Method for detecting ECSIT expression condition by adopting fluorescent probe

Abstract

The invention relates to the technical field of biological detection, in particular to a method for detecting ECSIT by adopting a fluorescent probe. The ECSIT gene is located in a No.19 chromosome-NC000019.10 of a wisdom, and the nucleotide sequence of the ECSIT gene is as shown in SEQ ID NO: 1; the nucleotide sequences of the PCR amplification primer of the ECSIT are shown as SEQ ID NO: 2 and SEQ ID NO: 3, the primer shown as SEQ ID NO: 2 is a forward PCR primer for amplifying cDNA (complementary deoxyribonucleic acid) corresponding to the ECSIT, and the primer shown as SEQ ID NO: 3 is a reverse PCR primer for amplifying cDNA corresponding to the ECSIT; the nucleotide sequence of the detection probe of cDNA corresponding to ECSIT is shown as SEQ ID NO.4, the 5'end of the probe is marked with a fluorescence reporter group FAM, and the 3 'end of the probe is marked with a fluorescence quenching group MGB. The detection probe has the advantages of high sensitivity, high accuracy and non-invasiveness, and is a safe and reliable gene detection means.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting ECSIT expression using a fluorescent probe. Background Art

[0002] The ECSIT gene is a conserved gene in humans and other species that encodes a signaling protein. The ECSIT protein is primarily associated with various biological processes, including intracellular signal transduction, immune response, and cellular metabolism. The ECSIT gene plays an important role in the human life process, participating in the regulation of key signaling pathways such as the NF-κB and Toll-like receptor pathways, as well as other signaling mechanisms involved in cellular immune responses. Therefore, the ECSIT gene plays a crucial role in the immune system, cellular energy metabolism, and cellular responses. It is also associated with cellular anti-inflammatory responses, oxidative stress, and the developmental processes of some cells. Gene mutations or disorders may lead to dysfunction of the immune system, thereby affecting the health of the organism.

[0003] Existing methods for detecting ECSIT expression still have significant shortcomings. Commonly used methods include Western blot, immunohistochemistry, and RNA sequencing. Western blot relies on the specificity and sensitivity of antibodies. If the antibody quality is not high or it cannot fully recognize the different forms of ECSIT protein, the accuracy and reliability of the results will be low. Although immunohistochemistry can observe the distribution of ECSIT in tissue sections, its resolution is limited and it cannot accurately quantify intracellular molecules. It still relies on the sensitivity and specificity of antibodies and is prone to false negatives and false positives. RNA sequencing technology requires high experimental costs, including sample preparation, sequencing, and data analysis, making it unsuitable for large-scale screening. At the same time, the large amount of data generated by RNA sequencing requires complex bioinformatics analysis, which is difficult to process and interpret data and has high technical requirements.

[0004] In view of the above problems, the present invention provides a solution. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting ECSIT expression using a fluorescent probe, which can accurately and quickly detect ECSIT expression in an organism and provide rapid and accurate genetic detection results for various complications caused by ECSIT deficiency, thereby designing treatment plans.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions: an ECSIT gene, which is located on chromosome 19 of Homo sapiens - NC_000019.10, and whose nucleotide sequence is shown in SEQ ID NO: 1;

[0007] Furthermore, the present invention provides a PCR amplification primer for ECSIT, the nucleotide sequences of the primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3, the primer shown in SEQ ID NO: 2 is a forward PCR primer for amplifying the cDNA corresponding to ECSIT, and the primer shown in SEQ ID NO: 3 is a reverse PCR primer for amplifying the cDNA corresponding to ECSIT;

[0008] Furthermore, the present invention provides a detection probe for cDNA corresponding to ECSIT, the nucleotide sequence of the probe is shown in SEQ ID NO: 4, the 5' end of the probe is labeled with a fluorescent reporter group FAM, and the 3' end of the probe is labeled with a fluorescent quencher group MGB;

[0009] Furthermore, the present invention provides a kit for detecting the expression of the ECSIT gene, wherein the nucleotide sequence of the ECSIT gene is shown in SEQ ID NO: 1;

[0010] The detection kit includes a PCR amplification primer for ECSIT, the nucleotide sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 3. The primer shown in SEQ ID NO: 2 is a forward PCR primer for amplifying the cDNA corresponding to ECSIT, and the primer shown in SEQ ID NO: 3 is a reverse PCR primer for amplifying the cDNA corresponding to ECSIT.

