fluorescent signal reaches a set threshold.

As used herein, the terms "comprising," "including," "covering," "including," "containing," "characterized by," "having," or any other variation thereof are intended to encompass non-exclusive inclusion. Meaning.

In one or more embodiments, the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3.

In one or more embodiments, the kit further includes specific primers for internal reference genes.

In one or more embodiments, the internal reference gene is GAPDH, the upstream primer of the internal reference gene is shown in SEQ ID NO: 4, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 5.

In one or more embodiments, the kit further includes: a reagent for extracting RNA from nasal polyp tissue or detached cells from nasal mucosa; a reagent for reverse transcription of total RNA into cDNA; using quantitative polymerase chain reaction Reagent for performing real-time quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA.

In one or more embodiments, the reagent for reverse transcription of total RNA into cDNA includes: a reverse transcription mixture and deRNase and deDNase water;

In one or more embodiments, the reagent for reverse transcription of the total RNA into cDNA includes: 1 μL to 40 μL of a reverse transcription mixed solution, and 0 μL to 160 μL of deRNase and deDNase water. Further preferably, the reagent for reverse transcription of total RNA into cDNA includes: 2 μL of a reverse transcription mixed solution, and 0 μL to 8 μL of deRNase and DNAse dehydrating water.

In one or more embodiments, a reagent for performing a real-time fluorescent quantitative PCR reaction of the ALOX15 gene and an internal reference gene in a cDNA using a quantitative polymerase chain reaction includes a PCR premix solution, double distilled water, machine fluorescence compensation, and a corrector. , The upstream primer of the ALOX15 gene, the downstream primer of the ALOX15 gene, the upstream primer of the internal reference gene, and the downstream primer of the internal reference gene.

In one or more embodiments, a reagent for performing a real-time fluorescent quantitative PCR reaction of the ALOX15 gene and an internal reference gene in the cDNA by quantitative polymerase chain reaction includes: 1 μL to 25 μL of a PCR master mix, and 0 μL to 50 μL of a double-distillation Water, 0 μL to 2 μL of machine fluorescence compensation and correction agent, 0.01 to 100 μM of the ALOX15 gene upstream primer, 0.01 to 100 μM of the ALOX15 gene downstream primer, 0.01 to 100 μM of the internal reference gene upstream primer, 0.01 to 100 μM of the internal reference gene Downstream primers; Further preferably, the reagent for performing real-time quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction includes: 5 μL of PCR premix, 0 μL to 10 μL of double distilled water, according to the total volume Make up to 10 μL with water, 0.2 μL machine fluorescence compensation and correction agent, 1 μM upstream primer for ALOX15 gene, 1 μM downstream primer for ALOX15 gene, 1 μM upstream reference gene for primer, and 1 μM downstream reference gene for primer.

In one or more embodiments, the reagent for extracting RNA from nasal polyp tissue can be selected from the following two reagents.

The first type includes: RNA extraction solution, chloroform, isopropanol, 65% to 90% ethanol, RNase and DNAse-free water; among these, preferably, 0.1 mL to 20 mL of RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other substances containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate or β-mercaptoethanol, the Trizol or the RNAiso Blood or the RNAiso Plus Or 0.1 to 0.5 times the volume of the other substance containing phenol, guanidine isothiocyanate, 8-quinolinol, guanidine isothiocyanate, or β-mercaptoethanol, and 0.5 to 3 times the volume of the chloroform Isopropyl alcohol, 0.5 to 5 times the volume of isopropyl alcohol, 65% to 90% ethanol, and 0.01mL to 5mL of RNase and DNase water; further preferably, for tissues from nasal polyps The reagents for RNA extraction can be selected from the following two reagents, the first includes: 1 mL of RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, isothiocyanate Guanidine acid or β-mercaptoethanol substance, 200 μL of chloroform, 200 μL of isopropanol, 200 μL volume concentration 65 to 90% ethanol, and RNA to DNA enzyme and 0.02mL of water to the enzyme;

Another type includes: the reagent for extracting RNA from nasal polyp tissue includes: a cell lysate, a first buffer solution for removing impurities from the purification column to which RNA is adsorbed, and an impurity for removing impurities from the purification column to which RNA is adsorbed And saline second buffer solution and deRNase and DNase water; the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; wherein the reagent for extracting RNA from nasal polyp tissue also includes DNase The reaction solution or the tool for extracting RNA from nasal polyp tissue also includes a genomic DNA adsorption column; the DNase reaction solution includes a DNase buffer solution, a recombinant DNase, and a double-distilled water of deRNase.

In one or more embodiments, the reagent for extracting RNA from nasal polyp tissue includes: 0.1 mL to 2 mL of a cell lysate for lysing cells and inhibiting RNA degradation, and 0.1 mL to 0.7 mL of a washing agent One buffer, 0.1 mL to 0.7 mL of second buffer for washing, 0.01 mL to 1 mL of RNase and DNase-free water, 0 to 10 μL of genomic DNA-recombinant DNase, and 0 to 10 μL of genomics DNA DNase buffer, 20 to 100 μL of RNase-free double-distilled water, the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; or includes 0.1 mL to 2 mL for lysing cells and inhibiting RNA degradation Cell lysate, 0.1 mL to 0.7 mL of first buffer for washing, 0.1 mL to 0.7 mL of second buffer for washing, 0.01 mL to 1 mL of RNase and DNAse-free water, said from the nose Tools for extracting RNA from polyp tissue include genomic DNA adsorption columns and RNA purification columns.

In one or more embodiments, the reagent for extracting RNA from nasal polyp tissue includes: 300 μL of a cell lysate for lysing cells and inhibiting RNA degradation, 500 μL of a first buffer for washing, and 600 μL of washing Use a second buffer, 0.02 mL of RNase and DNase-free water, 4 μL of genomic DNA-recombinant DNase, 5 μL of 10 × genomic DNA-removed DNase buffer, 41 μL of RNase-free double-distilled water The tool for extracting RNA from nasal polyp tissue includes an RNA purification column; or includes 300 μL of a cell lysate for lysing cells and inhibiting RNA degradation, 500 μL of a first buffer for washing, and 600 μL of a second buffer for washing Solution, 0.02 mL of RNase and DNase water, the tools for extracting RNA from nasal polyp tissue include genomic DNA adsorption column and RNA purification column.

In one or more embodiments, the nasal polyp tissue is nasal polyp tissue obtained from nasal pathological biopsy, or the nasal mucosa exfoliated cells are nasal polyp cells obtained by brushing or sticking to the surface of the nasal polyp.

In one or more embodiments, the ΔCt (Ct (ALOX15) -Ct (GAPDH)) analysis method is used to analyze the data result of the amplification product, and the cutoff value compared with the ΔCt is 1.675.

In one or more embodiments, the internal reference gene is GAPDH, and the upstream primer of the internal reference gene is shown in SEQ ID NO: 4, and the downstream primer is shown in SEQ ID NO: 5.

In one or more embodiments, the nasal cavity exfoliated cells are obtained by using a hair brush on the surface of the nasal polyp, and the hair brush after the nasal exfoliated cells are obtained is placed in a cell lysate and stored below 4 ° C.

In one or more embodiments, the method for extracting RNA from exfoliated cells in the nasal cavity includes two methods, wherein the first method includes the following steps:

Step 1: Dissolve the nasal cavity exfoliated cells in 100-2000 μL of cell lysate, add an equal volume of ethanol, mix well and add it to the RNA purification column. After centrifugation, remove the filtrate from the collection tube. The RNA purification column is placed in a collection tube;

Step 2: Add 300 μL to 700 μL of the first buffer to the RNA purification column obtained in step 1, and centrifuge to remove the first filtrate; continue to add 400 μL to 800 μL of the second buffer to the RNA purification column, After centrifugation, the second filtrate was removed, and an RNA purification column was eluted to obtain RNA.

In one or more embodiments, the method for extracting RNA from exfoliated cells in the nasal cavity further includes the following steps: adding 10 to 100 μL of a DNase reaction solution to the RNA purification column after removing the second filtrate, and After the standing treatment, 300 μL to 700 μL of the second buffer solution was added, and after centrifugation, the third filtrate was removed. After the RNA purification column was eluted, the RNA purity was measured using a spectrophotometer to obtain RNA;

The method for preparing the DNase reaction solution includes the following steps: a DNase buffer solution, a recombinant DNase, and double-distilled water from which the RNase is removed are mixed to obtain a DNase reaction solution. Preferably, the method for preparing the DNase reaction solution includes the following steps: 5 μL of 10 × DNase buffer solution, 4 μL of recombinant DNase, and 41 μL of RNase-free double-distilled water are mixed to obtain a DNase reaction solution.

In one or more embodiments, when the genomic content is low or the starting amount of the material is small when performing RNA extraction, the method for extracting RNA from nasal cavity exfoliated cells further includes the following steps: in step 1 After the nasal cavity exfoliated cells are dissolved in the cell lysate, they are first added to a genomic DNA adsorption column to take a filtrate, and then an equal volume of ethanol is added to the filtrate.

In one or more embodiments, in order to obtain a higher concentration of RNA, the method for extracting RNA from exfoliated cells in the nasal cavity further includes the following steps: adding an RNA purification column to be eluted and adding distilled water to remove RNA hydrolase Or diethyl pyrocarbonate treated water, left at room temperature, centrifuged, and eluted the RNA purification column, and measured the purity of the RNA using a spectrophotometer to obtain RNA.

Step 1 uses a cell lysate that can rapidly break down nasal cells and inhibit nucleases released by nasal cells; genomic DNA adsorption columns are used to remove genomic DNA; RNA purification columns in step 2 are used to enrich RNA; The collection tube is used for collecting the solution after removing the genomic DNA, the first buffer solution for removing impurities in the purification column to which the RNA is adsorbed, and the second buffer solution for removing impurities and salts in the RNA solution.

In one or more embodiments, the first method for extracting RNA from exfoliated nasal cells includes the following steps:

Step 1: Dissolve the nasal cavity exfoliated cells in 300 μL of cell lysate, add an equal volume of 70% ethanol, and mix the solution evenly with a pipette; immediately add the mixed solution to the RNA purification column at 12000 rpm , Centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;

Step 2: Add 500 μL of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 600 μL of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;

Step 3: Take 5 μL of 10 × DNase buffer, 4 μL of recombinant DNase, and 41 μL of RNase-free double-distilled water to obtain a DNase reaction solution. Add 50 μL of DNase to the RNA purification column after removing the second filtrate. The reaction solution was left at room temperature for 15 minutes, 350 μL of the second buffer solution was added, 12000 rpm, and centrifuged for 30 seconds to remove the third filtrate;

Step 4: Place the RNA purification column with the third filtrate removed in step 3 in a 1.5 mL RNA hydrolase-free collection tube. Add 50 μL of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. After standing at room temperature for 5 minutes, 12000 rpm, centrifugation for 2 minutes, the RNA purification column was eluted, and the OD260 / OD280 ratio of the RNA solution was measured using a spectrophotometer to obtain an RNA of 1.7 to 2.1.

In one or more embodiments, the method for extracting RNA from exfoliated cells in the nasal cavity includes the following steps:

Step 1: Take the genomic DNA adsorption column in a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 100-2000 μL of cell lysate, and add it to the genomic DNA adsorption column. Take the filtrate and add an equal volume of 70% ethanol, mixed well and added to the RNA purification column, 12000 rpm, centrifugation for 1min, the filtrate was removed, and the RNA purification column was placed in a 2mL collection tube;

Step 3: Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 μL of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. After standing at room temperature for 5 minutes, 12000 rpm, centrifugation for 2 minutes, the RNA purification column was eluted, and the OD260 / OD280 ratio of the RNA solution was measured using a spectrophotometer to obtain an RNA of 1.7 to 2.1.

In one or more embodiments, the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 mL to 20 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for lysis 1. Add chloroform 0.1-0.5 times the volume of the RNA extraction solution after shaking, mix by shaking, leave it at room temperature, centrifuge the centrifuge tube, take the supernatant, and add 0.5-3 times the volume of chloroform. Isopropyl alcohol, stand still, centrifuge after mixing, discard the supernatant, retain the first precipitate, and add 0.5 to 5 times the volume of 65% to 90% ethanol of the isopropyl alcohol to the first precipitate, After washing, mix and centrifuge, discard the supernatant, and retain the second pellet; cover the centrifuge tube, centrifuge again, remove the supernatant, and continue to add 0.01 to 5 mL of RNase and DNAase to the centrifuge tube The second precipitate was dissolved in water, and the purity of the RNA was measured by a spectrophotometer to obtain RNA.

