Application of mRNA (messenger Ribonucleic Acid) combination in predicting curative effect of myocardial peptide in treating myocardial ischemia-reperfusion injury

Abstract

The invention discloses an application of an mRNA combination in predicting the curative effect of myocardial peptide in treating myocardial ischemia-reperfusion injury, and belongs to the technical field of biology. The mRNA combination comprises Asb10 (Asb10) and Egr2 (Egr2); the nucleotide sequence of the Asb10 is as shown in SEQ ID NO. 1, and the nucleotide sequence of the Egr2 is as shown in SEQ ID NO. 2. Experiments prove that compared with a normal mouse, the expression of Asb10 in a model group is remarkably reduced, the expression of Egr2 is remarkably improved, and compared with the model group and a cardiomyopeptide group, the level of Asb10 is remarkably improved, and the level of Egr2 is remarkably reduced. The ROC curves of Asb10 and Egr2 after myocardial peptide treatment are constructed, and the AUC of Asb10 and Egr2 is 0.816 and 0.762 respectively. The invention provides a more effective monitoring means and a new thought for more stably predicting the curative effect of myocardial peptide on myocardial ischemia-reperfusion injury at low cost.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the application of mRNA combination in predicting the therapeutic effect of myocardial peptide in treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemia refers to a decrease in the blood perfusion of the heart, which leads to a decrease in cardiac oxygen supply, abnormal myocardial energy metabolism, and affects the normal functioning of the heart. Restoring blood perfusion after a period of myocardial ischemia will aggravate the damage to the myocardial structure and function, causing decreased cardiac function and malignant arrhythmias, namely myocardial ischemia-reperfusion injury. Studies have shown that myocardial peptides have a significant preventive and therapeutic effect on myocardial ischemia-reperfusion injury, and can reduce the release of myocardial enzymes and severe arrhythmias after ischemia-reperfusion. Specifically, myocardial peptides can reduce the release of myocardial enzymes after myocardial ischemia-reperfusion, and play a role in reducing myocardial damage; they can also reduce the incidence of severe ventricular arrhythmias in rats with ischemia-reperfusion injury by inhibiting the inward calcium current in ventricular myocytes.

[0003] Although the therapeutic effect of myocardial peptides on myocardial ischemia-reperfusion injury has been confirmed to a certain extent, there is a lack of clear and effective indicators to indicate its therapeutic effect in real time and accurately, so as to better monitor and evaluate the treatment. At present, the evaluation of the therapeutic effect of myocardial peptides in clinical practice mainly relies on some traditional indicators, such as myocardial enzyme activity, electrocardiogram changes, cardiac ultrasound, etc., but these indicators have certain limitations, such as (1) insufficient sensitivity: traditional indicators used to evaluate the therapeutic effect of myocardial peptides, such as myocardial enzyme activity, electrocardiogram changes, cardiac ultrasound, etc., often show obvious abnormalities only after myocardial damage reaches a certain degree. It may not be possible to detect early mild myocardial function improvement or damage repair in a timely and sensitive manner, resulting in missing the best treatment opportunity or misjudgment of the treatment effect. (2) weak specificity: traditional indicators are not unique to myocardial peptide treatment. Other factors such as myocardial infarction, myocarditis and other cardiovascular diseases may also cause similar changes in indicators. It is difficult to specifically reflect the therapeutic effect of myocardial peptides on myocardial ischemia-reperfusion injury, and it is impossible to accurately distinguish whether it is the therapeutic effect of myocardial peptides or the development of the disease itself or the influence of other drugs.

