Gene diagnostic biomarkers and kits for diagnosing refractory gastroesophageal reflux disease

Abstract

This invention relates to a gene diagnostic biomarker and kit for diagnosing refractory gastroesophageal reflux disease (GERD), belonging to the field of biomedical technology. The invention provides the application of a biomarker, NRF1 and / or NRF2, in the preparation of a diagnostic reagent for refractory GERD; a diagnostic reagent for diagnosing GERD, comprising reagents for detecting NRF1 and / or NRF2 expression; a diagnostic kit for diagnosing GERD, comprising reagents for detecting NRF1 and / or NRF2 expression; and the application of the reagent for detecting NRF1 and / or NRF2 expression in the preparation of a diagnostic reagent for refractory GERD, comprising primers for detecting NRF1 and / or NRF2 expression. This invention provides a reliable tool for the rapid and accurate diagnosis of refractory GERD.

Description

Technical Field

[0001] This invention relates to a gene diagnostic biomarker and kit for diagnosing refractory gastroesophageal reflux disease, belonging to the field of biomedical technology. Background Technology

[0002] Gastroesophageal reflux disease (GERD) is a group of diseases characterized by heartburn and / or acid reflux, caused by the reflux of stomach and duodenal contents into the esophagus. Globally, approximately 13.3% of the population experiences heartburn or acid reflux at least once a week, with the figure for Asian populations around 10%, only slightly lower than the average. Based on direct endoscopic observation and pathological interpretation of biopsied mucosal tissue, GERD can be mainly divided into three types: non-erosive reflux disease (NERD), erosive esophagitis (EE), and Barrett's esophagus (BE). These different subtypes of GERD are widely considered a spectrum of diseases, with NERD patients potentially progressing to EE over time and, after certain external stimuli or gene mutations, developing into BE. The occurrence of BE indicates that the patient has entered a cascade of tumor development, and some patients will inevitably end up with esophageal adenocarcinoma. Currently, there is no clearly effective method to prevent esophageal reflux disease (BE) from progressing to esophageal adenocarcinoma. Therefore, preventing esophageal mucosal damage and intestinal metaplasia in esophageal reflux disease (EE) patients is a crucial part of the tertiary prevention of cancer and has extremely high research value. At present, clinical treatment for GERD still mainly relies on proton pump inhibitors (PPIs). Most patients experience significant symptom relief and esophageal mucosal lesions can gradually lessen or even return to normal after regular PPI acid suppression therapy and follow-up. However, multiple studies have reported that approximately 30% of GERD patients are not sensitive to PPI treatment, and even after at least 8 weeks of double-dose PPI therapy, they do not experience symptom relief, and their esophageal mucosal lesions may even worsen. Therefore, research on refractory GERD has significant clinical and translational value.

[0003] Nuclear factor-erythroid 2-related factor 2 (NRF2) is an important antioxidant transcription factor that regulates the expression of hundreds of antioxidant genes and plays a central role in defending against toxicity and oxidative damage. Normally, when cells are stimulated by oxidative stress, the reactive oxygen species (ROS) response pathway is activated under the control of an inducible antioxidant program, which is regulated by NRF2 and its repressor protein Keap1, and performs antioxidant stress functions through downstream functional molecules such as HO-1 and NQO1. The NRF1 transcription factor activates the expression of several key metabolic genes that regulate cell growth and nuclear genes required for mitochondrial respiration, as well as mitochondrial DNA transcription and replication. Together with NRF2, NRF1 directly regulates the expression of several nuclear-encoded ECT proteins and indirectly regulates the biosynthetic coordination between the nuclear and mitochondrial genomes by activating MTTFA, MTTFB1, and MTTFB2. While NRF1 and NRF2 play crucial roles in many types of diseases, their role in gastroesophageal reflux disease (GERD) has been largely studied.

[0004] In the early stages of this study, mRNA biomarkers in reflux esophagitis tissue were analyzed using Gene Ontology (GO), Kyoto Gene, and the KEGG Encyclopedia. The analysis revealed that NRF1 and NRF2 showed a high expression trend in esophagitis tissue compared to normal esophageal mucosa. Based on this, the expression of NRF1 and NRF2 in the esophageal mucosa of patients with gastroesophageal reflux disease (GERD) was investigated. Esophageal mucosa samples from 20 clinically followed patients with refractory GERD and 20 with non-refractory GERD were examined. The results showed that NRF1 and NRF2 were significantly underexpressed in the refractory GERD mucosa, and their expression was negatively correlated with the expression level of tight junction proteins in the esophageal mucosa. These findings suggest that NRF1 and NRF2 could serve as independent factors for the early diagnosis of refractory GERD and could also be potential molecular targets for the treatment of refractory GERD. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problem of how to diagnose refractory gastroesophageal reflux disease at an early stage.