In one or more embodiments, the RNA extraction solution is Trizol, RNAiso Blood, RNAiso Plus or other phenol, guanidinium isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, and β-mercapto group. Any one or several reagents in ethanol.

In one or more embodiments, the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 to 20 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for dissolution and shaking After that, let stand at room temperature for 3 ~ 7min; add 40μL ～ 5mL of chloroform, shake and mix well, let stand at room temperature for 3 ～ 7min, centrifuge at 3 ℃ ～ 5 ℃, 10000 ～ 14000r / min for 10 ～ 20min; take the supernatant 40μL ～ 8mL Add equal volume of isopropanol, mix and let stand for 8 ~ 12min, centrifuge at 3 ℃ ～ 5 ℃, 10000 ～ 14000r / min for 10 ～ 20min, discard the supernatant and keep the first precipitate; Add 65% to 90% ethanol with the same volume as isopropanol, and centrifuge at 7000 to 14000 r / min for 10 to 20 minutes at 3 ° C to 5 ° C, discard the supernatant and retain the second pellet; cover the centrifuge tube, Centrifuge at 3 ° C to 5 ° C for 1 to 3 minutes at 7000 to 14000 r / min, remove the supernatant, and leave to stand for 10 to 20 minutes, and then add 0.01 to 5 mL of denanuclease and deDNase to the centrifuge tube. The second precipitation is described, and the RNA purity is measured by a spectrophotometer to obtain RNA.

In one or more embodiments, the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: After adding 1 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for dissolution and shaking, Let stand at room temperature for 5min; add 200μL of chloroform, shake and mix, stand at room temperature for 5min, and centrifuge at 12,000r / min for 15min at 4 ℃; take 200μL of supernatant, add 200μL of isopropanol, and let stand for 10min after mixing, and stand at 4 ℃ The supernatant was discarded by centrifugation at 12000 r / min for 15 min, and the first pellet was retained; 75% ethanol with an equal volume of isopropyl alcohol was added to the first pellet, and centrifuged at 7500 r / min for 15 min at 4 ° C, and the supernatant was discarded. Keep the second pellet; cover the centrifuge tube, centrifuge at 7 ° C, 7500 r / min for 2 min, remove the supernatant, and leave to stand for 15 min, then continue to add 50 μL of RNase and DNase to the centrifuge tube The second precipitate was dissolved, and the OD260 / OD280 ratio of the RNA solution was measured by a spectrophotometer to be 1.7 to 2.1 to obtain RNA.

In one or more embodiments, the method for reverse transcription of total RNA into cDNA includes the following steps: taking 1 to 3 μL of the reverse transcription mixture, 0 to 10 μL of de-hydrolase distilled water and the extracted RNA at a temperature of 37 ° C. A reverse transcription reaction occurred under the conditions for 15 min, and then a reverse transcriptase inactivation reaction occurred at a temperature of 84 ° C to obtain a reverse transcription product cDNA.

In one or more embodiments, the method for reverse transcription of total RNA into cDNA includes the following steps: taking 2 μL of the reverse transcription mixture, 8 μL of the deRNA hydrolase distilled water, and a total amount not exceeding 500 ng or The volume does not exceed 8 μL of total RNA, and the de-RNA hydrolase distilled water is used to make up to 10 μL. After gentle mixing, the reverse transcription reaction is performed under the following conditions: at 37 ° C, a reverse transcription reaction is performed for 15 minutes; at 85 Under the condition of ℃, the reverse transcriptase inactivation reaction was performed for 5 seconds; the product was left at 4 ℃. The reaction system can be scaled up according to requirements. A 10 μL reaction system can use a maximum of 500 ng of total RNA. Those skilled in the art can choose according to actual needs.

In one or more embodiments, the real-time quantitative PCR amplification includes the following steps:

Step 1: Prepare a real-time quantitative PCR reaction solution: include 1 μL to 25 μL of PCR premix, 0 μL to 10 μL of double distilled water to make up the total volume of water to 10 μL, 0 μL to 2 μL of machine fluorescence compensation and correction agent, 0.01 to 100 μM Upstream primer of ALOX15 gene, downstream primer of ALOX15 gene of 0.01 to 100 μM, upstream primer of internal reference gene of 0.01 to 100 μM, downstream primer of internal reference gene of 0.01 to 100 μM, 0.01 μL to 5 μL of cDNA;

Step 2: Real-time quantitative PCR detection using standard two-step PCR amplification standard program or three-step PCR amplification standard program;

Step 3: Calculate the expression of ALOX15 gene.

In one or more embodiments, a real-time quantitative PCR reaction solution is prepared: including 5 μL of a PCR premix, 2.8 μL of double distilled water to make up a total volume of water to 10 μL, 0.2 μL of a machine fluorescence compensation and correction agent, 0.5 μL of the ALOX15 upstream primer, 0.5 μL of the ALOX15 downstream primer, 0.5 μL of the internal reference gene upstream primer, 0.5 μL of the internal reference gene downstream primer, 1 ng / μL of the cDNA or 0.01 μL to 5 μL of all primers Mentioned RNA.

In one or more embodiments, the reaction conditions of the standard two-step PCR amplification procedure include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR reaction: at 95 The reaction was carried out at a temperature of 15 ° C for 15 seconds, and at a temperature of 60 ° C for 60 seconds, followed by annealing and extension, and 40 cycles were performed.

In one or more embodiments, the reaction conditions of the standard three-step PCR amplification procedure include the following steps: Stage 1: Pre-denaturation at 95 ° C for 2 minutes; Stage 2 PCR reaction: at 95 The reaction was performed at a temperature of 1 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute, and 40 cycles were thus performed. Finally, 72 ° C, annealed and extended for 7 minutes.

In one or more embodiments, the method for calculating the expression level of the ALOX15 gene is: calculating ^ΔCT or 2- ^ΔΔCT to calculate the expression level of the target gene; wherein ΔCT = (CT (ALOX15) -CT (GAPDH)), -ΔΔCT =-(ΔCT (CT (ALOX15) -CT (GAPDH))-average ΔCT of healthy control group); average ΔCT of healthy control group = △ CT (CT (ALOX15) -CT (GAPDH)) of each control group / Control sample size.

In one or more embodiments, the reagent for detecting ALOX15 includes a specific primer for the ALOX15 gene, and preferably the upstream primer of the specific primer for the ALOX15 gene is as shown in SEQ ID NO: 2 and the ALOX15 gene The downstream primers are shown in SEQ ID NO: 3.

In one or more embodiments, the reagent for detecting ALOX15 is selected from the group consisting of a primer, an antibody, an aptamer, a probe, or a combination thereof for ALOX15.

In one or more embodiments, detection of ALOX15 gene level by fluorescent PCR method is used to detect chronic sinusitis with nasal polyp subtypes.

In one or more embodiments, the amount of ALOX15 gene expression in nasal exfoliated cells is detected by fluorescent PCR.

In one or more embodiments, chronic sinusitis with nasal polyp subtype is determined according to △ Ct (Ct (ALOX15) -Ct (GAPDH)), Ct (ALOX15) is the Ct value of ALOX15 gene, and Ct (GAPDH) is Ct value of internal reference gene GAPDH.

In one or more embodiments, the ΔCt greater than or equal to 1.675 represents non-eosinophilic chronic sinusitis with nasal polyps, and the ΔCt less than 1.675 represents eosinophilic chronic sinusitis with nasal polyps.

In one or more embodiments, the chronic sinusitis with nasal polyp subtype is non-eosinophilic chronic sinusitis with nasal polyps or eosinophilic chronic sinusitis with nasal polyps.

Advantages and positive effects of the present disclosure include, but are not limited to, at least one of the following.

1. The present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtypes. The proteomics and transcriptomics methods are used to select the ALOX15 gene as a biomarker and apply it to the kit to achieve A kit was used to detect chronic sinusitis with nasal polyp subtypes, so that the final kit obtained included specific primers for the ALOX15 gene. Based on the specific primers, the kit of the present disclosure can quickly identify nasal polyp subtypes, and is more accurate than traditional pathological detection methods. The kit can simultaneously perform large-scale, rapid Testing, saving labor costs and medical treatment costs. And the systematic kit has high identification accuracy, which can fully reflect the histopathological characteristics. The effect of human error in the prior art is solved, and the disadvantages of misdiagnosis caused by the tissue section reflecting local characteristics of the tissue are avoided. The rapid, accurate, and comprehensive identification of nasal polyp subtypes through kits is essential for clinical diagnosis and treatment. In order to provide targeted treatment based on the inflammatory subtypes of nasal polyps as early as possible, it can effectively guide the drug treatment method for patients with chronic sinusitis and nasal polyps. And the determination of surgical methods, accurately predict the response to drug treatment, and judge the prognostic effect.

2. The kit provided by the present disclosure can detect nasal polyp cells from the surface of the nasal polyp by brushing or sticking, so as to determine the chronic sinusitis of the patient with nasal polyp subtypes, avoiding causing the patient The wound surface improves the safety of patient examination, and the operation is more convenient, saving labor costs and medical treatment costs.

3. The method for detecting the expression level of ALOX15 gene in nasal exfoliated cells provided by the present disclosure, using the effectively screened ALOX15 gene as a biomarker, and providing a method for detecting its gene expression level, thereby realizing the expression of ALOX15 gene in nasal exfoliated cells. The calculation can effectively obtain the expression level of ALOX15 gene, and the provided method is simple, fast, sensitive, and reproducible, which is suitable for wide application.

4. The method for detecting the expression of ALOX15 gene in nasal exfoliated cells provided by the present disclosure, wherein the ALOX15 gene can encode a lipoxygenase protein, and the encoded enzyme acts on various polyunsaturated fatty acid substrates to produce various biologically active lipids Medium, such as eicosanoid, heparin, lipoxin, etc. The arachidonic acid was converted into 12-eicosatetraenoic acid / 12-HPETE and 15-eicosatetraenoic acid / 15-HPETE. It also converts linoleic acid into 13-hydroperoxyoctadecadienoic acid. It can also act on (12S) -hydrogen peroxytetraenoic acid / (12S) -HPETE to produce heparin A3. The encoded enzyme and its reaction products can regulate inflammation and immune response. Diseases related to ALOX15 include softening of white matter around the ventricle. It helps clear apoptotic cells during inflammation by oxidizing membrane-bound phosphatidylethanolamine in macrophages, and inhibits autoimmune responses associated with inflammatory monocytes clearing apoptotic cells. At present, the ALOX15 gene has not been disclosed in the prior art, and the use of the gene has not been found. The method for the expression of ALOX15 gene in exfoliated nasal cells provided in the disclosure can be used to detect the expression of ALOX15 in exfoliated nasal cells, and it can be used to further provide a basis for the gene screening technology for detecting chronic sinusitis subtypes with nasal polyps, It provides a reliable basis for clinical guidance and drug treatment. The feasibility of the kit for detecting chronic sinusitis subtype with nasal polyps in clinical application is guaranteed.

5, nasal shedding method of detecting expression of genes in cells of the present disclosure provides ALOX15, using ΔCt or ² -ΔΔ relative quantification method ^Ct method according to actual needs, the expression of selected relatively constant reference gene, carried out with the number of reference gene Standardization. Calculate the expression of the target gene by measuring the difference between the Ct value of the target gene and the internal reference gene. The method is simple and fast, the detection accuracy is high, the detection cost can be reduced, and the detection time can be saved. The result is easy to interpret and so on. Greatly improved the experimental efficiency. In one or more embodiments, chronic sinusitis with nasal polyp subtype is detected by detecting the level of ALOX15 protein in nasal lesion tissue of sinusitis with nasal polyps.

In one or more embodiments, a nasal cavity tissue sample of chronic sinusitis with nasal polyps is obtained by, for example, nasal endoscopy, and the protein level of ALOX15 in the sample is determined by, for example, immunohistochemistry. Preferably, the protein level of ALOX15 in the sample is determined using an antibody against ALOX15.

In one or more embodiments, the use of an antibody against ALOX15 for detecting chronic sinusitis with nasal polyp subtypes is provided.

The embodiment of the present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtype, the kit includes a specific primer of ALOX15 gene.

The present disclosure obtains a kit for detecting chronic sinusitis with nasal polyp subtypes by using the ALOX15 gene as a biomarker through proteomics and transcriptomics methods through a large number of creative experiments and screenings, which are not currently provided in the existing technology. Any reports accordingly. Among them, the ALOX15 gene is a known gene, the gene ID is 246, the DNA sequence is shown in SEQ ID NO: 1, and the gene NM number is 001140.4.