[0004] In recent years, with the continuous development of molecular biology technology, more and more studies have shown that the mRNA expression levels of many genes will change significantly during myocardial ischemia-reperfusion injury. For example, some studies have found that some genes related to apoptosis, inflammatory response, oxidative stress, etc. are upregulated or downregulated after myocardial ischemia-reperfusion injury. The expression changes of these genes are closely related to the degree of myocardial injury and prognosis. As an intermediate product of gene expression, changes in mRNA expression levels can reflect the transcriptional regulation of genes in cells and have high sensitivity and specificity. By detecting the expression level of specific mRNA, we can deeply understand the physiological and pathological state of cells and provide important basis for the diagnosis, treatment and prognosis of diseases. However, there are also some defects in the technology of judging the efficacy by detecting the expression level of mRNA, such as: (1) Limitation of sample acquisition: Some current technologies for detecting mRNA expression levels require obtaining myocardial tissue samples, which is an invasive operation that may cause additional trauma and risks to patients. In addition, it is difficult to repeat multiple times in clinical practice, which limits the real-time monitoring of the dynamic changes of mRNA expression during treatment. (2) Complex technology and high cost: Although some advanced mRNA detection technologies, such as gene chips and RNA sequencing, can detect the expression levels of a large number of genes at the same time, the technical operations are complex and require professional equipment and technicians. The detection cost is also relatively high, which is not conducive to widespread promotion and application in clinical practice.

[0005] Therefore, finding mRNA markers that can specifically indicate the effect of myocardial peptides in treating myocardial ischemia-reperfusion injury and establishing a more sensitive, accurate and real-time treatment effect evaluation method are of great significance for optimizing the clinical application of myocardial peptides and improving the treatment effect of myocardial ischemia-reperfusion injury. Summary of the invention

[0006] The purpose of the present invention is to provide an application of an mRNA combination in predicting the efficacy of myocardial peptide in treating myocardial ischemia-reperfusion injury, so as to solve the problems existing in the above-mentioned prior art. The mRNA combination includes Asb10 and Egr2, and the expression level of the mRNA combination can indicate the effect of myocardial peptide in treating myocardial ischemia-reperfusion injury. The present invention provides a more effective monitoring means and a new idea for predicting the efficacy of myocardial peptide in treating myocardial ischemia-reperfusion injury at a low cost and more stably.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an application of an mRNA combination in preparing a product for predicting the efficacy of myocardial peptide in treating myocardial ischemia-reperfusion injury. The mRNA combination comprises Asb10 and Egr2; the nucleotide sequence of Asb10 is shown in SEQ ID NO.1, and the nucleotide sequence of Egr2 is shown in SEQ ID NO.2.

[0009] Optionally, the product includes a reagent, a kit or a gene chip.

[0010] Optionally, the product includes a reagent for detecting the expression level of the mRNA combination.

[0011] Optionally, the reagent includes RT-qPCR detection primers; the nucleotide sequence of the RT-qPCR detection primers is shown in SEQ ID NO.3-6.

[0012] The present invention also provides a product for predicting the efficacy of myocardial peptide in treating myocardial ischemia-reperfusion injury, the product comprising a reagent for detecting the expression level of mRNA combination;

[0013] The mRNA combination includes Asb10 and Egr2; the nucleotide sequence of Asb10 is shown in SEQ ID NO.1, and the nucleotide sequence of Egr2 is shown in SEQ ID NO.2.

[0014] Optionally, the reagent includes RT-qPCR detection primers; the nucleotide sequence of the RT-qPCR detection primers is shown in SEQ ID NO.3-6.

[0015] Optionally, the product includes a reagent, a kit or a gene chip.

[0016] The present invention discloses the following technical effects:

[0017] The present invention provides a new method for predicting the therapeutic effect of myocardial peptide on myocardial ischemia-reperfusion injury. The expression levels of two genes, Egr2 and Asb10, in peripheral blood are used, and combined analysis is performed to judge the therapeutic effect of myocardial peptide. Specifically, it is experimentally verified that compared with normal mice, the expression of Asb10 in the model group is significantly reduced, while compared with the model group, the myocardial peptide group significantly increases the level of Asb10. In addition, compared with normal mice, the expression of Egr2 in the model group is significantly increased, while compared with the model group, the myocardial peptide group significantly reduces the level of Egr2. And the ROC curves of Asb10 and Egr2 14 days after myocardial peptide treatment are constructed, and the AUCs of the two are 0.816 and 0.762, respectively, indicating that the two have high sensitivity and specificity for predicting the therapeutic effect of myocardial peptide, and can be used as markers of therapeutic effect. The present invention provides a more effective monitoring means and new ideas for predicting the therapeutic effect of myocardial peptide in treating myocardial ischemia-reperfusion injury at low cost and more stably. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of transcriptome analysis of target molecules; the left picture is Asb10, and the right picture is Egr2;

