Marker, primer combination, kit and application for epilepsy diagnosis / detection

Abstract

The invention discloses a marker, a primer combination and a kit for epilepsy diagnosis / detection and application, and belongs to the technical field of minute biology. Wherein the marker is one or a combination of two of a QSOX1 gene and an ABCC1 gene. The primer sequences contained in the primer combination are as shown in SEQ ID NO.1-6. The invention finds that the mRNA expression level of QSOX1 and ABCC1 in peripheral blood lymphocytes is obviously higher than that of a normal group in epilepsy patients, and verifies that the change of the expression level can reflect epilepsy conditions. Based on the detection kit or the primer combination developed by the invention, the mRNA level change of the peripheral blood lymphocytes QSOX1 and ABCC1 is detected, so that the epilepsy illness condition can be reflected, and the detection kit or the primer combination can also be used for early warning of epilepsy diagnosis. The detection result is more accurate and reliable, and the stability and repeatability are good.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a marker, a primer combination, a kit and uses for epilepsy diagnosis / detection. Background Art

[0002] Epilepsy is a chronic brain disease characterized by recurrent epileptic seizures. It is caused by abnormal discharges of brain neurons, and the onset of the disease is characterized by repetitive and transient nature. The causes of epilepsy include muscle contraction, cerebral cortex developmental disorders, brain tumors, head trauma, central nervous system infection, etc., and may be related to genetics. The onset of epilepsy is not limited to any age group, and it is relatively common in children and the elderly. According to statistics, epilepsy affects more than 70 million people worldwide. Epilepsy can occur in people of all ages, but it is more common in children and the elderly. Among pregnant women, the proportion of epileptic seizures is 0.3% to 0.7%.

[0003] Epilepsy is one of the most common paroxysmal diseases of the central nervous system. It has a high incidence rate, and long-term recurrent seizures can easily turn into refractory epilepsy, which seriously affects the patient's quality of life. Correct diagnosis is the prerequisite for effective treatment. Mastering effective diagnostic skills not only helps to improve the success rate of epilepsy diagnosis, but also can provide patients with the best subsequent treatment options. At present, the diagnosis of epilepsy is mainly based on the manifestations at the time of onset, and there is still a lack of consistent biological markers. Therefore, a large number of patients can only be diagnosed after the onset of the disease. Therefore, exploring the pathogenesis of epilepsy, further searching for biomarkers for early diagnosis, and safe and effective intervention measures are important issues that need to be urgently addressed. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a marker, primer combination, kit and use for epilepsy diagnosis / detection, which can be used for epilepsy condition judgment and early warning diagnosis, and its detection results are more accurate, reliable, stable and repeatable.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] The object of the present invention is to provide a marker for epilepsy diagnosis / detection, which is one or a combination of two of the QSOX1 and ABCC1 genes.

[0007] Furthermore, high expression of QSOX1 or ABCC1 genes suggests that the patient is suspected of having epilepsy.

[0008] Furthermore, the simultaneous high expression of QSOX1 and ABCC1 genes indicated that the patient suffered from epilepsy.

[0009] Another object of the present invention is to provide a primer combination for amplifying the above-mentioned marker, which includes a primer pair for amplifying QSOX1 and ABCC1 genes; wherein the primer pair sequence for amplifying QSOX1 is shown in SEQ ID NOs. 1 and 2; the primer pair sequence for amplifying ABCC1 is shown in SEQ ID NOs. 3 and 4.

[0010] Furthermore, it also includes a primer pair with the internal reference gene h-actinb as the target fragment; the sequence of the primer pair is shown in SEQ ID NO.5 and 6.