[0006] To achieve the objectives of this invention, the present invention provides the application of a biomarker in the preparation of a diagnostic reagent for diagnosing refractory gastroesophageal reflux disease, said biomarker comprising NRF1 and / or NRF2.

[0007] The present invention provides a diagnostic reagent for diagnosing refractory gastroesophageal reflux disease, the reagent comprising a reagent for detecting NRF1 and / or NRF2 expression.

[0008] The present invention provides a diagnostic kit for diagnosing refractory gastroesophageal reflux disease, the kit comprising reagents for detecting NRF1 and / or NRF2 expression.

[0009] The present invention provides the application of a reagent for detecting NRF1 and / or NRF2 expression in the preparation of a diagnostic agent for diagnosing refractory gastroesophageal reflux disease, the reagent comprising primers for detecting NRF1 and / or NRF2 expression.

[0010] Preferably, the primers include primer set one for detecting NRF1 expression and / or primer set two for detecting NRF2 expression; primer set one includes nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; primer set two includes nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0011] Preferably, the primers include primer set three for detecting the expression of the internal reference gene ACTB; the primer set three includes the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0012] Preferably, in the application, the ACTB gene is used as an internal reference gene, and based on RT-qPCR data, through 2 -ΔCt The expression levels of NRF1 and / or NRF2 genes in esophageal mucosa were analyzed, and the variance was calculated using GraphPad Prism7 software; where ΔCt = Ct value of NRF1 or NRF2 - Ct value of internal reference gene ACTB; the specimens were esophageal mucosa obtained during endoscopy of patients with gastroesophageal reflux disease.

[0013] Preferably, in the application, when NRF1 2 is detected in the sample to be tested... -ΔCt A value less than or equal to 0.015 indicates a higher risk of developing refractory gastroesophageal reflux disease. When NRF1 is detected in the sample, a value of 2... -ΔCt A value greater than 0.015 indicates a low likelihood of having refractory gastroesophageal reflux disease.

[0014] Preferably, in the application, when NRF2 in the sample to be tested is detected, -ΔCt A value less than or equal to 0.43 indicates a higher risk of developing refractory gastroesophageal reflux disease. When NRF2 is detected in the sample, a value of 2... -ΔCt A value greater than 0.43 indicates a low likelihood of having refractory gastroesophageal reflux disease.

[0015] Preferably, in the application, when NRF1 2 is detected in the sample to be tested... -ΔCt Values ​​less than or equal to 0.015 or NRF2 of 2 -ΔCtA value less than or equal to 0.43, including cases where both values ​​are simultaneously less than the corresponding cutoff values, indicates a higher risk of developing refractory gastroesophageal reflux disease; when NRF1 is detected in the test plasma at a level of 2... -ΔCt Values ​​greater than 0.015 and NRF2 of 2 -ΔCt A value greater than 0.43 indicates a low likelihood of having refractory gastroesophageal reflux disease.

[0016] This invention provides a PCR primer set for diagnosing refractory gastroesophageal reflux disease based on RT-qPCR, including forward and reverse PCR primers for NRF1, NRF2, and ACTB genes, as well as other reagents for detecting the expression levels of NRF1, NRF2, and ACTB genes; the reagents are the only key components of the kit for predicting the risk of refractory gastroesophageal reflux disease.

[0017] The complete kit can be configured in one of the following three ways:

[0018] Combination 1: Forward and reverse PCR primers for NRF1 and ACTB genes, and other reagents for detecting the expression levels of NRF1 and ACTB genes.

[0019] Combination 2: Forward and reverse PCR primers for NRF2 and ACTB genes, and other reagents for detecting the expression levels of NRF2 and ACTB genes.