[0019] Figure 2 The expression levels of Asb10 (left panel) and Egr2 (right panel) in peripheral blood mononuclear cells were detected by RT-qPCR;

[0020] Figure 3 HE staining was used to observe myocardial tissue pathology;

[0021] Figure 4 Masson staining is used to observe the fibers in myocardial tissue;

[0022] Figure 5 The full-automatic biochemical analyzer is used to detect the content of myocardial injury markers; the left picture shows the level of troponin; the right picture shows the level of myoglobin;

[0023] Figure 6 RT-qPCR was used to detect the expression levels of Asb10 (left figure) and Egr2 (right figure) in rat peripheral blood mononuclear cells;

[0024] Figure 7 ROC curves of Asb10 and Egr2 in the model group and 14 days after myocardial peptide treatment. DETAILED DESCRIPTION

[0025] The myocardial peptide used in the present invention is an injectable myocardial peptide provided by Dalian Zhenao Pharmaceutical Co., Ltd.

[0026] Example 1

[0027] 1. Construction of myocardial ischemia-reperfusion model

[0028] The myocardial infarction model was established by ligating the left anterior descending coronary artery. After anesthesia, the model group rats were prepared for neck and chest skin preparation, fixed in the supine position in the center of the operating table, and put on an inhalation mask. The electrodes of the multi-channel physiological recorder were inserted into the left and right upper and left lower limbs of the rats, and the standard II lead electrocardiogram was recorded. An oblique incision of about 1.5 cm was made in the third and fourth intercostal spaces of the left chest of the rats (the position where the heart beats most obviously can be felt by pressing the chest cavity with the hand), and the chest cavity was opened after the subcutaneous muscle tissue was separated layer by layer with hemostatic forceps, and the heart was exposed by traction hook. Under the field of view of a stereomicroscope, the pericardium was peeled off, and a 6 / 0 non-invasive suture needle and silk thread were held. The needle was inserted at the junction of the pulmonary artery cone and the left atrial appendage, 1 to 2 mm below the starting point of the coronary artery, and the depth was 1.0 to 1.5 mm for ligation. After the needle was inserted, a fishing line with a diameter of 0.53 mm and a length of 5 cm was placed on the top, and a slipknot was used for ligation. The chest cavity was cleaned with a dry cotton swab to squeeze out the residual gas in the chest cavity, and the muscles and skin were sutured layer by layer with 3 / 0 sutures, and the incision was locally disinfected. After 1 minute of observation, the rat was placed on a constant temperature electric blanket, and after 40 minutes, the fishing line was gently pulled out and then perfused.

[0029] Twenty rats were divided into groups according to the random number table method. The myocardial peptide drug group was continuously administered for 14 days, and blood was collected from the eyeballs 7 days after administration. The animals were killed and the myocardial tissue was dissected and processed. The study was divided into a control group of normal rats, a myocardial ischemia-reperfusion model group, and a myocardial peptide (56.7 mg / kg) treatment group, with 5 rats in each group, for a total of 15 rats.

[0030] 2. Peripheral blood transcriptome detection of differential gene expression

[0031] Three peripheral blood samples were collected from each group of animals for RNA-seq. The samples were diluted with PBS to double the volume, layered on 5 mL Ficoll-Paque Plus, and centrifuged at 400 × g for 30 minutes. The PBMC layer was aspirated and collected by centrifugation. The PBMC microspheres were treated with RBC lysis buffer for 15 minutes under light-free conditions, then centrifuged again and washed twice with PBS. Total RNA was extracted using the miRNeasy Kit and then treated with DNase I to avoid genomic DNA contamination. Oligo (dT) magnetic beads were used to enrich the mRNA with polyA structure in the total RNA, and the RNA was sheared to fragments of about 300 bp in length by ion shearing. A fragment with a length of 300 bp was selected (because the length of the linker is fixed, if the length of the interrupted fragment is shorter, the proportion of the linker sequence will be higher, thereby reducing the proportion of valid data; if the length of the interrupted fragment is longer, it will be unfavorable for the generation of clusters during sequencing). RNA was used as a template, and 6-base random primers and reverse transcriptase were used to synthesize the first chain of cDNA, and the first chain of cDNA was used as a template to synthesize the second chain of cDNA.