[0011] The specific sequences of the primer combinations are as follows:

[0012] QSOX1-F: 5'-AGACCACAGTTGCACCAACCA-3'; (SEQ ID NO.1)

[0013] QSOX1-R: 5'-TGCAGATTCCAGGTCAGCCAT-3'; (SEQ ID NO. 2)

[0014] ABCC1-F: 5'-AACTGGCTGGTTCGGATGTCA-3'; (SEQ ID NO.3)

[0015] ABCC1-R: 5'-AGCTGTCTCCTGGATTTGCCA-3'; (SEQ ID NO.4)

[0016] h-actinb-F: 5'-TGGCACCCAGCACAATGAA-3'; (SEQ ID NO.5)

[0017] h-actinb-R: 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'. (SEQ ID NO.6)

[0018] Another object of the present invention is to provide a kit comprising the above primer combination.

[0019] Furthermore, the kit also includes peripheral blood lymphocyte mRNA extraction solution, diluent, cleaning solution, reverse transcription reaction solution, standard positive template and SYBR Green real-time fluorescence quantitative PCR reaction solution.

[0020] Another object of the present invention is to provide the use of the above-mentioned markers, primer combinations or kits in the preparation of products for risk prediction, screening, prognosis assessment, treatment effect monitoring or recurrence monitoring of epilepsy.

[0021] Another object of the present invention is to provide use of the above markers in constructing an epilepsy animal model.

[0022] Another object of the present invention is to provide a method for detecting the expression of the above markers using real-time fluorescence quantitative PCR, specifically:

[0023] The RNA of the sample to be tested is extracted, and then amplified using the above primer combination or kit to detect the mRNA expression levels of QSOX1 and ABCC1.

[0024] Furthermore, the real-time fluorescence quantitative PCR reaction solution is composed of 2×ChamQ Universal SYBR qPCR MasterMix, 10 μM sense primer and antisense primer, and RNase Free H2O.

[0025] Beneficial effects of the present invention:

[0026] The present invention confirms that changes in the mRNA expression levels of QSOX1 and ABCC1 in peripheral blood lymphocytes can reflect the condition of epilepsy. The mRNA expression of peripheral blood lymphocytes of normal people was further detected, and the results showed that the mRNA expression levels of QSOX1 and ABCC1 in epilepsy patients were significantly higher than those in the normal group. Therefore, based on the peripheral blood lymphocyte mRNA fluorescence real-time quantitative RT-PCR special detection kit or primer combination developed by the present invention, the changes in the mRNA levels of QSOX1 and ABCC1 in peripheral blood lymphocytes can not only reflect the condition of epilepsy, but also serve as an early warning for epilepsy diagnosis. The test results are more accurate, reliable, stable and reproducible.

[0027] The present invention can not only reflect the condition of epilepsy, but also serve as an early warning for epilepsy diagnosis. It can detect the possibility of the patient suffering from the disease before obvious epilepsy symptoms appear, and can cooperate with the hospital to intervene early to prevent the further development of epilepsy. Compared with the current diagnosis of epilepsy based on seizure phenomena and accompanying symptoms, the present invention is more scientific and can be widely used in hospitals. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the screening result of differentially expressed genes related to ferroptosis;

[0029] Figure 2 It is the screening result of SVM-REF and LASSO models;

[0030] Figure 3 This is the result of gene co-localization analysis;

[0031] Figure 4 The ROC curve was used to analyze the AUC results of QSOX1 and ABCC1 genes for diagnosing epilepsy. DETAILED DESCRIPTION

[0032] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0033] Example 1 Screening of differentially expressed genes

[0034] The abnormally expressed genes in epilepsy were screened using public databases (GEO database) and various bioinformatics methods. The specific steps and results are as follows:

[0035] (1) Using the peripheral blood lymphocyte mRNA expression data of epilepsy patients and normal controls from GEO data GSE20977, we first removed batch effects and normalized the data, extracted the expression matrix of ferroptosis-related genes (obtained from the ferroptosis-related database FerrDb V2 website), and used the limma package of R language to obtain 16 ferroptosis-related differentially expressed genes, such as Figure 1 shown.