[0020] Combination 3: Forward and reverse PCR primers for NRF1, NRF2, and ACTB genes, and other reagents for detecting the expression levels of NRF1, NRF2, and ACTB genes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) This invention addresses the diagnosis of refractory gastroesophageal reflux disease (GERD). For the first time, we independently researched, designed, and optimized PCR primers for the NRF1 and NRF2 genes. The PCR primer combination (PCR primers for the NRF1, NRF2, and ACTB genes) was used to prepare a diagnostic kit for refractory GERD. The kit exhibits high amplification specificity and good amplification efficiency, providing a reliable tool for the rapid and accurate diagnosis of refractory GERD.

[0023] 2) This invention is the first to use RT-qPCR technology to detect the expression levels of NRF1 and / or NRF2 genes in the esophageal mucosa of subjects, analyze their diagnostic efficacy in patients with refractory gastroesophageal reflux disease (GERD), and design a novel diagnostic reagent for GERD. Previous literature reports that refractory GERD accounts for approximately 30% of all GERD patients, and is often diagnosed after poor response to PPI treatment. Clinically, there is a lack of methods and biomarkers for early diagnosis. Therefore, compared with traditional diagnostic methods, this invention is more timely, specific, and sensitive, enabling early diagnosis of refractory GERD.

[0024] 3) When this invention determines whether a subject has refractory gastroesophageal reflux disease (GERD) or is at risk of developing GERD by detecting the expression level of the NRF1 gene in the esophageal mucosa, the optimal cutoff value for NRF1 is 0.015, at which point the sensitivity is 65% and the specificity is 95%. When this invention determines whether a subject has refractory GERD or is at risk of developing GERD by detecting the expression level of the NRF2 gene in the esophageal mucosa, the optimal cutoff value for NRF2 is 0.43, at which point the sensitivity is 70% and the specificity is 90%. A kit that combines the detection of the expression levels of NRF1 and NRF2 genes in the esophageal mucosa to determine the risk of developing GERD has a specificity of 95% and a sensitivity of 75% when used in combination, and has high clinical application value. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the verification of the correlation between NRF1 and NRF2 gene expression levels and refractory gastroesophageal reflux disease in this invention.

[0026] Figure 2 This is a ROC curve diagram of an embodiment of the present invention;

[0027] Figure A shows the ROC curve of NRF1, with an AUC value of 0.795 and a cut-off value of 0.015; Figure B shows the ROC curve of NRF2, with an AUC value of 0.813 and a cut-off value of 0.43; Figure C shows the ROC curve of NRF1+NRF2, with an AUC value of 0.885. Detailed Implementation

[0028] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0029] This invention provides the use of a biomarker in the preparation of a diagnostic reagent for diagnosing refractory gastroesophageal reflux disease, the biomarker comprising NRF1 and / or NRF2.

[0030] The present invention provides a diagnostic reagent for diagnosing refractory gastroesophageal reflux disease, the reagent comprising a reagent for detecting NRF1 and / or NRF2 expression.

[0031] The present invention provides a diagnostic kit for diagnosing refractory gastroesophageal reflux disease, the kit comprising reagents for detecting NRF1 and / or NRF2 expression.

[0032] The present invention provides the application of a reagent for detecting NRF1 and / or NRF2 expression in the preparation of a diagnostic agent for diagnosing refractory gastroesophageal reflux disease, the reagent comprising primers for detecting NRF1 and / or NRF2 expression.

[0033] The primers described above include primer set one for detecting NRF1 expression and / or primer set two for detecting NRF2 expression; primer set one includes nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; primer set two includes nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0034] The primers described above include primer set three for detecting the expression of the internal reference gene ACTB; primer set three includes the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0035] In the above applications, the ACTB gene was used as an internal reference gene. Based on RT-qPCR data, the results were obtained through 2... -ΔCt The expression levels of NRF1 and / or NRF2 genes in esophageal mucosa were analyzed, and the variance was calculated using GraphPad Prism 7 software; where ΔCt = Ct value of NRF1 or NRF2 - Ct value of internal reference gene ACTB; the test specimens were esophageal mucosa tissues obtained during endoscopy of patients with gastroesophageal reflux disease.

[0036] In the above application, when NRF1 2 is detected in the sample to be tested -ΔCt A value less than or equal to 0.015 indicates a higher risk of developing refractory gastroesophageal reflux disease. When NRF1 is detected in the sample, a value of 2... -ΔCt A value greater than 0.015 indicates a low likelihood of having refractory gastroesophageal reflux disease.