In an alternative embodiment, the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3. Aiming at the upstream primer and the downstream primer determined in the kit of the present disclosure, the kit of the present disclosure has the highest accuracy and more effective in identifying nasal polyp subtypes, which makes the kit suitable for large-scale and rapid detection.

In an alternative embodiment, the kit further includes a reference gene. More preferably, the internal reference gene is GAPDH, the upstream primer of the internal reference gene is shown in SEQ ID NO: 4, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 5. The upstream and downstream primers of the internal reference gene determined by the kit of the present disclosure can effectively obtain the appropriate ΔCT value by displaying the expression of the ALOX15 gene compared to GAPDH when identifying the nasal polyp subtypes of the kit of the present disclosure. Identification of nasal polyp subtypes. And has a higher accuracy.

In an optional embodiment, the kit further comprises: a reagent for extracting RNA from nasal polyp tissue or detached cells from nasal mucosa; a reagent for reverse transcription of total RNA into cDNA; and a quantitative polymerase chain reaction for converting cDNA Reagents for the ALOX15 gene and internal reference genes for real-time quantitative PCR reactions. More preferably, the reagent for reverse transcription of total RNA into cDNA includes: a reverse transcription mixed solution and de-RNase and de-DNase water; using quantitative polymerase chain reaction to perform real-time ALOX15 gene and internal reference gene in cDNA The reagents for the quantitative PCR reaction include: PCR premix, double-distilled water, machine fluorescence compensation and correction agent, upstream primer of ALOX15 gene, downstream primer of ALOX15 gene, upstream primer of internal reference gene, and downstream primer of internal reference gene.

In addition, for the reagents for extracting RNA from nasal polyps, the following two reagents can be selected. The first includes: RNA extraction solution, chloroform, isopropanol, 65% to 90% ethanol, RNase and DNA. Enzyme water

Another type includes: the reagent for extracting RNA from nasal polyp tissue includes: a cell lysate, a first buffer solution for removing impurities from the purification column to which RNA is adsorbed, and an impurity for removing impurities from the purification column to which RNA is adsorbed And saline second buffer solution and deRNase and DNase water; the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; wherein the reagent for extracting RNA from nasal polyp tissue also includes DNase The reaction solution or the tool for extracting RNA from nasal polyp tissue also includes a genomic DNA adsorption column; the DNase reaction solution includes a DNase buffer solution, a recombinant DNase, and a double-distilled water of deRNase. Those skilled in the art can choose according to the actual preparation requirements.

Specifically, the reagent for reverse transcription of total RNA into cDNA includes: 1 μL to 40 μL of a reverse transcription mixed solution, and 0 μL to 160 μL of deRNase and DNAse dehydrating water. Further preferably, the reagent for reverse transcription of the total RNA into cDNA includes: 2 μL of a reverse transcription mixed solution, and 0 μL to 8 μL of deRNase and DNAase water (completed to 8 μL with water according to the amount of RNA). For the above-mentioned limited numerical ranges, the reverse transcription step can be realized, and for those skilled in the art, it can be selected according to actual needs.

Specifically, the reagents for performing real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction include: 1 μL to 25 μL PCR premix, 0 μL to 50 μL double-distilled water, and 0 μL to 2 μL machine Fluorescence compensation and correction agent, 0.01 to 100 μM upstream primer of ALOX15 gene, 0.01 to 100 μM downstream primer of ALOX15 gene, 0.01 to 100 μM internal reference gene upstream primer, 0.01 to 100 μM internal reference gene downstream primer; more preferably, The reagents for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction include: 5 μL of PCR premix, 0 μL to 10 μL of double distilled water (make up to 10 μL with water based on the total volume), 0.2 μL of machine fluorescence compensation and correction agent, 1 μM of the ALOX15 gene upstream primer, 1 μM of the ALOX15 gene downstream primer, 1 μM of the internal reference gene upstream primer, and 1 μM of the internal reference gene downstream primer. For the above-mentioned limited numerical ranges, the steps of real-time quantitative PCR amplification of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction can be realized, and those skilled in the art can choose according to actual needs.

Specifically, for the reagents for extracting RNA from nasal polyp tissue, the following two reagents can be selected. The first includes: 0.1 mL to 20 mL of RNA extraction solution Trizol or RNAiso, Blood or RNAiso Plus or other phenol, isothiocyanate Guanidine acid, 8-hydroxyquinoline, guanidine isothiocyanate, or β-mercaptoethanol, the Trizol or the RNAisoBlood or the RNAisoPlus or the other substances containing phenol, guanidine isothiocyanate, 8- Hydroxyquinoline, guanidine isothiocyanate, or β-mercaptoethanol are 0.1 to 0.5 times the volume of chloroform, 0.5 to 3 times the volume of isopropyl alcohol, and 0.5 to 5 times the volume of isopropyl alcohol. 65% to 90% ethanol, and 0.01mL to 5mL of RNase and DNase water;

Another type includes: 0.1 mL to 2 mL of a cell lysate for lysing cells and inhibiting RNA degradation, 0.1 mL to 0.7 mL of a first buffer for washing, 0.1 mL to 0.7 mL of a second buffer for washing, 0.01mL to 1mL of RNase and DNase-free water, 0 to 10 μL of genomic DNA-recombinant DNase, 0 to 10 μL of genomic DNA-removed DNase buffer, 20 to 100 μL of RNase-free double distillation Water, the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; or includes 0.1 mL to 2 mL of a cell lysate for lysing cells and inhibiting RNA degradation, and 0.1 mL to 0.7 mL of a first buffer for washing 0.1 to 0.7 mL of a second buffer solution for washing, 0.01 to 1 mL of RNase and DNase water, and the tools for extracting RNA from nasal polyp tissue include a genomic DNA adsorption column and an RNA purification column.

Further preferably, for the reagents for extracting RNA from nasal polyp tissue, the following two reagents can be selected, the first includes: 1 mL of RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other phenol, guanidine isothiocyanate , 8-hydroxyquinoline, guanidine isothiocyanate or β-mercaptoethanol, 200 μL of chloroform, 200 μL of isopropanol, 200 μL of 65% to 90% ethanol by volume, and 0.02 mL of deRNase and DNAse-free water; the names of the RNA extraction solutions are Trizol, RNAiso Blood, and RNAiso Plus are all trade names.

Another type includes: 300 μL of cell lysate for lysing cells and inhibiting RNA degradation, 500 μL of first buffer for washing, 600 μL of second buffer for washing, 0.02 mL of RNase and DNase Water, 4 μL of genomic DNA-removed recombinant DNase, 5 μL of 10 × genomic DNA-removed DNase buffer, 41 μL of de-RNase double-distilled water, the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; Alternatively, it includes 300 μL of a cell lysate for lysing cells and inhibits RNA degradation, 500 μL of a first buffer for washing, 600 μL of a second buffer for washing, 0.02 mL of RNase and DNase water, Tools for extracting RNA from nasal polyp tissue include genomic DNA adsorption columns and RNA purification columns. For the above-mentioned limited numerical ranges, the steps of extracting RNA from nasal polyp tissue can be realized, and those skilled in the art can choose according to actual needs.

The cell lysate described above is used to rapidly disrupt cells and inhibit the release of nucleases from cells. The first buffer is used to remove impurities from the purification column to which RNA is adsorbed, and the second buffer is used to remove the RNA that is adsorbed. The impurities and salts of the purification column, de-RNase and DNase-free water are used to dissolve the RNA.

In addition, the above-mentioned reagents for extracting RNA from nasal polyp tissue; reagents for reverse transcription of total RNA into cDNA; reagents for quantitative real-time PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction .

In an alternative embodiment, the nasal polyp tissue is nasal polyp tissue obtained from nasal pathological biopsy, or the nasal mucosa exfoliated cells are nasal polyp cells obtained by brushing or sticking to the surface of the nasal polyp. The brushing or sticking method is used to avoid wounds to the patient, improve the safety of patient examination, and make the operation more convenient, saving labor costs and medical treatment costs.

In an alternative embodiment, the ΔCt (Ct (ALOX15) -Ct (GAPDH)) analysis method is used to analyze the data result of the amplification product, and the limit value for comparison with the ΔCt is 1.675. The defined value can enable the kit provided by the present disclosure to achieve an accuracy rate of more than 75% when detecting chronic sinusitis with nasal polyp subtypes.

An embodiment of the present disclosure also provides an application of the ALOX15 gene as a biomarker in preparing a product for detecting chronic sinusitis with nasal polyp subtypes. The product may be a detection reagent, a chip or a kit. Although the above embodiments only describe the specific technical content of the kit, for those skilled in the art, based on the disclosure of the technical solution of the present application, combined with common general knowledge, the specific technical content of the detection reagents and chip products can be directly obtained. .

An embodiment of the present disclosure provides a method for detecting the expression level of ALOX15 gene in nasal exfoliated cells, including the following steps: extracting RNA from nasal exfoliated cells, reverse-transcribe total RNA into cDNA, and use quantitative polymerase chain reaction to convert the The ALOX15 gene and internal reference gene were amplified by real-time fluorescent quantitative PCR using specific primers of the ALOX15 gene and specific primers of the internal reference gene, respectively. The expression of ALOX15 gene was calculated based on the detection results of the amplified products.

The present disclosure uses proteomics and transcriptomics methods to screen a large number of creative experiments to obtain a subtype of chronic sinusitis with nasal polyps by calculating the expression of ALOX15 gene, and provides a simple and reliable method for calculating the expression of ALOX15 gene. , High accuracy. No corresponding reports have been provided in the existing technology. Among them, the ALOX15 gene is a known gene, the gene ID is 246, the DNA sequence is shown in SEQ ID NO: 1, and the gene NM number is 001140.4.

In an alternative embodiment, the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3. The design of the upstream and downstream primers of the ALOX15 gene has higher sensitivity. When detecting the expression of the ALOX15 gene, the results are more accurate and repeatable.

In an alternative embodiment, the internal reference gene is GAPDH, the upstream primer of the internal reference gene is shown in SEQ ID NO: 4, and the downstream primer is shown in SEQ ID NO: 5. According to the design of this feature, combining the upstream primer of the ALOX15 gene and the downstream primer of the ALOX15 gene to obtain a suitable ΔCT value for calculating the expression of the ALOX15 gene, and has a high accuracy rate.

In an alternative embodiment, the nasal cavity exfoliated cells are obtained by using a hair brush on the surface of the nasal polyps, and the hair brush after obtaining the nasal cavity exfoliated cells is placed in a cell lysate and stored below 4 ° C. Based on this method, the method of the present disclosure avoids wounds to patients, improves the safety of patient examination, and is more convenient to operate, saving labor costs and medical treatment costs.

In an alternative embodiment, the method for extracting RNA from exfoliated nasal cells includes two methods, wherein the first method includes the following steps:

Preferably, the method for extracting RNA from exfoliated cells in the nasal cavity further comprises the following steps: adding 10 to 100 μL of a DNase reaction solution to the RNA purification column after removing the second filtrate, and after standing treatment, adding 300 μL ~ 700 μL of the second buffer solution, after centrifugation, remove the third filtrate, take the RNA purification column and elute the RNA purity using a spectrophotometer to obtain RNA;

Preferably, the method for extracting RNA from nasal cavity exfoliated cells when the genome content is low or the starting amount of material is low when performing RNA extraction, further includes the following steps: in step 1, the nasal exfoliated cells are lysed After the cell lysate is added to a genomic DNA adsorption column to take a filtrate, an equal volume of ethanol is added to the filtrate.

Preferably, in order to obtain a higher concentration of RNA, the method for extracting RNA from nasal cavity exfoliated cells further includes the following steps: adding an RNA purification column to be eluted and adding distilled water to remove RNA hydrolase or diethyl pyrocarbonate treatment After the water was left at room temperature, the RNA purification column was eluted by centrifugation, and the RNA purity was measured by a spectrophotometer to obtain RNA.

Specifically, the first method for extracting RNA from exfoliated nasal cells includes the following steps:

Alternatively, the method for extracting RNA from exfoliated nasal cells includes the following steps:

Among them, step 1 uses a cell lysate to rapidly break down nasal cells and inhibit nuclease released by nasal cells; step 1 uses a genomic DNA adsorption column to remove genomic DNA; and an RNA purification column in step 2 is used for enrichment RNA; the collection tube is used to collect the solution after removing the genomic DNA, the first buffer is used to remove impurities from the purification column to which the RNA is adsorbed, and the second buffer is used to remove impurities and salts from the RNA solution.