[0032] After the library construction is completed, PCR amplification is used to enrich the library fragments, and then the library is selected according to the fragment size, and the library size is 450bp. Next, the library is quality checked by Agilent 2100Bioanalyzer, and then the total concentration of the library and the effective concentration of the library are detected. Then, according to the effective concentration of the library and the amount of data required for the library, the libraries containing different Index sequences (each sample is added with a different Index, and finally the offline data of each sample is distinguished according to the Index) are mixed in proportion. The mixed library is uniformly diluted to 2nM and denatured by alkali to form a single-stranded library. The second-generation sequencing technology (Next-Generation Sequencing, NGS) is used to perform paired-end (PE) sequencing on these libraries based on the Illumina sequencing platform.

[0033] Gene differential expression analysis: DESeq was used to perform differential analysis on gene expression, and the conditions for screening differentially expressed genes were: expression difference fold |log2FoldChange|>1, significant P-value<0.05. From the obtained differential gene list, the molecules downregulated in the model group vs. normal group were screened, and the molecules upregulated in the myocardial peptide treatment group vs. model group were screened. The molecules obtained from the two were intersected to obtain the target molecule Asb10; the molecules upregulated in the model group vs. normal group, and the molecules downregulated in the myocardial peptide treatment group vs. model group were screened, and the molecules obtained from the two were intersected to obtain the target molecule Egr2. The transcriptome differential analysis results of the two are shown as follows: Figure 1 shown.

[0034] In the above operation, the specific steps of using RT-qPCR technology to detect the expression difference of Asb10 and Egr2 in monocytes are as follows:

[0035] (1) Extraction of total RNA from monocytes: The total RNA from cells was extracted using a Trizol extraction kit. The specific steps are as follows:

[0036] 1) Collect cells, wash cells twice with 3 mL PBS, and discard the supernatant;

[0037] 2) Add 1 mL of Trizol to thoroughly homogenize the mixture and let it stand at room temperature for 5 minutes;

[0038] 3) Add 0.2 mL of chloroform, shake vigorously for 15 seconds, and let stand for 3 minutes;

[0039] 4) Centrifuge at 4°C, 12000 rpm for 10 min and collect the supernatant;

[0040] 5) Add 0.5 mL of isopropanol, mix well, and let stand on ice for 20-30 min;

[0041] 6) Centrifuge at 4°C, 12,000 rpm for 10 min and discard the supernatant;

[0042] 7) Add 1 mL of 75% ethanol to wash the precipitate. Centrifuge at 7500 g for 5 min at 4°C and discard the supernatant.

[0043] 8) Blow dry in a clean bench for about 5 minutes, and add an appropriate amount of RNase-free H2O to dissolve.

[0044] (2) cDNA synthesis: cDNA was synthesized using a first-strand cDNA synthesis kit. The specific steps are as follows:

[0045] For the reverse transcription reaction system, add the reagents listed in Table 1 into a 0.2 mL PCR tube.

[0046] Table 1 Reverse transcription reaction system

[0047]

[0048] After mixing, incubate at 37°C for 5 min, 42°C for 60 min, and 70°C for 10 min to terminate the reaction. Store the above solution at -20°C.

[0049] (3) Polymerase chain reaction (PCR)

[0050] 1) Primer synthesis

[0051] The primers used in real-time PCR were designed and synthesized by Primer Premier 5.0 software from Shanghai Bioengineering Co., Ltd., with the housekeeping gene Gapdh as the internal reference. The primer sequences are shown in Table 2.

[0052] Table 2 Primer information

[0053]

[0054] 2) Real time-PCR reaction system

[0055] Prepare the reaction solution according to the Real time-PCR reaction system (Table 3).

[0056] Table 3 Real time-PCR reaction system

[0057]

[0058] 3) PCR amplification conditions

[0059] Amplification conditions: 94°C for 10 min; 94°C for 20 sec, 55°C for 20 sec, 72°C for 20 sec, 40 cycles.