[0037] In the above applications, when NRF2 is detected in the sample to be tested, 2 -ΔCt A value less than or equal to 0.43 indicates a higher risk of developing refractory gastroesophageal reflux disease. When NRF2 is detected in the sample, a value of 2... -ΔCt A value greater than 0.43 indicates a low likelihood of having refractory gastroesophageal reflux disease.

[0038] In the above application, when NRF1 2 is detected in the sample to be tested -ΔCt Values ​​less than or equal to 0.015 or NRF2 of 2 -ΔCt A value less than or equal to 0.43, including cases where both values ​​are simultaneously less than the corresponding cutoff values, indicates a higher risk of developing refractory gastroesophageal reflux disease; when NRF1 is detected in the test plasma at a level of 2... -ΔCt Values ​​greater than 0.015 and NRF2 of 2 -ΔCt A value greater than 0.43 indicates a low likelihood of having refractory gastroesophageal reflux disease.

[0039] Example

[0040] I. Sample Collection:

[0041] Refractory Gastroesophageal Reflux Disease (RGERD) group: Esophageal mucosal tissues were collected from 20 patients clinically diagnosed with refractory gastroesophageal reflux disease at the Digestive Endoscopy Center of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine in 2020-2021. Among them, there were 11 males and 9 females, with an average age of 47.1±11.4 years.

[0042] Non-refractory gastroesophageal reflux disease (GERD) group: Esophageal mucosal tissues of 20 patients with non-refractory GERD clinically diagnosed with GERD at the Digestive Endoscopy Center of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in 2020-2021. Among them, there were 10 males and 10 females, with an average age of 46.7±10.6 years.

[0043] There were no statistically significant differences in gender and age between the refractory gastroesophageal reflux disease (RGERD) group and the non-refractory gastroesophageal reflux disease (GERD) group.

[0044] II. Design and Synthesis of PCR Primer Combinations for Diagnosing Refractory Gastroesophageal Reflux Disease

[0045] RT-qPCR amplification primers were designed based on the coding sequences of the human NRF1 and human NRF2 genes published in NIH-Genbank. After multiple adjustments and validations, the final primer sequences are as follows:

[0046] NRF1 gene:

[0047] Forward primer NRF1-F: 5'-GGCAACAGTAGCCACATTGGCT-3' (SEQ ID NO.1);

[0048] Reverse primer NRF1-R: 5'-GTCGTCTGGATGGTCATCTCAC-3' (SEQ ID NO.2);

[0049] NRF2 gene:

[0050] Forward primer NRF2-F: 5'-CACATCCAGTCAGAAACCAGTGG-3' (SEQ ID NO.3);

[0051] Reverse primer NRF2-R: 5'-GGAATGTCTGCGCCAAAAGCTG-3' (SEQ ID NO.4);

[0052] The primer sequences for the internal reference gene ACTB are as follows:

[0053] Forward primer ACTB-F: 5'-CACCATTGGCAATGAGCGGTTC-3' (SEQ ID NO.5);

[0054] Reverse primer ACTB-R: 5'-AGGTCTTTGCGGATGTCCACGT-3' (SEQ ID NO.6);

[0055] III. Correlation between NRF1 and NRF2 gene expression in esophageal mucosa and refractory gastroesophageal reflux disease:

[0056] Step 1: Processing of esophageal mucosal tissue samples;

[0057] Esophageal mucosal tissue samples were extracted from endoscopic biopsy specimens in patients with refractory gastroesophageal reflux disease (RGERD) and those with non-refractory gastroesophageal reflux disease (GERD). Samples were taken from undamaged esophageal mucosa at least 5 cm proximal to the squamocolumnar junction. The tissue samples were immediately immersed in RNAlater for preservation after extraction.

[0058] Step 2: Extraction of total RNA from esophageal mucosal tissues of patients with refractory gastroesophageal reflux disease (RGERD) and those with non-refractory gastroesophageal reflux disease (GERD):

[0059] Total RNA was extracted from the esophageal mucosal tissue samples obtained in step 1 using Trizol RNA extraction reagent (purchased from Thermo Fisher Scientific). The experimental procedures were performed according to the product instructions, as follows:

[0060] 2.1) Transfer the esophageal mucosal biopsy specimen from the RNA Later solution to a 1.5 mL sterile enzyme-free EP tube pre-filled with 1 mL Trizol.