[0011] The kit includes an ECSIT detection probe and a cDNA detection probe corresponding to ECSIT, wherein the nucleotide sequence of the probe is shown in SEQ ID NO: 4, the 5' end of the probe is labeled with a fluorescent reporter group FAM, and the 3' end of the probe is labeled with a fluorescent quencher group MGB;

[0012] The kit also includes an ECSIT expression protein standard, the corresponding amino acid sequence of which is shown in SEQ ID NO: 5;

[0013] Furthermore, the kit includes RNA extraction reagent, reverse transcription reaction system, genomic DNA reaction system and PCR reaction solution;

[0014] Furthermore, the present invention provides a method for detecting ECSIT using a fluorescent probe, comprising the following steps:

[0015] S1: Draw 5 ml of venous blood from a fasting patient into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000 rpm / min for 5-10 minutes. Transfer the supernatant to an enzyme-free EP tube. Centrifuge again at 1000 rpm / min for 2-4 minutes. Transfer the supernatant to a new enzyme-free EP tube to obtain a serum sample. Store at -80°C until use.

[0016] S2: Take 1 ml of serum sample into an EP tube, add 2 ml of Trizol, mix by pipetting repeatedly, and let it stand on ice for 10 minutes. After standing, add 200 μl of chloroform, mix and shake until uniform, and let it stand on ice for 10 minutes;

[0017] S3: After standing, place the sample in a centrifuge and centrifuge at 10,000 rpm for 10 minutes at 4°C. Carefully aspirate the supernatant and quickly transfer it to a new 1.5 ml EP tube without enzyme. Immediately add 0.5 ml of isopropanol, mix well, and let it stand on ice for 10 minutes.

[0018] S4: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 minutes. After centrifugation, discard the supernatant and add 1 ml of alcohol and centrifuge at 4°C, 10,000 rpm / min for 10 minutes. After centrifugation, discard the supernatant, dry, and add 30-100 μl of DEPC to dissolve.

[0019] S5: Use DNA digestion enzyme to remove residual genomic DNA. The total reaction volume is 10 ml. The reaction solution is digested at 37°C for 40 minutes and then inactivated at 85°C for 3 minutes.

[0020] The specific reaction system is as follows:

[0021] mRNA 6ul (2ug)

[0022] DNase I 1 μl

[0023] 10×Buffer 1ul

[0024] H2O (RNase-free) 2ul

[0025] S6: mRNA was reverse transcribed to obtain cDNA according to the reverse transcription system. The reaction program was 37°C for 15 min, 42°C for 30 min, 85°C for 3 min, and 4°C for 1.5 min.

[0026] The specific reaction system is as follows:

[0027] RNA template 1ul (100ng-5ug)

[0028] 5×Buffer 4ul

[0029] RNase I (20U / ul) 1ul

[0030] 10mM dNTP Mix 2ul

[0031] RT MIX (200U / ul) 1ul

[0032] Random primer 1ul

[0033] S7: amplify the obtained cDNA by fluorescent quantitative PCR;

[0034] The specific reaction system is as follows:

[0035] cDNA 2ul

[0036] Forward primer (500nM) 1ul

[0037] Reverse primer (500nM) 1ul

[0038] Probe 2ul

[0039] 2X PCR MIX 25ul

[0040] ddH2O 19ul

[0041] The PCR amplification program is as follows, with a total of 40 cycles of amplification-denaturation, amplification-annealing, and amplification-extension:

[0042] Pre-denaturation at 95°C for 3 minutes

[0043] Denaturation at 95°C for 10 seconds

[0044] Annealing at 58°C for 15 seconds

[0045] Extension at 72°C for 40 seconds

[0046] Complete extension at 72°C for 10 min

[0047] Cooling to 4°C for 15 seconds

[0048] S8: The expression of ECSIT in the test sample can be determined by analyzing the fluorescent signal amplified by fluorescent quantitative PCR in S7.