In an optional embodiment, the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 mL to 20 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells, lysing, shaking and adding 0.1 to 0.5 times the volume of the chloroform of the RNA extraction solution, shake and mix, and stand at room temperature, centrifuge the centrifuge tube, take the supernatant, and add 0.5 to 3 times the volume of chloroform in isopropyl alcohol. After mixing, let stand, centrifuge, discard the supernatant, retain the first pellet, add 0.5 to 5 times the volume of 65% to 90% ethanol of the isopropanol volume to the first pellet, wash and mix Centrifuge, discard the supernatant, and retain the second pellet; cover the centrifuge tube, centrifuge again, remove the supernatant, and continue to add 0.01 to 5 mL of RNase and DNAase dehydration solution to the centrifuge tube. The second precipitation is described, and the RNA purity is measured by a spectrophotometer to obtain RNA.

Preferably, the RNA extraction solution is any one or more of Trizol, RNAisoBlood, RNAisoPlus or other phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate and β-mercaptoethanol. Of reagents.

In a preferred embodiment, the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 to 20 mL of RNA extraction solution to a centrifuge tube containing nasal exfoliated cells for dissolution, shaking, and room temperature Let stand for 3 ~ 7min; add 40μL ~ 5mL of chloroform, shake and mix well, let stand at room temperature for 3 ~ 7min, centrifuge at 3 ℃ ～ 5 ℃, 10000 ～ 14000r / min for 10 ～ 20min; take the supernatant 40μL ～ 8mL, add etc. The volume of isopropanol was mixed and left to stand for 8-12 minutes, centrifuged at 3 ° C to 5 ° C, 10,000 to 14000 r / min for 10 to 20 minutes, the supernatant was discarded, and the first precipitate was retained; Propanol equal volume of 65% to 90% ethanol was centrifuged at 3 ° C to 5 ° C, 7000 to 14000r / min for 10 to 20min, the supernatant was discarded, and the second pellet was retained; the centrifuge tube was tightly closed at 3 ° C ~ 5 ° C, centrifuge at 7000 ~ 14000r / min for 1 ~ 3min, remove the supernatant, and let stand for 10 ~ 20min, then continue to add 0.01 ~ 5mL of deRNase and deDNase water to the centrifuge tube to dissolve the second Precipitate and measure RNA purity using a spectrophotometer to obtain RNA.

Specifically, the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 1 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for dissolution, shaking, and standing at room temperature for 5 min; adding 200 μL of chloroform, Mix by shaking, let stand at room temperature for 5 minutes, and centrifuge at 12,000 r / min for 15 min at 4 ° C. Take the supernatant 200 μL, add 200 μL isopropanol, mix and let stand for 10 min, and centrifuge at 4 ° C for 15 min at 12000 r / min to discard the supernatant. , Keep the first pellet; add 75% ethanol with equal volume to isopropyl alcohol to the first pellet, centrifuge at 7500 r / min for 15min at 4 ° C, discard the supernatant, and keep the second pellet; Centrifuge the tube at 4 ° C, 7500 r / min for 2 minutes, remove the supernatant, and leave it to stand for 15 minutes, and then add 50 μL of RNase and DNAse-free water to the centrifuge tube to dissolve the second precipitate and use spectrophotometry. The ratio of the OD260 / OD280 of the RNA solution was 1.7 to 2.1 to obtain RNA.

Preferably, the method for reverse transcription of total RNA into cDNA includes the following steps: 1 to 3 μL of the reverse transcription mixed solution, 0 to 10 μL of de-hydrolase-distilled water and the extracted RNA undergo a reverse transcription reaction at a temperature of 37 ° C. After 15 min, a reverse transcriptase inactivation reaction occurred at a temperature of 84 ° C to obtain a reverse transcription product cDNA.

In an alternative embodiment, the method for reverse transcription of total RNA into cDNA includes the following steps: taking 2 μL of the reverse transcription mixture, 8 μL of the deRNA hydrolase distilled water, and a total amount not exceeding 500 ng or a volume not exceeding When the total RNA exceeds 8 μL, the de-hydrolase enzyme distilled water is used to make up to 10 μL; the reverse transcription reaction is performed after gentle mixing, the conditions are as follows: at 37 ° C, the reverse transcription reaction is performed for 15 minutes; at 85 ° C, Under the conditions, an inactivation reaction of reverse transcriptase was performed for 5 seconds; the product was left at 4 ° C.

In an optional embodiment, the real-time quantitative PCR amplification includes the following steps:

Step 3: Calculate the expression of ALOX15 gene.

Specifically, a real-time quantitative PCR reaction solution was prepared: including 5 μL of a PCR premix, 2.8 μL of double distilled water to make up a total volume of water to 10 μL, 0.2 μL of a machine fluorescence compensation and correction agent, and 0.5 μL of the upstream ALOX15 gene. Primer, 0.5 μL of the downstream primer of the ALOX15 gene, 0.5 μL of the upstream primer of the internal reference gene, 0.5 μL of the downstream primer of the internal reference gene, 1 ng / μL of the cDNA.

In an optional embodiment, the reaction conditions of the standard two-step PCR amplification procedure include the following steps: first stage: pre-denaturing at 95 ° C for 30 seconds; second stage PCR reaction: at 95 ° C Under the conditions, the reaction is performed for 15 seconds, and the reaction is performed at 60 ° C for 60 seconds, and the annealing is extended, so that 40 cycles are performed;

The reaction conditions of the standard three-step PCR amplification procedure include the following steps: first stage: pre-denaturation at 95 ° C for 2 minutes; second stage PCR reaction: at 95 ° C, reaction for 1 minute, at The reaction is performed at 55 ° C for 1 minute, and at 72 ° C for 1 minute, so that 40 cycles are performed; finally, 72 ° C, 7 minutes of annealing and extension;

The method for calculating the expression level of ALOX15 gene is: Calculate ^ΔCT or 2 ^-ΔΔCT to calculate the expression level of the target gene; where ΔCT = (CT (ALOX15) -CT (GAPDH)), -ΔΔCT =-(ΔCT (treated specimen CT (ALOX15) -CT (GAPDH))-average ΔCT of healthy control group; average ΔCT of healthy control group = ΣCT (CT (ALOX15) -CT (GAPDH)) / control sample number per control group. Relative quantification method using ΔCT method or 2- ^ΔΔCt method, selecting internal reference genes with relatively constant expression, normalizing with the number of internal reference genes, and calculating the target gene expression by measuring the difference between the Ct value of the target gene and the internal reference gene. It is simple and fast, and the detection accuracy is high, which can reduce the detection cost and save the detection time. The result is easy to interpret and so on. Greatly improved the experimental efficiency.

Specifically for the same subject, the expression of ALOX15 and the reference gene are different. The reference gene is a gene that is more stable in vivo and usually does not change with diseases. Therefore, the comparison with the reference gene can reflect the relative abundance of the target gene and the reference gene. , You can use ΔCT method. For different subjects, the expression of ALOX15 and the reference gene can be different using the 2- ^ΔΔCt method.

Application of the above method for detecting the expression level of ALOX15 gene in nasal exfoliated cells in the preparation of a kit for detecting a chronic sinusitis subtype with nasal polyps.

In order to more clearly introduce the kit for detecting chronic sinusitis with nasal polyp subtypes provided by the embodiments of the present disclosure and the application of ALOX15 gene as a biomarker, and a method for detecting nasal cavity shedding provided by the embodiments of the present invention The method and application of ALOX15 gene expression in cells will be described below with reference to specific examples.

Examples

Example 1:

A kit for detecting chronic sinusitis with nasal polyp subtypes, comprising the following reagents:

Reagents for RNA extraction from nasal polyp tissue: 20mL RNA extraction solution Trizol or RNAiso Blood or RNAisoPlus or other products containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, β-mercaptoethanol, etc. Substances that can rapidly disrupt cells and inhibit nucleases released by the cells; 2mL of chloroform; 20mL of isopropanol; 40mL of 65-90% ethanol; 5mL of RNase and DNAse-free water;

Reagent for reverse transcription of extracted RNA into cDNA: 40 μL of reverse transcription mixture (containing reverse transcription enzymes, RNase inhibitors, random 6-nucleotide primers, polythymine, T repeat oligonucleotides , Deoxyribonucleotide triphosphate mixture, buffer solution, etc.), 160 μL of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;

Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction: 25 μL of premixed solution (containing enzymes and buffers required for PCR), 0 to 50 μL of double distilled water (based on total Make up volume to 50 μL with water), 0 to 2 μL of dye (for fluorescence compensation and correction of the machine), 100 μM upstream primer of ALOX15 gene, 100 μM downstream primer of ALOX15 gene, 100 μM upstream primer of internal reference gene, 100 μM The downstream primer of the internal reference gene, 10 μg positive control, 10 μg negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).

Example 2:

Reagents for RNA extraction from nasal polyps: 1 mL of RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other substances containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, β-mercaptoethanol, etc. Substances that can rapidly disrupt cells and inhibit the release of nucleases from cells; 0.2mL of chloroform; 0.2mL of isopropanol; 0.2mL of 65-90% ethanol; 0.05mL of RNase and DNAse-free water;

Reagent for reverse transcription of extracted RNA into cDNA: 2 μL of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitor, random 6 nucleotide primers, polythymine, T repeat oligonucleotide , Deoxyribonucleotide triphosphate mixture, buffer solution, etc.), 7 μL of RNase and DNAse-free water; of which RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;

Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction: 5 μL of premix (containing enzymes and buffers required for PCR), 0-10 μL of double-distilled water (based on total Make up volume to 10 μL with water), 0 to 2 μL of dye (for fluorescence compensation and correction of the machine), 50 μM upstream primer of ALOX15 gene, 50 μM downstream primer of ALOX15 gene, 50 μM upstream primer of internal reference gene, 50 μM The downstream primer of the internal reference gene, 5 μg positive control, 5 μg negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).

Example 3:

Reagent for RNA extraction from nasal polyp tissue: 0.1mL RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, β-mercaptoethanol And other substances that can rapidly break down cells and inhibit the nuclease released by the cells; 0.05mL of chloroform; 0.015mL of isopropanol; 0.0075mL of 65-90% ethanol; 0.01mL of RNase and DNase-free water ;

Reagent for reverse transcription of total RNA into cDNA: 1 μL of reverse transcription mixture (containing enzymes required for reverse transcription, RNase inhibitors, random 6 nucleotide primers, polythymine, T repeat oligonucleotides, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0-10 μL of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;

Quantitative polymerase chain reaction reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA: 1 μL of premixed solution (containing enzymes and buffers required for PCR), 0 to 10 μL of double-distilled water (based on total Make up volume to 10 μL with water), 0 to 2 μL of dye (for fluorescence compensation and correction of the machine), 1 μM upstream primer of ALOX15 gene, 1 μM downstream primer of ALOX15 gene, 1 μM upstream primer of internal reference gene, 1 μM The downstream primer of the internal reference gene, 1 μg positive control, 1 μg negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).

Example 4:

A kit for detecting chronic sinusitis with nasal polyp subtypes, comprising the following reagents and tools:

Reagents for RNA extraction from nasal polyp tissue: 100 μL of cell lysate (RL buffer with 50 x dithiothreitol (DTT)), genomic DNA adsorption column for removing genomic DNA, and for collecting and removing genomic DNA A collection tube for the solution, an RNA purification column for enriching RNA, 0.1 mL of a first buffer for removing impurities from the purification column to which the RNA is adsorbed, and 0.1 mL of a second buffer for removing impurities and salts from the RNA solution Solution, centrifuge tube for collecting RNA, 0.01 mL of RNase and DNase water for dissolving RNA;

Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction: 25 μL premix (containing enzymes and buffers required for PCR), 0-10 μL double-distilled water (based on total Make up volume to 10 μL with water), 0 to 2 μL of dye (for fluorescence compensation and correction of the machine), 0.01 μM upstream primer of ALOX15 gene, 0.01 μM downstream primer of ALOX15 gene, 0.01 μM upstream of reference gene Primer, 0.01 μM of the internal reference gene downstream primer, 1 μg positive control, 1 μg negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).