[0061] 2.2) Use a tissue homogenizer to homogenize the tissue for 1 minute at a frequency of 70 Hz, and let it stand at room temperature for 10 minutes.

[0062] 2.3) Take 200 μL of chloroform per tube, slowly and evenly add it to each sample, shake vigorously for 30 seconds, and let stand at room temperature for 3 minutes.

[0063] 2.4) Pre-cool the centrifuge to 4°C and centrifuge for 15 minutes (12,000 RPM). After centrifugation, the liquid in the centrifuge tube was clearly separated into 3 layers.

[0064] 2.5) Transfer the upper aqueous phase to a sterile enzyme-free EP tube, record the specific volume of the liquid (approximately 300-400 μL), add isopropanol to make up the liquid to a total volume of 1000 μL, invert and mix the sample, and let it stand at room temperature for 10 minutes.

[0065] 2.6) Pre-cool the centrifuge to 4°C and centrifuge for 15 minutes (12,000 rpm). After centrifugation, a white precipitate will be visible at the bottom of the EP tube; remove any excess liquid.

[0066] 2.7) Add 1000 μL of 75% ethanol to each sample, allowing the white precipitate to float, and shake gently to mix.

[0067] 2.8) Pre-cool the centrifuge to 4°C and centrifuge for 5 minutes (7500 rpm). Remove the supernatant and place in a well-ventilated area at room temperature for 10 minutes to allow the surface ethanol to evaporate.

[0068] 2.9) Take 10 μL of DEPC water to dissolve the white precipitate in the tube, shake to mix, and centrifuge at low speed until all the liquid in the tube reaches the bottom. The solution can then be used to detect RNA concentration. After that, store the solution in a -80℃ freezer.

[0069] 2.10) After extracting total RNA, the RNA concentration and purity were measured. The sample was placed on ice to prevent degradation. Using a DeNovis micro-volume spectrophotometer, 1 μL of RNA sample was taken from the EP tube containing the RNA sample and dropped onto the spectrophotometer's detection position. The OD 260 / 280 value was measured. Ideally, the OD 260 / 280 value of the RNA sample should be between 1.8 and 2.0. Values ​​below 1.8 or above 2.0 are considered unacceptable.

[0070] Step 3: Reverse transcription to synthesize cDNA;

[0071] Place all reagents and qualified RNA samples on ice. The Ⅲ1st Strand cDNASynthesis SuperMix reverse transcription kit is used to remove residual genomic DNA. cDNA is obtained by reverse transcription (20 μL system) and stored at -20°C.

[0072] 3.1) Residual genomic DNA removal:

[0073] Prepare the following mixture in an enzyme-free centrifuge tube, mix well, and incubate for 120 seconds at 42°C.

[0074] Element Dosage DEPC water To 15μL 5×gDNA digester mixture 3μL Total RNA 1μg

[0075] 3.2) Preparation of reverse transcription reaction system (20 μL system):

[0076] Add directly to the sample after DNA removal After mixing with SuperMix Plus, gently blow and whisk until well combined.

[0077]

[0078] 3.3) Reverse transcription procedure

[0079] temperature time 25℃ 5 minutes 55℃ 15 minutes 85℃ 5 minutes

[0080] Step 4: RT-qPCR

[0081] use qRT-PCR was performed using the SYBR Green Master Mix qPCR kit. First, the premix in the kit was thoroughly mixed. A 20 μL reaction mixture was prepared (10 μL SYBR Green Master premix, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.4 μL 50×High ROX, 1 μL cDNA, and 7.8 μL sterile distilled water). Three wells were designed for each sample for the experiment, with ACTB as an internal control. The 22 -ΔΔCt The value represents the relative expression level of the target gene. The reaction is carried out under the following conditions:

[0082]

[0083]

[0084] The denaturation-annealing process is repeated 40 times.

[0085] Step 5: Agarose gel electrophoresis was used to verify the specificity of the NRF1 and NRF2 gene primers;

[0086] Gel preparation: Prepare TAE buffer; dissolve 1g agarose in 60ml IX TAE buffer, mix thoroughly, and heat to boiling. After cooling, add 6ug GRgreen II nucleic acid dye. Mix the above liquids and pour into the electrophoresis tank to allow gelation.