[0049] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: the present invention provides an ECSIT gene, and designs PCR amplification primers and fluorescent detection probes for cDNA reverse transcribed from mRNA expressed by the ECSIT gene. This can provide rapid and accurate genetic detection results for various complications caused by ECSIT expression deficiency, thereby designing treatment plans. The present invention has the advantages of high sensitivity, high accuracy, and non-invasiveness, and is a safe and reliable genetic detection method. DETAILED DESCRIPTION

[0050] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments.

[0051] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0052] Example 1

[0053] 1: Five male and female unilateral ECSIT gene knockout mice and five male and female normal mice were purchased from the Medical Laboratory Animal Base of Nanjing Medical University and artificially housed in a mouse house. Female ECSIT gene knockout mice were labeled as ♀knockout, and male ECSIT gene knockout mice were labeled as ♂knockout. Normal female mice were labeled as ♀normal, and normal male mice were labeled as ♂normal.

[0054] 2: The mice were divided into groups, with one male and one female in each group. The specific grouping is shown in Table 1. The number of offspring born by mice in different groups was recorded after normal feeding for 180 days. The number of offspring born by mice is shown in Table 2.

[0055] 3. After normal feeding, add adenine to the mouse feed at a rate of 2.5g per kg of feed for 21 days. After the feeding period, remove two ♀ knockout mice, two ♂ knockout mice, two ♀ normal mice, and two ♂ normal mice. After cervical dislocation, use a syringe to draw 1ml of cardiac blood into a blood collection tube without anticoagulant. Remove the mouse kidneys and prepare slices.

[0056] 4: After the mouse heart blood has been allowed to stand for 30 minutes, place it in a centrifuge and centrifuge it at 1000 rpm / min for 10 minutes. The supernatant is transferred to an enzyme-free EP tube. Centrifuge it again at 1000 rpm / min for 4 minutes. The supernatant is transferred to a new enzyme-free EP tube to obtain a serum sample. 1 ml of serum sample is placed in an EP tube, 2 ml of Trizol is added, and the sample is mixed by pipetting repeatedly. The sample is allowed to stand on ice for 10 minutes. After the sample is allowed to stand, 200 μl of chloroform is added, and the sample is mixed and shaken until uniform. The sample is allowed to stand on ice for 10 minutes.

[0057] 5. After standing, place the sample in a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 10 minutes. Carefully aspirate the supernatant and quickly transfer it to a new 1.5 ml EP tube without enzyme. Immediately add 0.5 ml of isopropanol, mix well, and let it stand on ice for 10 minutes.

[0058] 6: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 minutes. After centrifugation, discard the supernatant and add 1 ml of alcohol. Centrifuge at 4°C, 10,000 rpm / min for 10 minutes. After centrifugation, discard the supernatant, dry, and add 50 μl of DEPC to dissolve.

[0059] 7: Use DNA digestion enzyme to remove residual genomic DNA. The total reaction volume is 10 ml. The reaction solution is digested at 37°C for 40 minutes and then inactivated at 85°C for 3 minutes.

[0060] The specific reaction system is as follows:

[0061] mRNA 6ul (2ug)

[0062] DNase I 1 μl

[0063] 10×Buffer 1ul

[0064] H2O (RNase-free) 2ul

[0065] 8: Reverse transcribe the mRNA to obtain cDNA using the reverse transcription system. The reaction procedure is 37°C for 15 min, 42°C for 30 min, 85°C for 3 min, and 4°C for 1.5 min.