Example 5:

Reagent for RNA extraction from nasal polyp tissue: 0.3 mL of cell lysate (RL buffer with 50 x dithiothreitol (DTT)), RNA purification column for enriching RNA, 0.5 mL for removing adsorbed The first buffer of impurities in the RNA purification column, 0.6 mL of the second buffer for removing impurities and salts from the RNA solution, 4 μL of genomic DNA-removing recombinant DNase, 5 μL of genomic DNA-removing DNase buffer , 41 μL double-distilled water for RNase, centrifuge tube for collecting RNA, 0.05 mL of RNase and DNase water for dissolving RNA;

Reagent for reverse transcription of total RNA into cDNA: 2 μL of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitor, random 6 nucleotide primers, polythymine, T repeat oligonucleotide, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0-10 μL of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;

Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction: 5 μL of premix (containing enzymes and buffers required for PCR), 0-10 μL of double-distilled water (based on total Make up volume to 10 μL with water), 0 to 2 μL of dye (for fluorescence compensation and correction of the machine), 10 μmol / L upstream primer of ALOX15 gene, 10 μmol / L downstream primer of ALOX15 gene, 10 μmol / L internal reference The upstream primer of the gene, the downstream primer of the internal reference gene at 10 μmol / L, 1 μg positive control, 1 μg negative control, the positive control was a plasmid containing ALOX15, and the negative control was an empty plasmid (plasmid vector).

Example 6:

Reagents for RNA extraction from nasal polyps: 2 mL of cell lysate (RL buffer with 50 x dithiothreitol (DTT)), a genomic DNA adsorption column for removing genomic DNA, and after collecting and removing genomic DNA A collection tube for the solution, an RNA purification column for enriching RNA, 0.7 mL of a first buffer for removing impurities from the purification column to which the RNA is adsorbed, and 0.7 mL of a second buffer for removing impurities and salts from the RNA solution Liquid, centrifuge tube for collecting RNA, 1 mL of RNase and DNase water for dissolving RNA;

Reagent for reverse transcription of total RNA into cDNA: 2 μL of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitor, random 6 nucleotide primers, polythymine, T repeat oligonucleotide, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0 ~ 8μL of RNase and DNase water (make up to 8μL with water according to the amount of RNA); RNase and DNase-free water is used for replenishment. Uniform system, dissolve and dilute RNA;

Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction: 5 μL of premix (containing enzymes and buffers required for PCR), 0-10 μL of double distilled water (based on total Make up volume to 10 μL with water), 0 to 2 μL of dye (for fluorescence compensation and correction of the machine), 1 μM upstream primer of ALOX15 gene, 1 μM downstream primer of ALOX15 gene, 1 μM upstream primer of internal reference gene, 1 μM Downstream primers for the internal reference gene, 1 μg positive control, 1 μg negative control.

In the above-mentioned Examples 4 to 6, the manufacturer of the first buffer RWA buffer used was Takara Company, No. 9767; the manufacturer of the second buffer RWB buffer was Takara Company, No. 9767.

The kits provided in Examples 1 to 6 of the present disclosure can all realize the detection of chronic rhinosinusitis with nasal polyp subtypes. The following are specific test experiments of the kits for detecting chronic rhinosinusitis with nasal polyp subtypes:

Experiments on detection of nasal polyp subtypes:

Experimental Example 1:

1. Experimental method:

(1) Collection and processing of samples:

After randomly selecting 78 CRSwNP patients to flush the nasal cavity with normal saline, nasal polyps were obtained under nasal endoscope. Nasal polyps were cut into tissues with a diameter of about 0.5 cm, immersed in RNA stabilization and storage solution (RNAlater), stored at 4 ° C for a short period of time, and then transferred to a storage temperature below -20 ° C for a long time.

(2) Extraction of RNA:

Step 1: Weigh the tissue immersed in RNA stabilization and storage solution (RNAlater). Weigh about 0.01g of tissue into a magnetic beaded centrifuge tube, place it in liquid nitrogen, and grind it on a homogenizer. (3000r, 5min) (or manual grinding). Add 1mL Trizol to the test tube containing the tissue cells to dissolve it, collect it in a centrifuge tube, shake it thoroughly, and leave it at room temperature for 5 minutes; then add 200 μL of chloroform (trichloromethane) to the RNA extraction reagent group, and shake vigorously to mix. Let stand at room temperature for 5 minutes.

Step 2: 12,000 rpm, 4 ° C, and centrifuge for 15 minutes.

Step 3: Take the supernatant to obtain a volume of about 200 μL, add it to a centrifuge tube, and add an equal amount (about 200 μL) of isopropanol to the above RNA extraction reagent group. After mixing, let stand for 10 minutes, 12000 rpm, and centrifuge at 4 ° C for 15 minutes. Discard the supernatant and retain the pellet.

Step 4: Add about 200 µL of 75% ethanol (equivalent to about 150 µL of anhydrous ethanol and 50 µL of DNAse and RNase water) of the above-mentioned RNA extraction reagent group (equivalent to isopropanol) to wash the pellet and mix. Centrifuge at 7500 rpm, 4 ° C for 15 minutes. Discard the supernatant and retain the pellet.

Step 5: Cap the centrifuge tube tightly and centrifuge at 7500 rpm and 4 ° C for 2 minutes.

Step 6: Open the lid, discard the supernatant, and leave it in a fume hood for 15 minutes.

Step 7: Add 0.02 mL of RNA hydrolase-free and DNA hydrolase-free (RNase-free and DNase-free) water-soluble precipitates to the above-mentioned RNA extraction reagent group.

Step 8: Measure the RNA concentration with a spectrophotometer, and the OD260 / OD280 ratio is between 1.7-2.1.

(3) Preparation of cDNA by reverse transcription: Prepare a reverse transcription (RT) reaction solution on ice, including the following reagents: 2 μL of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitors, random 6-core Primers, polythymine, T repeat oligonucleotides, triphosphate deoxyribonucleotide mixtures, buffers, etc.), not more than 500ng or 8μL of total RNA, 0-8μL deRNase and DNA Enzyme water (make up to 8 μL with water according to the amount of RNA); take the extracted total RNA and the above-mentioned reverse transcription reaction solution and add them to the reaction system to perform the reverse transcription reaction to obtain a cDNA template, where the reaction system can be scaled up as required and 10 μL reaction The system can use up to 500ng of total RNA;

The reverse transcription reaction conditions are as follows:

37 ° C for 15 minutes (reverse transcription reaction)

84 ° C for 5 seconds (inactivation of reverse transcriptase)

The product was left at 4 ° C.

(4) Preparation of real-time quantitative PCR reaction solution: including 5 μL of premix (containing enzymes and buffers required for PCR), 0.2 μL of machine fluorescence compensation and correction agent, 1 ng / μL cDNA or positive control or negative control, 0.5 μL upstream primer of ALOX15 gene, 0.5 μL downstream primer of ALOX15 gene, 0.5 μL upstream primer of internal reference gene, 0.5 μL downstream primer of internal reference gene, and 2.8 μL of double-distilled water;

(5) Real-time quantitative PCR detection:

Reaction conditions:

Stage 1: Pre-denaturation: 95 ° C, 30 seconds.

Phase 2: PCR reaction: 95 ° C, 15 seconds; 60 ° C, 1 minute annealing extension, for a total of 40 cycles; Phase 2: Melting curve: 60 ° C gradually rises to 95 ° C, the rate is 0.1 ° C / second, Collecting fluorescence;

After the reaction, confirm that the amplification curves of real-time quantitative PCR are shown in Figures 1 to 4, and the melting curves are shown in Figures 5 to 8, read the Ct values of ALOX15 and GAPDH, and use the △ Ct analysis method (ALOX15 Ct value minus Ct value of GAPDH) was analyzed, and GAPDH was used as a reference gene.

(6) Data analysis:

Step 1: Judgment of the quality control of the experiment: The positive control Ct value <20 and the negative control Ct value> 38 are regarded as valid experiments, otherwise the experiments are invalid.

Step 2: Judgment of typing: The Ct value of the target gene minus the Ct value of the reference gene. According to the ROC curve, the optimal cutoff value of the Ct value of ALOX15 is 1.675. If the Ct value of ALOX15 is greater than 1.675, it is a non-eosinophil. Cellular chronic sinusitis with nasal polyps; if the Ct value of ALOX15 is less than 1.675, it is a typical eosinophilic chronic sinusitis with nasal polyps.

2. Experimental results: The results of detecting nasal polyp subtypes based on the kits provided above are shown in Table 1:

Table 1 Obtaining results of nasal polyp subtypes obtained from the kits and histopathological methods provided in this disclosure

受试者编号Subject number	△Ct值△ Ct value	依据界值判断的结果Judgment results based on thresholds	病理结果 Pathological results
11	3.0293.029	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
22	2.6162.616	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
33	3.1883.188	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type

44	6.0526.052	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
55	1.6951.695	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
66	1.3131.313	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
77	5.4375.437	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
88	1.4751.475	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
99	7.7787.778	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1010	4.2924.292	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
1111	2.2842.284	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1212	4.1984.198	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
1313	3.3833.383	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1414	3.6843.684	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1515	3.3023.302	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1616	4.1444.144	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
1717	3.7143.714	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1818	3.5563.556	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
1919	4.1364.136	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
2020	2.1862.186	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
21twenty one	4.0624.062	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
22twenty two	4.4204.420	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
23twenty three	0.3130.313	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
24twenty four	2.9992.999	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
2525	2.8922.892	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
2626	3.0483.048	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
2727	2.1522.152	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
2828	0.3870.387	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
2929	1.5471.547	嗜酸性粒细胞型Eosinophil type		嗜酸性粒细胞型Eosinophil type
3030	2.4012.401	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
3131	1.6551.655	嗜酸性粒细胞型Eosinophil type		嗜酸性粒细胞型Eosinophil type
3232	0.5470.547	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3333	0.2820.282	嗜酸性粒细胞型Eosinophil type		嗜酸性粒细胞型Eosinophil type
3434	1.8291.829	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
3535	-0.895-0.895	嗜酸性粒细胞型Eosinophil type		嗜酸性粒细胞型Eosinophil type
3636	2.1522.152	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
3737	3.2723.272	非嗜酸性粒细胞型Non-eosinophil type		嗜酸性粒细胞型Eosinophil type
3838	2.5372.537	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
3939	3.9683.968	非嗜酸性粒细胞型Non-eosinophil type		嗜酸性粒细胞型Eosinophil type
4040	3.1583.158	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
4141	0.1830.183	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4242	-0.933-0.933	嗜酸性粒细胞型Eosinophil type	非嗜酸性粒细胞型Non-eosinophil type

4343	-0.021-0.021	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4444	0.2960.296	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4545	-1.137-1.137	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4646	3.2023.202	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
4747	0.4420.442	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4848	0.7650.765	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4949	2.2532.253	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
5050	0.7690.769	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
5151	2.4222.422	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
5252	0.7490.749	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
5353	-3.834-3.834	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
5454	1.9301.930	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
5555	0.7670.767	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
5656	0.7780.778	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
5757	0.0610.061	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
5858	4.6404.640	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
5959	-0.547-0.547	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6060	0.0190.019	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6161	0.3550.355	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6262	0.2510.251	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6363	0.5890.589	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6464	3.6643.664	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
6565	2.4542.454	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
6666	0.2240.224	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6767	3.1173.117	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
6868	1.6371.637	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
6969	1.4891.489	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7070	2.0732.073	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
7171	1.3171.317	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7272	0.2060.206	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7373	-1.086-1.086	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7474	-0.012-0.012	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7575	1.6091.609	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7676	-3.187-3.187	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7777	0.7320.732	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
7878	1.1521.152	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type

The ROC curve of the above 78 samples is shown in Figure 9. Using this kit to predict the classification of chronic sinusitis with nasal polyps, the accuracy is 78.2%.

Experimental example two:

1. Experimental method:

(1) Collection and processing of samples:

After randomly selecting 47 CRSwNP patients to flush the nasal cavity with normal saline, press the surface of the nasal polyps under a nasal endoscope with a brush (Copan) for 30 seconds, rotate 3-4 times, brush the surface of the polyp, and place the brush on the lysis In the liquid, store at 4 ° C for a short time (no more than 24 hours), or transfer to a long-term storage below -20 ° C.