[0087] Sample preparation: Add 2 μL of 10X Loading buffer to the DNA obtained after RT-qPCR reaction and mix well;

[0088] Electrophoresis detection: Add 5 μL of DNA marker to the first well of the agarose gel, and add the mixed sample to the remaining wells in sequence. Then perform electrophoresis at 110V for 15 minutes and observe the electrophoresis results on a UV spectrometer.

[0089] Step 6: Experimental Results and Analysis:

[0090] The RT-qPCR amplification curves of the refractory gastroesophageal reflux disease (RGERD) group and the non-refractory gastroesophageal reflux disease (GERD) group showed clear inflection points and good overall parallelism, with flat baselines and no upward slope, indicating similar amplification efficiencies in each reaction tube. The large slope of the exponential phase of the curves indicated high amplification efficiency. The melting curves of the amplified products were all single-peaked, indicating that only one amplification product was produced, representing specific amplification. Based on the RT-qPCR data, through 2... -ΔΔCt The expression levels of NRF1 and NRF2 genes in the esophageal mucosa tissues of the two groups were analyzed, and the variance was calculated using GraphPad Prism 7 software. In both the refractory gastroesophageal reflux disease (RGERD) group and the non-refractory GERD group, the internal reference gene was ACTB. For the refractory GERD group, ΔCt = Ct value of the refractory GERD group - Ct value of the internal reference gene ACTB; for the non-refractory GERD group, ΔCt = Ct value of the non-refractory GERD group - Ct value of the internal reference gene ACTB; -ΔCt = -(refractory GERD group - non-refractory GERD group ΔCt). Substituting the -ΔCt value into the 2... -ΔΔCt If the expression level of NRF1 or NRF2 in the GERD group is set to 1, then the resulting 2 -ΔΔCt The values ​​represent the relative expression levels of NRF1 or NRF2 in the RGERD group. Then, ROC curves are plotted using R language (RStudio) to calculate the cut-off values ​​for NRF1 and NRF2 (for optimal specificity and sensitivity). -ΔCt The results showed that the expression levels of NRF1 and NRF2 genes in the esophageal mucosa tissue of the refractory gastroesophageal reflux disease (GERD) group were significantly lower than those of the non-refractory GERD group, and *P<0.05. Figure 2 In esophageal mucosal tissue samples from patients with refractory gastroesophageal reflux disease (RGERD), the cutoff value of NRF1 was 0.015, and the AUC value was 0.795; the cutoff value of NRF2 was 0.43, and the AUC value was 0.813; the combined detection of NRF1 and NRF2 had a sensitivity of 75%, a specificity of 95%, and an AUC value of 0.885.

[0091] Applications of this invention:

[0092] How to use:

[0093] 1) The sample to be tested was taken from an undamaged esophageal mucosa at least 5 cm proximal to the squamocolumnar junction. The tissue sample was immediately immersed in RNAlater for preservation after extraction.

[0094] 2) Extraction of total RNA from the esophageal mucosal tissue to be tested:

[0095] Total RNA was extracted from the esophageal mucosal tissue samples obtained in step 1) using Trizol RNA extraction reagent (purchased from Thermo Fisher Scientific). The experimental procedures were performed according to the product instructions, as follows:

[0096] 2.1) Transfer the esophageal mucosal biopsy specimen from the RNA Later solution to a 1.5 mL sterile enzyme-free EP tube pre-filled with 1 mL Trizol.

[0097] 2.2) Use a tissue homogenizer to homogenize the tissue for 1 minute at a frequency of 70 Hz, and let it stand at room temperature for 10 minutes.

[0098] 2.3) Take 200 μL of chloroform per tube, slowly and evenly add it to each sample, shake vigorously for 30 seconds, and let stand at room temperature for 3 minutes.

[0099] 2.4) Pre-cool the centrifuge to 4°C and centrifuge for 15 minutes (12,000 RPM). After centrifugation, the liquid in the centrifuge tube was clearly separated into 3 layers.

[0100] 2.5) Transfer the upper aqueous phase to a sterile enzyme-free EP tube, record the specific volume of the liquid (approximately 300-400 μL), add isopropanol to make up the liquid to a total volume of 1000 μL, invert and mix the sample, and let it stand at room temperature for 10 minutes.