[0066] The specific reaction system is as follows:

[0067] RNA template 1ul (100ng-5ug)

[0068] 5×Buffer 4ul

[0069] RNase I (20U / ul) 1ul

[0070] 10mM dNTP Mix 2ul

[0071] RT MIX (200U / ul) 1ul

[0072] Random primer 1ul

[0073] 9: Amplify the obtained cDNA by fluorescent quantitative PCR;

[0074] The specific reaction system is as follows:

[0075] cDNA 2ul

[0076] Forward primer (500nM) 1ul

[0077] Reverse primer (500nM) 1ul

[0078] Probe 2ul

[0079] 2X PCR MIX 25ul

[0080] ddH2O 19ul

[0081] The PCR amplification program is as follows, with a total of 40 cycles of amplification-denaturation, amplification-annealing, and amplification-extension:

[0082] Pre-denaturation at 95°C for 3 minutes

[0083] Denaturation at 95°C for 10 seconds

[0084] Annealing at 58°C for 15 seconds

[0085] Extension at 72°C for 40 seconds

[0086] Complete extension at 72°C for 10 min

[0087] Cooling to 4°C for 15 seconds

[0088] Table 1. Grouping of mice

[0089] Group 1 1 ♀ knockout mouse ♂1 knockout mouse Group 2 1 ♀ knockout mouse ♂1 normal mouse Group 3 ♀1 normal mouse ♂1 knockout mouse control group ♀1 normal mouse ♂1 normal mouse

[0090] Table 2. Number of offspring born to mice

[0091] Grouping Number of babies Number of survivors time Group 1 3 17 Rearing time: 180 days Group 2 4 34 Rearing time: 180 days Group 3 4 32 Rearing time: 180 days control group 6 38 Rearing time: 180 days

[0092] Analysis of Tables 1 and 2 indicates that complete knockout of the ECSIT gene is embryonic lethal, leading to a significant decrease in the number of offspring born in the first group. This finding is consistent with genetic laws and is therefore considered reliable. Analysis of the number of offspring born and the number of surviving offspring in the second, third, and control groups indicates that knockout of the ECSIT gene negatively impacts the reproductive potential of mice. Observation of kidney sections from mice in different groups indicates that ECSIT knockout mice experience greater damage from chronic kidney injury, with more pronounced renal inflammation.

[0093] Table 3. Expression of ECSIT genes in mice (using ACTB as a control gene)

[0094] Grouping Relative expression level of ECSIT gene ♀ knockout mice 0.74 ♂knockout mice 0.69 Normal mice 1.65 ♂Normal mice 1.58

[0095] Example 2

[0096] 100 volunteers aged ≥18 years with no history of genetic diseases were recruited for sample collection, including 38 normal volunteers, 14 patients with systemic lupus erythematosus, 22 patients with diabetes, 11 patients with Parkinson's disease, and 15 patients with breast cancer;

[0097] 5 ml of venous blood was drawn from a fasting patient into a blood collection tube without anticoagulant. After standing for 30 minutes, the blood sample was placed in a centrifuge and centrifuged at 1000 rpm / min for 5-10 minutes. The supernatant was transferred to an enzyme-free EP tube. The sample was centrifuged again at 1000 rpm / min for 2-4 minutes. The supernatant was transferred to a new enzyme-free EP tube to obtain a serum sample, which was stored at -80°C until use.

[0098] The volunteer serum samples were subjected to the following procedures;

[0099] 1. Take 1 ml of serum sample into an EP tube, add 2 ml of Trizol, pipette repeatedly to mix, let it stand on ice for 10 minutes, add 200 μl of chloroform after standing, mix and shake until uniform, let it stand on ice for 10 minutes;

[0100] 2: After the sample is allowed to stand, place it in a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 10 minutes. Carefully aspirate the supernatant and quickly transfer it to a new enzyme-free 1.5 ml EP tube. Immediately add 0.5 ml of isopropanol, mix well, and let it stand on ice for 10 minutes.

[0101] 3: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 minutes. After centrifugation, discard the supernatant and add 1 ml of alcohol. Centrifuge at 4°C, 10,000 rpm / min for 10 minutes. After centrifugation, discard the supernatant, dry it, and add 30-100 μl of DEPC to dissolve it.

[0102] 4. Use DNA digestion enzyme to remove residual genomic DNA. The total reaction volume is 10 ml. The reaction solution is digested at 37°C for 40 minutes and then inactivated at 85°C for 3 minutes.