[0036] (2) The 16 ferroptosis-related differentially expressed genes were analyzed using two machine model algorithms, SVM-REF and LASSO. The two algorithms screened out characteristic genes related to epilepsy respectively. Then, the characteristic genes screened out by the two algorithms were intersected to obtain 9 genes, such as Figure 2 shown.

[0037] (3) The co-localization analysis of the 9 genes obtained showed that QSOX1 and ABCC1 genes were significantly associated with epilepsy, such as Figure 3 As shown. The differentially expressed genes were plotted into ROC curves (see Figure 4 ),Depend on Figure 4 It can be seen that the areas under the curve (AUC) are 0.839 and 0.964, respectively, both greater than 0.65, which are statistically significant, indicating that the QSOX1 and ABCC1 genes screened by the present invention are significantly associated with epilepsy, and it is effective to use the expression levels of these two genes to predict epilepsy.

[0038] Example 2

[0039] 1. A total of 23 samples were collected, including 11 epilepsy patients, all diagnosed as epilepsy patients by clinical manifestations of epilepsy, and 12 normal samples. The sample sources and inclusion criteria are as follows:

[0040] (1) Selection criteria

[0041] Patients diagnosed with epilepsy.

[0042] (2) Exclusion criteria

[0043] ① Suffering from other mental illnesses (such as schizophrenia) and other neurodevelopmental disorders;

[0044] ② Suffering from genetic metabolic diseases;

[0045] ③ Suffering from severe neurological diseases, history of craniocerebral injury and other major physical diseases;

[0046] 2. Detection of QSOX1 and ABCC1 gene expression

[0047] (1) The total RNA of human peripheral blood lymphocytes from the above samples was extracted using RNA extraction reagent (Trizol), and the specific process was as follows:

[0048] Use a disposable anticoagulated vacuum tube for blood collection. The blood volume is generally 1-2mL and stored at room temperature. Take fresh anticoagulated whole blood and dilute the whole blood with an equal volume of diluent. Add lymphocyte separation solution (5mL) to a sterile plastic centrifuge tube. Slowly add the diluted blood sample to the lymphocyte separation solution, ensuring that the blood sample is located on the upper layer of the separation solution to prevent blood cells from spreading to the lower layer. Use a centrifuge and centrifuge at 2000rpm for 20min at room temperature. After centrifugation, there is a thin and dense white film between the plasma layer and the separation solution layer, which is the mononuclear cell (including lymphocytes and monocytes) layer. Use a pipette to carefully aspirate the white film layer into another centrifuge tube. Use a washing solution (5mL) to mix the aspirated cells by inverting. Centrifuge again at 1500rpm for 10min and discard the supernatant. Repeat washing 1-2 times, discard the supernatant, add 1mL Trizol and let stand on ice for 30 minutes to fully extract RNA. After standing on ice for 30 minutes, add 200μL chloroform, shake vigorously for 15s, stand at room temperature for 5 minutes, centrifuge at 12000rpm 4℃ for 10 minutes, take the supernatant to a new Ep tube, add an equal volume of isopropanol, mix gently, stand at room temperature for 10 minutes, centrifuge at 12000rpm 4℃ for 10 minutes, and remove the supernatant. Add 1mL 75% anhydrous ethanol (pre-cooled in advance), invert and mix, centrifuge at 12000rpm 4℃ for 10 minutes, and remove the supernatant. Repeat ethanol washing twice. Invert and dry. Preheat RNaseFreeH2O to 55℃, add 30μL to each tube to dissolve RNA, and measure the concentration.

[0049] (2) Using total RNA extracted from human peripheral blood lymphocytes, cDNA of each mRNA sample was obtained by reverse transcription;

[0050] Reverse transcription reaction solution configuration: 4×gDNA wiper Mix 4μL, RNA sample 1μg, RNase FreeH2O 7.2μL; 42℃, 2 minutes; 5×HiScriptШqRT SuperMix 4μL, 37℃, 15 minutes; 85℃, 5 seconds.