[0101] 2.6) Pre-cool the centrifuge to 4°C and centrifuge for 15 minutes (12,000 rpm). After centrifugation, a white precipitate will be visible at the bottom of the EP tube; remove any excess liquid.

[0102] 2.7) Add 1000 μL of 75% ethanol to each sample, allowing the white precipitate to float, and shake gently to mix.

[0103] 2.8) Pre-cool the centrifuge to 4°C and centrifuge for 5 minutes (7500 rpm). Remove the supernatant and place in a well-ventilated area at room temperature for 10 minutes to allow the surface ethanol to evaporate.

[0104] 2.9) Take 10 μL of DEPC water to dissolve the white precipitate in the tube, shake to mix, and centrifuge at low speed until all the liquid in the tube reaches the bottom. The solution can then be used to detect RNA concentration. After that, store the solution in a -80℃ freezer.

[0105] 2.10) After extracting total RNA, the RNA concentration and purity were measured. The sample was placed on ice to prevent degradation. Using a DeNovis micro-volume spectrophotometer, 1 μL of RNA sample was taken from the EP tube containing the RNA sample and dropped onto the spectrophotometer's detection position. The OD 260 / 280 value was measured. Ideally, the OD 260 / 280 value of the RNA sample should be between 1.8 and 2.0. Values ​​below 1.8 or above 2.0 are considered unacceptable.

[0106] 3) Reverse transcription to synthesize cDNA

[0107] Place all reagents and qualified RNA samples on ice. The Ⅲ1st Strand cDNASynthesis SuperMix reverse transcription kit is used to remove residual genomic DNA. cDNA is obtained by reverse transcription (20 μL system) and stored at -20°C.

[0108] 3.1) Residual genomic DNA removal:

[0109] Prepare the following mixture in an enzyme-free centrifuge tube, mix well, and incubate for 120 seconds at 42°C.

[0110] Element Dosage DEPC water To 15μL 5×gDNA digester mixture 3μL Total RNA 1μg

[0111] 3.2) Preparation of reverse transcription reaction system (20 μL system):

[0112] Add directly to the sample after DNA removal After mixing with SuperMix Plus, gently blow and whisk until well combined.

[0113]

[0114] 3.3) Reverse transcription procedure

[0115] temperature time 25℃ 5 minutes 55℃ 15 minutes 85℃ 5 minutes

[0116] 4) RT-qPCR

[0117] use qRT-PCR was performed using the SYBR Green Master Mix qPCR kit. First, the premix in the kit was thoroughly mixed. A 20 μL reaction mixture was prepared (10 μL SYBR Green Master premix, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.4 μL 50×High ROX, 1 μL cDNA, and 7.8 μL sterile distilled water). Three wells were designed for each sample for the experiment, with ACTB as an internal control. The 22 -ΔΔCt The value represents the relative expression level of the target gene. The reaction is carried out under the following conditions:

[0118] step temperature time Pre-variation 95℃ 300 seconds transsexual 95℃ 10 seconds Annealing / Extension 60℃ 30 seconds Melting curve stage default default

[0119] 40 cycles of denaturation-annealing

[0120] 5) Agarose gel electrophoresis was used to verify the specificity of the NRF1 and NRF2 gene primers;

[0121] Gel preparation: Prepare TAE buffer; dissolve 1g agarose in 60ml IX TAE buffer, mix thoroughly, and heat to boiling. After cooling, add 6ug GRgreen II nucleic acid dye. Mix the above liquids and pour into the electrophoresis tank to allow gelation.

[0122] Sample preparation: Add 2 μL of 10X Loading buffer to the DNA obtained after RT-qPCR reaction and mix well;

[0123] Electrophoresis detection: Add 5 μL of DNA marker to the first well of the agarose gel, and add the mixed sample to the remaining wells in sequence. Then perform electrophoresis at 110V for 15 minutes and observe the electrophoresis results on a UV spectrometer.