[0060] (4) Real-Time PCR Data Processing

[0061] After PCR amplification, the real-time fluorescence quantitative PCR instrument automatically analyzes the results, adjusts the threshold and baseline according to the negative control to determine the Ct value of each sample, and determines whether the Ct value is valid according to the melting curve. The results are exported and 2 -△△CT The expression difference of the target gene between the control group and each concentration group was analyzed by the following calculation formula: △Ct = Ct target gene - Ct internal reference. The △Ct of each group was subtracted from the △Ct control to calculate the average △Ct value of the control group, that is, △△Ct = △Ct sample - △Ct control average. Then the 2 -△△CT The value is the relative expression level of the gene in each group.

[0062] Test results such as Figure 2 As shown, compared with the control group, Asb10 was significantly downregulated and Egr2 was significantly upregulated in the I / R model group; compared with the I / R model group, Asb10 was significantly upregulated and Egr2 was significantly downregulated.

[0063] 3. Hematoxylin-eosin (HE) staining

[0064] HE staining was used to observe the pathological changes of myocardial tissue sections. The experimental process was as follows: myocardial tissues were obtained after the rats in each group were killed.

[0065] (1) Tissue dehydration: Dehydrate the tissue using gradient alcohol;

[0066] (2) Tissue transparency: Tissue blocks must be transparent after being dehydrated with alcohol. The transparent agent (xylene) can be mixed with the dehydrating agent and paraffin at the same time. After it replaces the dehydrating agent, the paraffin can penetrate the tissue smoothly;

[0067] (3) Wax dipping: The transparent tissue block is dipped into three cylinders of paraffin wax (60°C) in sequence;

[0068] (4) Embedding: Embedding is to wrap the wax-soaked tissue block in a paraffin block. The temperature of the embedding wax should be slightly higher than the wax soaking temperature to ensure that the tissue block and the embedding paraffin are completely integrated;

[0069] (5) Slicing and baking: The embedded wax block is sliced ​​using a Leica pathology slicer. The sliced ​​tissue slices are placed in a 40°C water bath for spreading. The anti-slip slide is inserted into the water surface at an angle to pick up the slices. The slices are attached to the appropriate position on the slide and baked in a 60°C oven for 3 hours.

[0070] (6) Dewaxing sections: Place the paraffin sections in xylene I (20 min) - xylene II (20 min) - xylene III (20 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% alcohol (5 min) - 90% alcohol (5 min) - 80% alcohol (5 min) - 70% alcohol (5 min), and then rinse with distilled water for 5 min;

[0071] (7) Stain the sections with Mayer's hematoxylin (the staining background is clean and no differentiation is required) for 5 min, then wash with tap water to turn blue.

[0072] (8) The sections were stained with 1% water-soluble eosin solution for 5 min and then washed with tap water for 30 s;

[0073] (9) After rinsing the slices in water, place the slices in 95% alcohol for 30 seconds, 95% alcohol for 1 minute, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes.

[0074] (10) Sealing: Drop neutral gum next to the tissue and cover it with a coverslip. Lay one side flat first, then gently put down the other side to avoid bubbles. Lay the sealed slices flat in a fume hood to dry.

[0075] (11) Microscopic examination and photography: The dried slices can be observed or images can be collected under a microscope.

[0076] The results are as follows Figure 3 As shown in the results, in the myocardial tissue of the model group rats, the arrangement of myocardial cells was irregular, the ratio of nuclei to cytoplasm was unbalanced, and the infiltration of inflammatory cells increased significantly. After treatment with myocardial peptide, the arrangement of myocardial cells showed a clear trend of regularization.

[0077] 4. Masson staining

[0078] Masson staining technique was used to detect changes in muscle fibers and collagen fibers in myocardial tissue. The experimental process is as follows.

[0079] (1) The tissue is fixed in Bouin's solution or Zenker's solution, rinsed in running water overnight, and dehydrated and embedded as usual.

[0080] (2) Dewaxing the sections and rinsing them with water: 1) After dewaxing, treat the sections with 0.5% iodine alcohol for 10 minutes; 2) Wash with water; 3) Treat with 5% sodium thiosulfate for 5 minutes; 4) Rinse with running water for 10 minutes.