(2) Extraction of RNA:

Step 1: Add 1mL Trizol to the test tube containing the detached cells, dissolve it, shake it thoroughly, and let it stand at room temperature for 5 minutes, then add 200 μL of chloroform (trichloromethane), mix with vigorous shaking, and let it stand at room temperature for 5 minutes;

Step 2: 12,000 rpm, 4 ° C, and centrifuge for 15 minutes;

Step 3: Take the supernatant to obtain a volume of about 200 μL, add to the centrifuge tube, and add the same amount of isopropyl alcohol (about 200 μL) as chloroform. After mixing, let stand for 10 minutes, 12000 rpm, and centrifuge at 4 ° C for 15 minutes. Discard the supernatant and keep the pellet;

Step 4: Add approximately 200 μL of 75% ethanol (a mixture of approximately 150 μL of absolute ethanol and 50 μL of DNAse and RNAse water) to the RNA extraction reagent group (equivalent to isopropyl alcohol), and mix well. 7500 rpm, 4 ° C, centrifugation for 15 minutes, discard the supernatant and retain the pellet;

Step 5: Cap the centrifuge tube tightly at 7500 rpm, 4 ° C, and centrifuge for 2 minutes;

Step 6: Open the lid, discard the supernatant, and leave it in a fume hood for 15 minutes;

Step 7: Add 0.02 mL of RNA-hydrolase-free and DNA-hydrolase-free (RNase-free and DNase-free) water-soluble precipitates to the above-mentioned RNA extraction reagent group;

Step 8: Measure the RNA concentration with a spectrophotometer. The OD260 / OD280 ratio is preferably 1.7 to 2.1.

(3) Preparation of cDNA by reverse transcription: same as Experimental Example 1;

(4) Preparation of real-time quantitative PCR reaction solution: same as Experimental Example 1;

(5) Real-time quantitative PCR detection: same as Experimental Example 1. After the reaction, confirm that the amplification curve of real-time quantitative PCR is shown in Figs. 10 to 12 and the melting curve is shown in Figs. 13 to 15;

(6) Data analysis: The Ct value of the target gene minus the Ct value of the reference gene. According to the results of previous experiments, the optimal cutoff value for the Ct value of ALOX15 is 1.675. If the Ct value of ALOX15 ≥ 1.675, it is a non-eosinophil. Cellular chronic sinusitis with nasal polyps; if the Ct value of ALOX15 is less than 1.675, it is a typical eosinophilic chronic sinusitis with nasal polyps;

2. Experimental results: The calculation results are shown in Table 2.

Table 2 Results of nasal polyp subtype detection using the kit provided in the present disclosure

受试者编号Subject number	△Ct值△ Ct value	依据界值判断的病理结果Pathological results based on cutoffs	病理结果 Pathological results
11	3.4133.413	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
22	0.4910.491	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
33	2.772.77	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
44	0.7110.711	嗜酸性粒细胞型Eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
55	7.177.17	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
66	1.8181.818	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
77	2.9522.952	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型 Non-eosinophil type
88	0.1670.167	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
99	2.0142.014	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1010	1.831.83	非嗜酸性粒细胞型Non-eosinophil type	嗜酸性粒细胞型Eosinophil type
1111	2.0152.015	非嗜酸性粒细胞型Non-eosinophil type		嗜酸性粒细胞型Eosinophil type
1212	3.6653.665	非嗜酸性粒细胞型Non-eosinophil type	非嗜酸性粒细胞型Non-eosinophil type

1313	1.7571.757	非嗜酸性粒细胞型Non-eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1414	0.2180.218	嗜酸性粒细胞型Eosinophil type		非嗜酸性粒细胞型Non-eosinophil type
1515	2.3292.329	非嗜酸性粒细胞型Non-eosinophil type		嗜酸性粒细胞型Eosinophil type
1616	1.1071.107	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
1717	0.3210.321	嗜酸性粒细胞型Eosinophil type		嗜酸性粒细胞型Eosinophil type
1818	0.8980.898	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
1919	0.3940.394	嗜酸性粒细胞型Eosinophil type		嗜酸性粒细胞型Eosinophil type
2020	0.4830.483	嗜酸性粒细胞型Eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
21twenty one	0.6190.619	嗜酸性粒细胞型Eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
22twenty two	0.8690.869	嗜酸性粒细胞型Eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
23twenty three	1.551.55	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
24twenty four	1.021.02	嗜酸性粒细胞型Eosinophil type	非嗜酸性粒细胞型Non-eosinophil type
2525	-0.121-0.121	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
2626	-0.709-0.709	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
2727	-0.303-0.303	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
2828	-0.196-0.196	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
2929	-1.375-1.375	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3030	-2.017-2.017	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3131	-0.42-0.42	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3232	-2.01-2.01	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3333	-1.25-1.25	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3434	-1.26-1.26	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3535	-0.26-0.26	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3636	-2.312-2.312	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3737	-1.309-1.309	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3838	-0.786-0.786	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
3939	-1.363-1.363	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4040	-0.375-0.375	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4141	-0.072-0.072	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4242	-0.088-0.088	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4343	-0.789-0.789	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4444	-0.891-0.891	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4545	-0.581-0.581	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4646	-1.423-1.423	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type
4747	-1.128-1.128	嗜酸性粒细胞型Eosinophil type	嗜酸性粒细胞型Eosinophil type

According to the data results provided in Table 2, the accuracy of the kit in Experimental Example 2 of the present disclosure for detecting chronic sinusitis with nasal polyp subtypes was 80.9%.

Experimental example three:

1. Test method:

(1) Collection and processing of samples:

After a patient rinses the nasal cavity with normal saline, press the surface of the nasal polyps under a nasal endoscope with a brush (Copan) for 30 seconds, rotate 3-4 times, brush the surface of the polyp, and place the brush in the lysate. Stored at ℃ for a short time (no more than 24 hours), or transferred to long-term storage below -20 ° C.

(2) RNA extraction:

Step 1: Place the genomic DNA adsorption column (genomic DNA Spinner Column) on a 2mL collection tube (Collection Tube);

Step 2: Transfer the lysate (cell lysate) containing the detached cells into a genomic DNA adsorption column;

Step 3: 12,000 rpm, centrifuge for 1 minute;

Step 4: Discard the genomic DNA adsorption column and retain the filtrate in the 2mL collection tube;

Step 5: Add 300 μL of 70% ethanol to the above step 4 (precipitation may occur at this time), and use a pipette to mix the solution uniformly;

Step 6: Immediately transfer all the mixed solution (including the precipitate) into an RNA purification column (containing a 2 mL collection tube);

Step 7: 12,000 rpm, centrifuge for 1 minute, and discard the filtrate. Place the RNA purification column back into the 2mL collection tube;

Step 8: Add 500 μL of the first buffer (Buffer RWA) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;

Step 9: Add 600 μL of the second buffer (Buffer RWB) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate.

Step 10: Place the RNA purification column on a 1.5 mL RNAase-free collection tube (RNase Free Colletion Tube), and add 50 μL of RNA hydrolysis-free distilled water (RNase Free dH2O) or 0.1% to the center of the RNA purification column membrane. Diethyl pyrocarbonate (DEPC) treated water, and allowed to stand at room temperature for 5 minutes;

Step 11: Centrifuge at 12,000 rpm and elute RNA for 2 minutes with de-RNase and de-DNase water;

Step 12: Measure the RNA concentration with a spectrophotometer, and the OD260 / OD280 ratio is 2.0.

(4) Preparation of real-time PCR reaction solution:

Step 1: Configure 45μL of SYBR Green 1 pre-mixed solution; and ROX: 1.8μL, mix and divide into 3 parts, respectively A.11.7μL; B.11.7μL; C.23.4μL, add 1ng / Solution A was obtained from μL positive control, solution B was added to B with 1ng / μL negative control, and solution C was added from C with 2ng to obtain solution C (SYBR Green Green 1 premix and ROX are products of Takara Company, article number RR820A);

Step 2: Configure 8 groups of parallel holes;

The first and second parallel wells: solution A, specific primers for ALOX15 gene, 3.8 μL sterilized double distilled water;

The third and fourth parallel wells: solution B, specific primers for ALOX15 gene, 3.8 μL sterilized double distilled water;

5th and 6th parallel wells: C solution, specific primers for ALOX15 gene, 3.8 μL sterilized double distilled water;

7th and 8th parallel wells: C solution, specific primers for GAPDH gene, 3.8 μL sterilized double distilled water;

Step 3: Seal the plate with clear plastic film, centrifuge, and perform the PCR operation.

Step 4: Two-step PCR standard procedure:

(5) Real-time quantitative PCR detection:

Reaction conditions:

Stage 1: Pre-denaturation: 95 ° C, 30 seconds.

Phase 2: PCR reaction: 95 ° C, 15 seconds; 60 ° C, 1 minute annealing extension, 40 cycles were performed. ;

After the reaction, confirm that the amplification curve of real-time quantitative PCR is shown in FIG. 16 and the melting curve is shown in FIG. 17. Value) for analysis, using GAPDH as a reference gene.

(6) Data analysis: same as Experimental Example 1;

2. Experimental results: The average Ct of positive control wells: 16.2; the average Ct of negative control wells: 39.7; the average Ct of sample ALOX15: 19.4; the average Ct of sample GAPDH: 16.4; the difference: 19.4-16.4 is 3, greater than 1.675, and it is non-tropic Acid granulocytic chronic sinusitis with nasal polyps.

Experimental Example 4:

The kits provided in Examples 1 to 6 of the present disclosure can all realize the detection of chronic rhinosinusitis with nasal polyp subtypes. Taking Example 5 as an example, the effect of the kit for detecting chronic rhinosinusitis with nasal polyp subtypes is performed as follows. Detection experiment:

(1) Collection and processing of samples:

After a patient rinses the nasal cavity with saline, a polyp is removed under nasal endoscopy. Polyps were cut into tissues with a diameter of about 0.5 cm, immersed in RNA stabilization and storage solution (RNAlater), stored at 4 ° C for a short period of time, and then transferred to long-term storage below -20 ° C.

(2) RNA extraction:

Step 1: Weigh the tissue immersed in RNA stabilization and storage solution (RNAlater). Weigh about 0.01g of tissue into a magnetic beaded centrifuge tube, place it in liquid nitrogen, and grind it on a homogenizer. (3000r, 5min) (or manual grinding); add 0.3mL cell lysate, 12,000 rpm, centrifuge for 15 minutes;

Step 2: Aspirate the supernatant, add 70% ethanol (70% absolute ethanol and 30% DEPC or RNase and DNase water) equal to the volume of the supernatant, and use a pipette to mix the solution uniformly;

Step 3: Transfer all the mixed solution (including the precipitate) to the RNA purification column (including the 2mL collection tube) immediately;

Step 4: 12,000 rpm, centrifuge for 1 minute, and discard the filtrate. Put the RNA purification back into the 2mL collection tube;

Step 5: Add 500 μL of the first buffer solution (Buffer RWA) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;

Step 6: Add 600 μL of a second buffer (Buffer RWB) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;

Step 7: Preparation of DNase I reaction solution: Take 5 μL of 10 × DNase I buffer, 4 μL of recombinant DNase I ((RNase-free, 5U / μL), 41 μL of RNase-free Double distilled water into a new 1.5mL tube (no RNase) and mix well;

Step 8: Add 50 μL DNase I reaction solution to the center of the RNA purification column membrane, and leave it at room temperature for 15 minutes;

Step 9: Add 350 μL of a second buffer to the center of the RNA purification column membrane, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;

Step 10: Repeat step 6;

Step 11: Reposition the RNA purification column on a 2mL collection tube, 12,000 rpm, and centrifuge for 2 minutes;

Step 12: Place the RNA purification column on a 1.5 mL RNAase-free collection tube (RNase Free Colletion Tube), and add 50 μL of RNAase-free distilled water (RNase Free HdO) or 0.1% to the center of the RNA purification column membrane. Diethyl pyrocarbonate (DEPC) treated water, and allowed to stand at room temperature for 5 minutes;

Step 13: Centrifuge at 12,000 rpm, and elute RNA for 2 minutes with RNase and DNase-free water;

Step 14: Measure the RNA concentration with a spectrophotometer, and the OD260 / OD280 ratio is 2.0;

(4) Preparation of a real-time quantitative PCR reaction solution: the same as in Experiment Example 3;

(5) Real-time quantitative PCR detection:

The reaction conditions used a standard three-step PCR amplification procedure:

Stage 1: Pre-denaturation: 95 ° C, 2 minutes;

Phase 2: PCR reaction: 95 ° C, 1 minute; 55 ° C, 1 minute, 72 ° C for 1 minute, a total of 40 cycles were performed, and finally 72 ° C, 7 minutes annealing extension.