[0103] The specific reaction system is as follows:

[0104] mRNA 6ul (2ug)

[0105] DNase I 1 μl

[0106] 10×Buffer 1ul

[0107] H2O (RNase-free) 2ul

[0108] 5: Reverse transcribe the mRNA to obtain cDNA according to the reverse transcription system. The reaction program is 37℃ for 15min, 42℃ for 30min, 85℃ for 3min, and 4℃ for 1.5min;

[0109] The specific reaction system is as follows:

[0110] RNA template 1ul (100ng-5ug)

[0111] 5×Buffer 4ul

[0112] RNase I (20U / ul) 1ul

[0113] 10mM dNTP Mix 2ul

[0114] RT MIX (200U / ul) 1ul

[0115] Random primer 1ul

[0116] 6: Amplify the obtained cDNA by fluorescent quantitative PCR;

[0117] The specific reaction system is as follows:

[0118] cDNA 2ul

[0119] Forward primer (500nM) 1ul

[0120] Reverse primer (500nM) 1ul

[0121] Probe 2ul

[0122] 2X PCR MIX 25ul

[0123] ddH2O 19ul

[0124] The PCR amplification program is as follows, with a total of 40 cycles of amplification-denaturation, amplification-annealing, and amplification-extension:

[0125] Pre-denaturation at 95°C for 3 minutes

[0126] Denaturation at 95°C for 10 seconds

[0127] Annealing at 58°C for 15 seconds

[0128] Extension at 72°C for 40 seconds

[0129] Complete extension at 72°C for 10 min

[0130] Cooling to 4°C for 15 seconds

[0131] 7: The expression of ECSIT in the test sample can be determined by analyzing the fluorescence signal amplified by fluorescence quantitative PCR, with ACTB as the control gene.

[0132] Table 4. Relative expression levels of ECSIT in volunteers

[0133] Grouping ECSIT relative expression level Normal volunteers 1.56±0.17 Patients with systemic lupus erythematosus 0.98±0.73 Diabetic patients 0.95±0.41 Parkinson's disease patients 1.06±0.55 Breast cancer patients 0.82±0.33

[0134] Analysis of Table 4 shows that the relative expression level of ECSIT in serum samples of normal volunteers is 1.56±0.17, and the relative expression level of ECSIT in serum samples of patients with systemic lupus erythematosus, diabetes, Parkinson's disease, and breast cancer is 0.5-0.8 times that of the relative expression level of ECSIT in serum samples of normal volunteers. Compared with normal volunteers, the expression levels of ECSIT in patients with systemic lupus erythematosus, diabetes, Parkinson's disease, and breast cancer are significantly reduced. Among them, patients with systemic lupus erythematosus are affected by the lack of ECSIT expression, resulting in the inability to properly regulate the immune system, thus leading to an overactive autoimmune response; Parkinson's disease patients suffer from mitochondrial dysfunction due to the lack of ECSIT expression, which in turn affects nerve cells; diabetic patients suffer from mitochondrial dysfunction and energy metabolism due to the lack of ECSIT expression, and thus diabetic patients caused by the lack of ECSIT are also likely to develop Parkinson's disease; breast cancer patients are affected by the lack of ECSIT expression, resulting in abnormal cell metabolism and immune escape, thereby inducing breast cancer.

[0135] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A PCR amplification primer for ECSIT, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO: 2 and SEQ ID NO:

3. The primer shown in SEQ ID NO: 2 is a forward PCR primer for amplifying the cDNA corresponding to ECSIT, and the primer shown in SEQ ID NO: 3 is a reverse PCR primer for amplifying the cDNA corresponding to ECSIT.

2. A detection probe for cDNA corresponding to ECSIT, characterized in that: The nucleotide sequence of the probe is shown in SEQ ID NO:

4. The 5' end of the probe is labeled with a fluorescent reporter group FAM, and the 3' end of the probe is labeled with a fluorescent quencher group MGB.

3. A kit for detecting ECSIT gene expression, characterized in that: The nucleotide sequence of the ECSIT gene is shown in SEQ ID NO:

1.