[0051] (3) SYBR Green reaction solution preparation: 2×ChamQ Universal SYBR qPCR MasterMix 10 μL, forward primer and antisense primer (10 μM) 0.4 μL each, cDNA template 2 μL, RNase Free H2O 7.2 μL, a total of 20 μL. The primer sequences are shown in Table 1.

[0052] Table 1 Primer sequences

[0053]

[0054]

[0055] (4) The reaction steps of the fluorescent real-time quantitative PCR system are as follows:

[0056] Stage 1: DNA polymerase activation and pre-denaturation, 95°C, 30s, 1 cycle;

[0057] Stage 2: cyclic reaction, 95℃ for 5s, 60℃ annealing for 34s, 40 cycles;

[0058] Stage 3: Melting curve, 95℃15s, 60℃1min, 95℃15s, 1 cycle.

[0059] 3. Data analysis: relative quantification - 2 -△△Ct Law.

[0060] The results are shown in Tables 2 and 3. As can be seen from Tables 2 and 3, the expression levels of QSOX1 and ABCC1 detected by the kit of the present invention in epilepsy patients were significantly higher than those in the normal group, and only one patient in each group did not meet the diagnosis results of the autism rating scale, and the detection efficiency was 90.91%.

[0061] Table 2 QSOX1 expression levels

[0062]

[0063]

[0064] Table 3 ABCC1 expression levels

[0065]

[0066]

[0067] 4. Based on the test results in Tables 2 and 3, the diagnostic criteria for epilepsy were constructed (Table 4). As shown in Table 4, when any one of the two genes, QSOX1 and ABCC1, is dysregulated (i.e., the expression of QSOX1 or ABCC1 is higher than the diagnostic threshold), it can be judged as mild (with no obvious symptoms) or suspected epilepsy, and clinical characteristics need to be combined to further determine whether epilepsy is present.

[0068] If both genes are dysregulated, that is, the expression levels of QSOX1 and ABCC1 are higher than the diagnostic threshold, epilepsy can be diagnosed and the symptoms are obvious.

[0069] Table 4 Diagnostic criteria for epilepsy

[0070]

[0071] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A marker for epilepsy diagnosis / detection, characterized in that: The marker is one or a combination of two of the QSOX1 and ABCC1 genes.

2. The marker according to claim 1, characterized in that High expression of QSOX1 or ABCC1 genes suggests that the patient may suffer from epilepsy.

3. The marker according to claim 1, characterized in that The simultaneous high expression of QSOX1 and ABCC1 genes indicates that the patient suffers from epilepsy.

4. A primer combination for amplifying the marker according to any one of claims 1 to 3, characterized in that: It comprises primer pairs for amplifying QSOX1 and ABCC1 genes; wherein the primer pair sequences for amplifying QSOX1 are shown as SEQ ID NOs.1 and 2; and the primer pair sequences for amplifying ABCC1 are shown as SEQ ID NOs.3 and 4.

5. The primer combination according to claim 4, characterized in that: It also includes a primer pair with the internal reference gene h-actinb as the target fragment; the sequence of the primer pair is shown in SEQ ID NO.5 and 6.

6. A kit, characterized in that: Comprising the primer combination described in claim 5.

7. The kit according to claim 6, characterized in that The kit also includes peripheral blood lymphocyte mRNA extraction solution, diluent, cleaning solution, reverse transcription reaction solution, standard positive template and SYBR Green real-time fluorescence quantitative PCR reaction solution.

8. Use of the marker according to any one of claims 1 to 3, the primer combination according to claim 4 or 5, or the kit according to claim 6 or 7 in the preparation of a product for risk prediction, screening, prognosis assessment, treatment effect monitoring, or recurrence monitoring of epilepsy.

9. Use of the marker according to any one of claims 1 to 3 in constructing an epilepsy animal model.

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