After the reaction, confirm that the amplification curve of real-time quantitative PCR is shown in FIG. 18 and the melting curve is shown in FIG. 19. Read the Ct values of ALOX15 and GAPDH, and use the △ Ct analysis method (Ct value of ALOX15 minus Ct of GAPDH). Value) for analysis, using GAPDH as a reference gene.

(6) Data analysis: same as Experimental Example 1;

2. Experimental results: average Ct of positive control wells: 16.7; average Ct of negative control wells: 38.4; average Ct of sample ALOX15: 19.2; average Ct of sample GAPDH: 16.4; difference: 2.8, greater than 1.675, non-eosinophils Chronic sinusitis with nasal polyps.

In summary, the present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtypes. The screening uses the ALOX15 gene as a biomarker and applies it to the kit to realize the use of the kit to detect chronic The method of sinusitis with nasal polyp subtypes can quickly, accurately and comprehensively identify patients with nasal polyp subtypes through the kit, in order to carry out targeted treatment based on the inflammatory subtypes of nasal polyps as soon as possible, and effectively guide chronic sinusitis. The determination of drug treatment methods and surgical methods for patients with nasal polyps, accurately predict the response to drug treatment, and judge the prognostic effect.

Example 7

Collection and processing of samples:

After a patient rinses the nasal cavity with normal saline, press the surface of the nasal polyps under a nasal endoscope with a brush (Copan) for 30 seconds, rotate 3 to 4 times, brush the surface of the polyp, and place the brush in the lysate. 4 Stored at ℃ for a short time (no more than 24 hours), or transferred to long-term storage below -20 ° C.

A method for detecting the expression of ALOX15 gene in exfoliated nasal cells, comprising the following steps:

Step 1: Extract RNA from exfoliated nasal cells:

Step 1: Take the genomic DNA adsorption column into a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 300 μL of cell lysate, and add it to the genomic DNA adsorption column. Take the filtrate and add an equal volume of 70% to the filter. Ethanol, after mixing, added to the RNA purification column, 12000 rpm, centrifugation for 1min, the filtrate was removed, and the RNA purification column was placed in a 2mL collection tube;

Step 3: Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 μL of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. Leave at room temperature for 5 minutes, 12000 rpm, centrifuge for 2 minutes, elute the RNA purification column, and use a spectrophotometer to measure the OD260 / OD280 ratio of the RNA solution to 2.0 to obtain RNA;

Step 2: Prepare cDNA by reverse transcription, including the following steps: take 2 μL of the reverse transcription mixture, 0-8 μL of the deRNA hydrolase distilled water (make up to 8 μL with water according to the amount of RNA), and the total amount does not exceed 500ng Or the total RNA volume is not more than 8 μL, and the de-RNA hydrolase distilled water is used to make up to 10 μL. After gentle mixing, reverse transcription reaction is performed under the following conditions: at 37 ° C, a reverse transcription reaction is performed for 15 minutes; Under the condition of 85 ° C, the reverse transcriptase inactivation reaction was performed for 5 seconds; the product was left at 4 ° C.

Step 3: Real-time quantitative PCR amplification detection, including the following steps:

Step 1: Prepare a real-time PCR reaction solution: include 1 μL of PCR premix, 0-10 μL of double distilled water (to make up to 10 μL with water based on the total volume), 0.2 μL of machine fluorescence compensation and correction agent, 1 μM of ALOX15 Gene upstream primer, 1 μM ALOX15 gene downstream primer, 1 μM internal reference gene upstream primer, 1 μM internal reference gene downstream primer, 0.01 μL of the cDNA, 1 μg positive control, 1 μg negative control, positive control contains ALOX15 plasmid, the negative control is an empty plasmid (plasmid vector);

Step 2: Standard procedure for two-step PCR amplification: The reaction conditions for the standard procedure for two-step PCR amplification include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR reaction : Reaction was performed at 95 ° C for 15 seconds, and at 60 ° C, reaction was performed for 60 seconds, followed by annealing extension, and 40 cycles were performed.

Step 4: Calculate the expression of the ALOX15 gene:

After the reaction, confirm the amplification curve and melting curve of real-time quantitative PCR, read the Ct values of ALOX15 and GAPDH, select △ Ct analysis method (Ct value of ALOX15 minus Ct value of GAPDH) for analysis, and use GAPDH as the internal reference gene; Positive control Ct value <20 and negative control Ct value> 38 are considered valid, otherwise the test is invalid;

ΔCT method was used to compare the expression of ALOX15 and the reference gene: the average CT value of ALOX15 was 20.9, the average CT value of GAPDH was 18.9, and the ΔCT value was 2.0.

Example 8:

Collection and processing of samples:

Step 1: Extract RNA from exfoliated nasal cells:

Step 1: Take the genomic DNA adsorption column into a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 100 μL of cell lysate, add it to the genomic DNA adsorption column, centrifuge at 12000 rpm for 60s, and take the filtrate. Add an equal volume of 70% ethanol to the filter, mix it and add it to the RNA purification column, 12000 rpm, centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;

Step 2: Add 300 μL of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 seconds to remove the first filtrate; continue to add 400 μL of the second buffer to the RNA purification column Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;

Step 2: Reverse transcription to prepare cDNA, including the following steps: take 1 μL of the reverse transcription mixture, 0 to 10 μL of the deRNA hydrolase distilled water, and total RNA not exceeding 500 ng or 8 μL in volume. Make up the RNA hydrolase distilled water to make up to 10 μL; gently mix the reverse transcription reaction under the following conditions: at 37 ° C for 15 minutes, and at 85 ° C for 5 seconds Reverse transcriptase inactivation reaction; the product was left at 4 ° C.

Step 1: Prepare a real-time quantitative PCR reaction solution: include 25 μL of PCR premix, 0 to 10 μL of double distilled water (to make up to 10 μL with water based on the total volume), and 0 to 2 μL of dye (for fluorescence compensation of the machine) And correction), 0.01 μM ALOX15 gene upstream primer, 0.01 μM ALOX15 gene downstream primer, 0.01 μM internal reference gene upstream primer, 0.01 μM internal reference gene downstream primer, 5 μL cDNA, 1 μg positive control, 1 μg negative Control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector);

Step 4: Calculate the expression of the ALOX15 gene:

After the reaction, confirm the amplification curve and melting curve of real-time quantitative PCR, read the Ct values of ALOX15 and GAPDH, select △ Ct analysis method (Ct value of ALOX15 minus Ct value of GAPDH) for analysis, and use GAPDH as the reference gene Positive control Ct value <20 and negative control Ct value> 38 are considered valid, otherwise the test is invalid;

ΔCT method was used to compare the expression difference of ALOX15 and internal reference genes: the average CT value of ALOX15 was 19.5, the average CT value of GAPDH was 18.0, and the ΔCT value was 1.5.

Example 9:

Collection and processing of samples:

Step 1: Extract RNA from exfoliated nasal cells:

Step 1: Take the genomic DNA adsorption column into a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 2000 μL of cell lysate, add it to the genomic DNA adsorption column, centrifuge at 12000 rpm for 60 seconds, and take the filtrate. Add an equal volume of 70% ethanol to the filter, mix it and add it to the RNA purification column, 12000 rpm, centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;

Step 2: Add 700 μL of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 800 μL of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;

Step 2: Reverse transcription to prepare cDNA, including the following steps: take 3 μL of the reverse transcription mixture, 0-8 μL of RNase and DNAse-free water (make up to 8 μL with water according to the amount of RNA), and the total amount does not exceed 500ng or total volume of no more than 8μL, the deRNA hydrolase distilled water was made up to 10μL; the reverse transcription reaction was performed after gentle mixing, the conditions were as follows: at 37 ° C, a reverse transcription reaction was performed for 15 minutes; Under the condition of 85 ° C, the reverse transcriptase inactivation reaction was performed for 5 seconds; the product was left at 4 ° C.

Step 1: Prepare a real-time PCR reaction solution: include 5 μL of premixed solution (containing enzymes and buffers required for PCR), 0-10 μL of double-distilled water (make up to 10 μL with water based on the total volume), and 0 to 2 μL of Dye (for fluorescence compensation and correction of the machine), 1 μM upstream primer of ALOX15 gene, 1 μM downstream primer of ALOX15 gene, 1 μM upstream reference gene of primer, 1 μM downstream reference gene of primer, 2 μL of cDNA, 1 μg Positive control, 1 μg negative control;

Step 2: Standard procedure for two-step PCR amplification: The reaction conditions for the standard procedure for two-step PCR amplification include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR : Reaction was performed at 95 ° C for 15 seconds, and at 60 ° C, reaction was performed for 60 seconds, followed by annealing extension, and 40 cycles were performed.

Step 4: Calculate the expression of the ALOX15 gene:

ΔCT method was used to compare the expression of ALOX15 and internal reference genes: the average CT value of ALOX15 was 20.1, the average CT value of GAPDH was 17.9, and the ΔCT value was 2.2.

In Examples 7 to 9 of the present disclosure, the preferred manufacturer of the first buffer RWA buffer used is Takara Company, article number 9767; the manufacturer of the second buffer RWB buffer is Takara Company, article number 9767. However, the protection scope of the present application is not limited to the first buffer solution and the second buffer solution described above. Those skilled in the art can choose according to the actual application needs.

Example 10:

Collection and processing of samples:

After 78 cases of nasal cavity were rinsed with saline, nasal endoscope was used to press the surface of the nasal polyps with a brush (Copan) for 30 seconds, rotated 3-4 times to brush the surface of the polyp, and the brush was placed in a subsequent place. The lysate is stored at 4 ° C for short-term storage (no more than 24 hours), or transferred to long-term storage below -20 ° C.

Step 1: Extract RNA from exfoliated nasal cells: add 1mL RNA extraction solution to the centrifuge tube containing detached nasal cells, dissolve and shake, and let stand at room temperature for 5min; add 200μL of chloroform, mix by shaking, and let stand at room temperature for 5min. Centrifuge at 12000 r / min for 15 min at 4 ° C. Take 200 μL of the supernatant, add 200 μL isopropanol, mix and let stand for 10 min. Centrifuge at 4 ° C, 12000 r / min for 15 min. Discard the supernatant and retain the first pellet. Add 75% ethanol with the same volume as isopropyl alcohol to the first pellet, centrifuge at 7500 r / min for 15 min at 4 ° C, discard the supernatant, and retain the second pellet; cover the centrifuge tube, and set the tube to 7500 r / min at 4 ° C. Centrifuge for 2 min, remove the supernatant, and leave to stand for 15 min. Continue to add 50 μL of deRNase and deDNase water to the centrifuge tube to dissolve the second precipitate. Use a spectrophotometer to measure the OD260 / OD280 ratio of the RNA solution. 1.8, get RNA;

Step 2: The steps for preparing cDNA by reverse transcription are the same as in Example 7.

Step 3: The detection steps of real-time quantitative PCR amplification are the same as those in Example 7.

Step 4: Calculate the expression of the ALOX15 gene:

Step 1: Calculate the average ΔCT of the healthy control group: In this example, subjects 1 to 10 are healthy control groups. The calculation method is as follows: average ΔCT (CT (ALOX15) -CT (GAPDH)) = (1.754 + 1.655 + 1.829 + 2.2 + 1.308 + 1.449 + 1.234 + 1.343 + 1.49 + 1.695 / 10 = 1.5975;

Step 2: Calculate the relative expression of the subject:

Based on 1.5975, determine the relative expression level of each subject (represents the expression level of the gene in the subject relative to the average value of a healthy control group, and the value represents the multiple of change. For example, 1.5 is equivalent to the subject's expression level It is 1.5 times the average of the healthy control group). The calculation results are shown in Table 3.

The calculation formula is 2 ^-ΔΔCT , -ΔΔCT =-(ΔCT (subject CT (ALOX15)-CT (GAPDH))-average ΔCT (average ΔCT of healthy control group)).

Table 3 Calculation results of ALOX15 gene expression method according to the method provided in Example 8

Example 11:

Collection and processing of samples:

Before obtaining the cells, the patient was instructed to flush the nasal cavity with normal saline, and under a nasal endoscope, press the surface of the nasal polyp with a brush (Copan) for 30 seconds, rotate 3-4 times, brush the surface of the polyp, and place the brush in the lysate , Short-term storage at 4 ° C (not more than 24 hours), or transfer to long-term storage below -20 ° C.