[0124] 6) Experimental Results and Analysis:

[0125] When using only the combination of NRF1 and ACTB

[0126] <![CDATA[2 of NRF1 -ΔCt value]]> Risk of developing refractory gastroesophageal reflux disease ≤0.015 high ＞0.015 Low

[0127] When using only the combination of NRF2 and ACTB

[0128]

[0129]

[0130] When using a combination of NRF1, NRF2, and ACTB

[0131] <![CDATA[2 of NRF1 -ΔCt value]]> <![CDATA[The value of NRF2 -ΔCt > Risk of developing refractory gastroesophageal reflux disease ≤0.015 ≤0.43 high ≤0.015 ＞0.43 high ＞0.015 ≤0.43 high ＞0.015 ＞0.43 Low

[0132] like Figure 1 The diagram shown is a flowchart for verifying the correlation between NRF1 and NRF2 gene expression levels and refractory gastroesophageal reflux disease in this invention.

[0133] like Figure 2 The figure shown is a ROC curve diagram of an embodiment of the present invention;

[0134] Figure A shows the ROC curve of NRF1, with an AUC value of 0.795 and a cut-off value of 0.015; Figure B shows the ROC curve of NRF2, with an AUC value of 0.813 and a cut-off value of 0.43; Figure C shows the ROC curve of NRF1+NRF2, with an AUC value of 0.885.

[0135] When this invention determines whether a subject has refractory gastroesophageal reflux disease (GERD) or is at risk of developing GERD by detecting the expression level of the NRF1 gene in the esophageal mucosa tissue, the optimal cutoff value for NRF1 is 0.015, at which point the sensitivity is 65% and the specificity is 95%.

[0136] This invention determines whether a subject has refractory gastroesophageal reflux disease (GERD) or is at risk of developing GERD by detecting the expression level of the NRF2 gene in the esophageal mucosa tissue of the subject. The optimal cutoff value for NRF2 is 0.43, at which point the sensitivity is 70% and the specificity is 90%.

[0137] A kit that uses combined detection of the expression levels of NRF1 and NRF2 genes in the esophageal mucosa of subjects to determine whether subjects are at risk of having refractory gastroesophageal reflux disease. The specificity of the combined diagnosis is 95% and the sensitivity is 75%, which has high clinical application value.

[0138] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The use of a reagent for detecting NRF1 and / or NRF2 expression in the preparation of a diagnostic agent for diagnosing refractory gastroesophageal reflux disease, characterized in that, The reagents include primers for detecting NRF1 and / or NRF2 expression.

2. The application according to claim 1, characterized in that, The primers include primer set one for detecting NRF1 expression and / or primer set two for detecting NRF2 expression; primer set one includes nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; primer set two includes nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO:

4.

3. The application according to claim 2, characterized in that, The primers include primer set three for detecting the expression of the internal reference gene ACTB; primer set three includes the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO:

6.

4. The application according to claim 3, characterized in that, In this application, the ACTB gene is used as an internal reference gene, and based on RT-qPCR data, it is determined through 2... -ΔCt The expression levels of NRF1 and / or NRF2 genes in esophageal mucosa were analyzed, and the variance was calculated using GraphPad Prism 7 software; where ΔCt = Ct value of NRF1 or NRF2 - Ct value of internal reference gene ACTB; the specimens were esophageal mucosa obtained during endoscopy of patients with gastroesophageal reflux disease.

5. The application according to claim 4, characterized in that, In the aforementioned application, when NRF1 2 is detected in the sample to be tested... -ΔCt A value less than or equal to 0.015 indicates a high risk of developing refractory gastroesophageal reflux disease. When NRF1 is detected in the sample, a value of 2... -ΔCt A value greater than 0.015 indicates a low risk of refractory gastroesophageal reflux disease.

6. The application according to claim 4, characterized in that, In the aforementioned application, when NRF2 is detected in the sample to be tested, 2 -ΔCt A value less than or equal to 0.43 indicates a high risk of developing refractory gastroesophageal reflux disease. When NRF2 is detected in the sample, a value of 2... -ΔCt A value greater than 0.43 indicates a low risk of refractory gastroesophageal reflux disease.

7. The application according to claim 4, characterized in that, In the aforementioned application, when NRF1 2 is detected in the sample to be tested... -ΔCt Values ​​less than or equal to 0.015 or NRF2 of 2 -ΔCt A value less than or equal to 0.43, including cases where both values ​​are less than the corresponding cutoff values, indicates a high risk of developing refractory gastroesophageal reflux disease; when NRF1 is detected in the test plasma at a level of 2... -ΔCt Values ​​greater than 0.015 and NRF2 of 2 -ΔCt A value greater than 0.43 indicates a low risk of refractory gastroesophageal reflux disease.

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