[0081] (3) Stain with Weiger's iron hematoxylin for 5-10 minutes.

[0082] (4) Rinse with running water.

[0083] (5) 1% hydrochloric acid alcohol differentiation.

[0084] (6) Rinse with running water for several minutes.

[0085] (7) Dye with Ponceau acid fuchsin solution for 5-10 minutes.

[0086] (8) Rinse briefly with distilled water.

[0087] (9) Treat with 1% phosphomolybdic acid aqueous solution for about 5 minutes.

[0088] (10) Without washing with water, directly stain with aniline blue or green solution for 5 minutes.

[0089] (11) Treat with 1% glacial acetic acid for 1 minute.

[0090] (12) Dehydrate with 95% alcohol multiple times.

[0091] (13) Dehydrate with anhydrous alcohol, make transparent with xylene, and seal with neutral gum.

[0092] The results are as follows Figure 4 As shown: In the myocardial tissue sections of the model group rats, compared with the sections of normal rats, it can be found that the collagen fibers are arranged in a disordered state, the number of muscle fibers is reduced, and the infiltration of inflammatory factors is more serious. After receiving myocardial peptide treatment, the muscle fibers in the myocardial tissue have recovered to a certain extent, the number of collagen fibers has decreased, and the degree of inflammatory infiltration has also been alleviated.

[0093] 5. Automatic biochemical analyzer to detect serum markers of myocardial injury

[0094] Follow the instrument manual to prepare samples, load samples, and acquire data. The results are as follows: Figure 5 As shown, with the treatment of cardiomyopeptide, the levels of serum troponin T (TnT) and myoglobin (MYO), markers of myocardial injury in rats, gradually decreased, which shows that cardiomyopeptide treatment has a protective effect on myocardial injury.

[0095] 6. RT-qPCR detection of changes in the expression levels of Asb10 and Egr2 in peripheral blood mononuclear cells in the model group

[0096] 14 rats were randomly modeled, and the mRNA expressions of Asb10 and Egr2 were detected after modeling and after myocardial peptide treatment according to the procedures described in "2. Detection of differential gene expression in peripheral blood transcriptome". Figure 6 As shown, with the treatment of cardiomyopeptide, Asb10 in rat peripheral blood mononuclear cells gradually increased from 0 to 14 days, while Egr2 gradually decreased.

[0097] 7. ROC analysis of Asb10 and Egr2 in peripheral blood as myocardial peptides for the treatment of myocardial ischemia-reperfusion

[0098] ROC curves were constructed to compare the AUCs of Asb10 and Egr2 14 days after myocardial peptide treatment. Figure 7 As shown in the figure, 14 days after myocardial peptide treatment, the AUCs of Asb10 and Egr2 were 0.816 and 0.762, respectively, indicating that the two have high sensitivity and specificity for predicting the efficacy of myocardial peptide and can be used as markers of efficacy.

[0099] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of mRNA combination in the preparation of a product for predicting the efficacy of myocardial peptide in treating myocardial ischemia-reperfusion injury, characterized in that: The mRNA combination includes Asb10 and Egr2; the nucleotide sequence of Asb10 is shown in SEQ ID NO.1, and the nucleotide sequence of Egr2 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The products include reagents, kits or gene chips.

3. The use according to claim 1, characterized in that The product includes reagents for detecting the expression level of the mRNA combination.

4. The use according to claim 3, characterized in that The reagents include RT-qPCR detection primers; the nucleotide sequences of the RT-qPCR detection primers are shown in SEQ ID NO.3-6.

5. A product for predicting the efficacy of myocardial peptide in treating myocardial ischemia-reperfusion injury, characterized in that: The product includes reagents for detecting the expression level of mRNA combinations; The mRNA combination includes Asb10 and Egr2; the nucleotide sequence of Asb10 is shown in SEQ ID NO.1, and the nucleotide sequence of Egr2 is shown in SEQ ID NO.

2.

6. The product according to claim 5, characterized in that The reagents include RT-qPCR detection primers; the nucleotide sequences of the RT-qPCR detection primers are shown in SEQ ID NO.3-6.

7. The product according to claim 5, characterized in that The products include reagents, kits or gene chips.

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