Step 1: Extract RNA from the nasal cavity exfoliated cells: Add 20mL RNA extraction solution to the centrifuge tube containing the nasal cavity exfoliated cells, dissolve and shake, and let stand at room temperature for 7min; add 10mL of chloroform, shake and mix, and leave at room temperature for 7min Centrifuge at 5 ° C, 14000r / min for 20min; take 20mL of supernatant, add 20mL of isopropanol, mix well and let stand for 12min, centrifuge at 5 ° C, 14000r / min for 20min, discard the supernatant, and retain the first precipitate; 40 mL of 90% ethanol was added to the first pellet, centrifuged at 14000 r / min for 3 min at 5 ° C, the supernatant was discarded, and the second pellet was retained; the centrifuge tube was capped, and centrifuged at 5 ° C, 14000 r / min for 3 min After removing the supernatant, let it stand for 20 minutes, and then continue to add 5 mL of RNase and DNase water to the centrifuge tube to dissolve the second precipitate, and use a spectrophotometer to measure the RNA solution OD260 / OD280 ratio of 2.1 to obtain RNA;

Step 3: The preparation of the real-time quantitative PCR reaction solution in the real-time quantitative PCR amplification detection step is the same as in Example 7. A standard three-step method for PCR amplification is adopted. The reaction conditions of the standard three-step PCR standard procedure include The following steps: Phase 1: Pre-denaturation at 95 ° C for 2 minutes; Phase 2 PCR reaction: Reaction at 95 ° C for 1 minute, at 55 ° C for 1 minute, at 72 ° C Under the conditions, the reaction was performed for 1 minute, and thus 40 cycles were performed; finally, 72 ° C, 7 minutes of annealing extension.

Step 4: Calculate the expression of the ALOX15 gene:

Average Ct of positive control wells: 16.7; average Ct of negative control wells: 38.4; average Ct of sample ALOX15: 21.5; average Ct of sample GAPDH: 19.4; difference: 2.1. The expression of ALOX15 in this patient was 0.23 times (1/2 ^2.1 ) of GAPDH.

Example 12:

Collection and processing of samples:

Before obtaining the cells, the patient was instructed to flush the nasal cavity with normal saline, and under a nasal endoscope, press the surface of the nasal polyp with a brush (Copan) for 30 seconds, rotate 3-4 times, brush the surface of the polyp, and place the brush in the lysis solution , Short-term storage at 4 ° C (not more than 24 hours), or transfer to long-term storage below -20 ° C.

Step 1: Extract RNA from nasal cavity exfoliated cells. Add 0.1mL RNA extraction solution to the centrifuge tube containing nasal exfoliated cells, dissolve and shake, and let stand at room temperature for 7min. Add 0.03mL of chloroform, shake and mix, and leave at room temperature. Centrifuge at 7min, and centrifuge at 14000r / min for 20min at 5 ℃; take 20mL of supernatant, add 0.015mL of isopropanol, mix and let stand for 12min, centrifuge at 5 ℃, 14000r / min for 20min, discard the supernatant, and keep the first Precipitation; adding 0.0075 mL of 90% ethanol to the first precipitate, centrifuging at 14000r / min for 3min at 5 ° C, discarding the supernatant, and retaining the second precipitate; capping the centrifuge tube at 5 ° C, 14000r Centrifuge for 3min / min, remove the supernatant, and let stand for 20min, then add 0.01mL deRNase and deDNase water to the centrifuge tube to dissolve the second precipitate, and measure the RNA solution OD260 / OD280 with a spectrophotometer The ratio is 2.1 to obtain RNA;

Step 4: Calculate the expression of the ALOX15 gene:

The average Ct of positive control wells: 15.9; the average Ct of negative control wells: 38.7; the average Ct of sample ALOX15: 17.8; the average Ct of sample GAPDH: 16.4; the difference: 17.8-16.4 was 1.4. The expression of ALOX15 in this patient is 0.51 times (1/2 ^1.4 ) of GAPDH.

Industrial applicability:

The present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtypes. The proteomics and transcriptomics methods are used to select the ALOX15 gene as a biomarker and apply it to the kit to realize the use of reagents. The kit is used to detect chronic sinusitis with nasal polyp subtypes, so that the final obtained kit includes a specific primer for the ALOX15 gene. Based on the specific primers, the kit of the present disclosure can quickly identify nasal polyp subtypes, and is more accurate than traditional pathological detection methods. The kit can simultaneously perform large-scale, rapid Testing, saving labor costs and medical treatment costs. And the systematic kit has high identification accuracy, which can fully reflect the histopathological characteristics. The effect of human error in the prior art is solved, and the disadvantages of misdiagnosis caused by the tissue section reflecting local characteristics of the tissue are avoided. The rapid, accurate, and comprehensive identification of nasal polyp subtypes through kits is essential for clinical diagnosis and treatment. In order to provide targeted treatment based on the inflammatory subtypes of nasal polyps as early as possible, it can effectively guide the drug treatment method for patients with chronic sinusitis and nasal polyps. And the determination of surgical methods, accurately predict the response to drug treatment, and judge the prognostic effect.

The kit provided by the present disclosure can detect nasal polyp cells from the surface of the nasal polyp by brushing or sticking, so as to determine the chronic sinusitis of the patient with nasal polyp subtypes, thereby avoiding wounds to the patient. The safety of patient examination is improved, the operation is more convenient, and the labor cost and medical treatment cost are saved.

The method for detecting the expression level of ALOX15 gene in nasal exfoliated cells provided by the present disclosure uses the effectively selected ALOX15 gene as a biomarker to provide a method for detecting the expression level of the gene and realize calculation of the expression level of ALOX15 gene in nasal exfoliated cells. It can effectively obtain the expression level of ALOX15 gene. The method provided is simple and fast, high sensitivity, good reproducibility, and is suitable for wide application.

The present disclosure provides a method for detecting the expression level of ALOX15 gene in nasal exfoliated cells, wherein the ALOX15 gene can encode a lipoxygenase protein, and the encoded enzyme acts on various polyunsaturated fatty acid substrates to produce various biologically active lipid mediators, For example, eicosanoid, heparin, lipoxin and so on. The arachidonic acid was converted into 12-eicosatetraenoic acid / 12-HPETE and 15-eicosatetraenoic acid / 15-HPETE. It also converts linoleic acid into 13-hydroperoxyoctadecadienoic acid. It can also act on (12S) -hydrogen peroxytetraenoic acid / (12S) -HPETE to produce heparin A3. The encoded enzyme and its reaction products can regulate inflammation and immune response. Diseases related to ALOX15 include softening of white matter around the ventricle. It helps clear apoptotic cells during inflammation by oxidizing membrane-bound phosphatidylethanolamine in macrophages, and inhibits autoimmune responses associated with inflammatory monocytes clearing apoptotic cells. At present, the ALOX15 gene has not been disclosed in the prior art, and the use of the gene has not been found. The method for the expression of ALOX15 gene in exfoliated nasal cells provided in the disclosure can be used to detect the expression of ALOX15 in exfoliated nasal cells, and it can be used to further provide a basis for the gene screening technology for detecting chronic sinusitis subtypes with nasal polyps, It provides a reliable basis for clinical guidance and drug treatment. The feasibility of the kit for detecting chronic sinusitis subtype with nasal polyps in clinical application is guaranteed.

Nasal shedding was detected ALOX15 gene expression in cells of the present disclosure provides, according to actual needs or relative quantitation method using ΔCt ² -ΔΔCt method, the expression of selected relatively constant reference gene, normalized by the number of reference genes by The difference between the Ct value of the target gene of the sample and the internal reference gene is calculated to calculate the expression amount of the target gene. The method is simple and fast, the detection accuracy is high, the detection cost can be reduced, and the detection time can be saved. The result is easy to interpret and so on. Greatly improved the experimental efficiency.

Claims

A kit for detecting chronic sinusitis with nasal polyp subtype, wherein the kit includes a specific primer for ALOX15 gene.
The kit for detecting chronic sinusitis with nasal polyp subtype according to claim 1, wherein the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3 shown.
The kit for detecting chronic sinusitis with nasal polyp subtypes according to any one of claims 1 or 2, wherein the kit further comprises specific primers for internal reference genes.
The kit for detecting chronic sinusitis with nasal polyp subtype according to claim 3, wherein the internal reference gene is GAPDH, and the upstream primer of GAPDH is shown in SEQ ID NO: 4, and the GAPDH The downstream primers are shown in SEQ ID NO: 5.
The kit for detecting chronic sinusitis with nasal polyps subtype according to any one of the preceding claims, wherein the kit further comprises: a reagent for extracting RNA from nasal polyp tissue or detached cells from nasal mucosa Reagent for reverse transcription of total RNA into cDNA; Reagent for performing real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction.
The kit for detecting chronic sinusitis with nasal polyp subtype according to claim 5, wherein,

The reagent for reverse transcription of total RNA into cDNA includes: a reverse transcription mixed solution and de-RNase and de-DNase water;

The reagents for real-time fluorescence quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction include: PCR premix, double distilled water, machine fluorescence compensation and correction agent, upstream primer of ALOX15 gene, and ALOX15 gene. Downstream primer, upstream primer of internal reference gene and downstream primer of internal reference gene.
Use of ALOX15 gene as a biomarker for detecting chronic sinusitis with nasal polyp subtypes.
Use of a kit according to any one of claims 1-6 for detecting chronic sinusitis with nasal polyp subtypes.
Use of the reagent for detecting ALOX15 for detecting chronic sinusitis with nasal polyp subtype.
The use according to claim 9, wherein the reagent for detecting ALOX15 includes a specific primer for ALOX15 gene, for example, an upstream primer of the specific primer for ALOX15 gene is shown in SEQ ID NO: 2 and the ALOX15 gene The downstream primers are shown in SEQ ID NO: 3.
The use according to claim 9, wherein the reagent for detecting ALOX15 is selected from the group consisting of a primer, an antibody, an aptamer, a probe, or a combination thereof for ALOX15.
A method for detecting the expression of ALOX15 gene in exfoliated nasal cells, comprising the following steps:

Extract RNA from exfoliated nasal cells,

Reverse transcription of total RNA into cDNA,

Quantitative polymerase chain reaction was used to amplify the ALOX15 gene and the reference gene in the cDNA using the specific primers of the ALOX15 gene and the specific primers of the reference gene for real-time quantitative PCR amplification, and

The expression of ALOX15 gene was calculated based on the detection results of the amplified products.
The method for detecting the expression level of ALOX15 gene in exfoliated nasal cells according to claim 12, wherein:

The upstream primer of ALOX15 gene is shown in SEQ ID NO: 2, and the downstream primer of ALOX15 gene is shown in SEQ ID NO: 3;

The internal reference gene is GAPDH, the upstream primer of the internal reference gene is shown in SEQ ID NO: 4, and the downstream primer is shown in SEQ ID NO: 5.
The method for detecting the expression level of ALOX15 gene in nasal exfoliated cells according to claim 12 or 13, wherein the nasal exfoliated cells are obtained by using a hair brush on the surface of the nasal polyp, and the brush after obtaining the nasal exfoliated cells is placed in the cell The lysate is stored below 4 ° C.
A method for detecting a chronic sinusitis subtype with nasal polyps, comprising detecting the expression level of ALOX15 gene in nasal exfoliated cells by the method according to any one of claims 12-14.
A method for detecting a chronic sinusitis subtype with nasal polyps, comprising detecting and detecting the expression level of ALOX15 gene in nasal cavity exfoliated cells, for example, using the kit according to any one of claims 1-6 to detect the cavity exfoliated cells Expression of ALOX15 gene.
The kit according to any one of claims 1-6, the use according to claim 7, the use according to claim 8, the use according to any of claims 9-11, and the claim 15 The method of claim 16 or claim 16, wherein the expression level of ALOX15 gene in nasal exfoliated cells is detected by fluorescent PCR.
The kit, use or method according to claim 17, wherein the chronic sinusitis with nasal polyp subtype is determined according to △ Ct (Ct (ALOX15) -Ct (GAPDH)), and Ct (ALOX15) is the Ct value of ALOX15 gene , Ct (GAPDH) is the Ct value of the internal reference gene GAPDH.
The kit, use or method according to claim 18, wherein △ Ct is greater than or equal to 1.675 representing non-eosinophilic chronic sinusitis with nasal polyps, and △ Ct is less than 1.675 representing eosinophilic chronic Sinusitis with nasal polyps.
The kit according to any one of claims 1-6, the use according to claim 7, the use according to claim 8, the use according to any of claims 9-11, and the claim 15 The method of claim 16 or claim 16, wherein the chronic sinusitis with nasal polyp subtype is non-eosinophilic chronic sinusitis with nasal polyps or eosinophilic chronic sinusitis with nasal polyps.