4. The detection kit for ECSIT gene expression according to claim 4, characterized in that: The detection kit includes a PCR amplification primer for ECSIT, the nucleotide sequence of the primer is shown in SEQ ID NO: 2 and SEQ ID NO: 3, the primer shown in SEQ ID NO: 2 is a forward PCR primer for amplifying the cDNA corresponding to ECSIT, and the primer shown in SEQ ID NO: 3 is a reverse PCR primer for amplifying the cDNA corresponding to ECSIT.

5. The detection kit for ECSIT gene expression according to claim 4, characterized in that: The kit includes a cDNA detection probe corresponding to ECSIT, the nucleotide sequence of the probe is shown in SEQ ID NO: 5, the 5' end of the probe is labeled with a fluorescent reporter group ROX, and the 3' end of the probe is labeled with a fluorescent quencher group MGB.

6. The detection kit for ECSIT gene expression according to claim 4, characterized in that: The kit also includes an ECSIT expression protein standard, the corresponding amino acid sequence of which is shown in SEQ ID NO:

5.

7. The ECSIT detection kit according to claim 4, characterized in that: The kit comprises an RNA extraction reagent, a reverse transcription reaction system, a genomic DNA reaction system and a PCR reaction solution.

8. A method for detecting ECSIT expression using a fluorescent probe, characterized in that: The following steps are involved: S1: Draw 5 ml of venous blood from a fasting patient into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000 rpm / min for 5-10 minutes. Transfer the supernatant to an enzyme-free EP tube. Centrifuge again at 1000 rpm / min for 2-4 minutes. Transfer the supernatant to a new enzyme-free EP tube to obtain a serum sample. Store at -80°C until use. S2: Take 1 ml of serum sample into an EP tube, add 2 ml of Trizol, mix by pipetting repeatedly, and let it stand on ice for 10 minutes. After standing, add 200 μl of chloroform, mix and shake until uniform, and let it stand on ice for 10 minutes; S3: After standing, place the sample in a centrifuge and centrifuge at 10,000 rpm for 10 minutes at 4°C. Carefully aspirate the supernatant and quickly transfer it to a new 1.5 ml EP tube without enzyme. Immediately add 0.5 ml of isopropanol, mix well, and let it stand on ice for 10 minutes. S4: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 minutes. After centrifugation, discard the supernatant and add 1 ml of alcohol and centrifuge at 4°C, 10,000 rpm / min for 10 minutes. After centrifugation, discard the supernatant, dry, and add 30-100 μl of DEPC to dissolve. S5: Use DNA digestion enzyme to remove residual genomic DNA. The total reaction volume is 10 ml. The reaction solution is digested at 37°C for 40 minutes and then inactivated at 85°C for 3 minutes. The specific reaction system is as follows: mRNA 6ul (2ug) DNase I 1 μl 10×Buffer 1ul H2O (RNase-free) 2ul S6: mRNA was reverse transcribed to obtain cDNA according to the reverse transcription system. The reaction program was 37°C for 15 min, 42°C for 30 min, 85°C for 3 min, and 4°C for 1.5 min. The specific reaction system is as follows: RNA template 1ul (100ng-5ug) 5×Buffer 4ul RNase I (20U / ul) 1ul 10mM dNTP Mix 2ul RT MIX (200U / ul) 1ul Random primer 1ul S7: amplify the obtained cDNA by fluorescent quantitative PCR; The specific reaction system is as follows: cDNA 2ul Forward primer (500nM) 1ul Reverse primer (500nM) 1ul Probe 2ul 2X PCR MIX 25ul ddH2O 19ul The PCR amplification program is as follows, with a total of 40 cycles of amplification-denaturation, amplification-annealing, and amplification-extension: Pre-denaturation at 95°C for 3 minutes Denaturation at 95°C for 10 seconds Annealing at 58°C for 15 seconds Extension at 72°C for 40 seconds Complete extension at 72°C for 10 min Cooling to 4°C for 15 seconds S8: The expression of ECSIT in the test sample can be determined by analyzing the fluorescent signal amplified by fluorescent quantitative PCR in S7.