Application of SNORA58 in the preparation of products for diagnosing esophageal cancer

Abstract

The present invention provides the use of SNORA58 in the preparation of a product for diagnosing esophageal cancer. Through research, the present invention discovered that the expression level of SNORA58 is associated with esophageal cancer and that SNORA58 is abnormally highly expressed in esophageal cancer patients. Using SNORA58 as a biomarker for diagnosing esophageal cancer, by detecting the expression level of SNORA58 in a subject's sample, it is possible to determine whether the subject has esophageal cancer and the prognosis of the patient, thereby facilitating early diagnosis and screening of esophageal cancer. Using SNORA58 as a target molecule for the treatment of esophageal cancer, by silencing SNORA58, the sensitivity of esophageal cancer patients to radiotherapy is increased, the efficacy of subsequent treatment for esophageal cancer patients is improved, and the survival of esophageal cancer patients is thereby prolonged. This is of great significance in the clinical treatment of esophageal cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical biological detection technology, and in particular to the application of SNORA58 in preparing products for diagnosing esophageal cancer. Background Art

[0002] Esophageal cancer is a regionally prevalent malignant tumor that poses a threat to human health. Esophageal squamous cell carcinoma (ESCC), also known as ESCC, is the most common pathological type of esophageal cancer in my country, accounting for over 90% of all cases. Esophageal cancer has an insidious onset, and the vast majority of patients are already in the middle or late stages at the time of initial diagnosis, resulting in a very poor prognosis. Neoadjuvant radiotherapy has shown significant effects in the treatment of middle or late-stage esophageal cancer in recent years. However, some patients are still insensitive to radiotherapy or chemotherapy, which leads to continued disease progression and seriously affects their survival. Therefore, effective diagnosis and dynamic monitoring of patients with early-stage esophageal cancer will be extremely important for the effective treatment of esophageal cancer.

[0003] Currently, the detection methods for blood samples of esophageal cancer patients mainly include the following three methods: (1) serum protein mass spectrometry analysis; (2) serum protein chip; (3) serological test strips. The above three existing detection methods for blood samples of esophageal cancer patients mainly target protein molecules and have certain limitations. Serum protein mass spectrometry analysis is complex and expensive for sample processing. The results require professional analysis. The entire detection cycle is long, and the specificity for unidentified peptides cannot be guaranteed, resulting in poor universality. Serum protein chip detection is expensive, the experimental method is complex, and the operating requirements for the test personnel are high. Serological test strips mainly target three specific antigens (peripheral blood tumor antigens such as CEA, squamous cell antigen, and CYFRA21). Its experimental process is cumbersome and the detection sensitivity is poor.

[0004] Small nucleolar RNAs (snoRNAs) are a class of small, noncoding RNAs widely distributed in the nucleoli of eukaryotic cells. Their aberrant expression has been found to be closely associated with the development and progression of human diseases, particularly cancer, neurodegenerative diseases, and viral infections. SnoRNAs are stable in blood, sputum, and urine, making them promising molecular biomarkers for disease diagnosis. Some snoRNAs have been used in blood tests for cancer patients, including those for non-small cell lung cancer, pancreatic cancer, colorectal cancer, prostate cancer, and clear cell renal cell carcinoma. However, there are currently no reports or applications of snoRNAs in blood tests for patients with esophageal squamous cell carcinoma. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of SNORA58 in the preparation of a product for diagnosing esophageal cancer. The expression level of SNORA58 is correlated with the prognosis of esophageal cancer patients. Therefore, SNORA58 can be used as a biomarker for early diagnosis of esophageal cancer patients.

[0006] According to a first aspect of the present invention, there is provided use of SNORA58 in preparing a product for diagnosing esophageal cancer.

[0007] SNORA58 (small nucleolar RNA, H / ACA box 58) is located on the long arm of chromosome 3 (3q22.1) in the human genome. It was identified in 2004 by Arnold M. Kiss et al., who also determined the specific sequence where it binds to target genes. A review of relevant literature has revealed no reports of SNORA58 in human disease.

[0008] The present invention, through snoRNA PCR chip testing of esophageal squamous cell carcinoma and adjacent tissues, found that SNORA58 is highly expressed in esophageal squamous cell carcinoma tissue and patient plasma, and is associated with poor patient prognosis. The present invention uses SNORA58 as a biomarker for the diagnosis of esophageal cancer and applies it to the preparation of products for the diagnosis of esophageal cancer. This allows for the diagnosis and dynamic detection of esophageal cancer, facilitating the screening and diagnosis of early-stage esophageal cancer patients and thereby improving the subsequent treatment outcomes for patients. Furthermore, because SNORA58 can be stably present in blood, sputum, and urine, its application as a biomarker in the diagnosis of esophageal cancer results in stable test results with high accuracy and sensitivity.

[0009] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0010] Preferably, the above product can diagnose esophageal cancer by measuring the expression level of SNORA58 in a sample.

[0011] Preferably, the sample includes at least one of blood, sputum, urine, and tissue fluid.

[0012] Preferably, the above-mentioned product includes a product for diagnosing esophageal cancer by detecting the level of SNORA58 using RT-qPCR, blot hybridization, in situ hybridization, array hybridization, gene chip or next-generation sequencing.

[0013] Preferably, the product comprises a formulation, a kit and / or a chip.

[0014] According to a second aspect of the present invention, a kit for diagnosing esophageal cancer is provided, the kit comprising primers and / or probes targeting SNORA58.

[0015] The kit involved in this protocol contains primers and / or probes specific for SNORA58. These primers and / or probes specifically bind to SNORA58 and can be used to detect SNORA58 expression levels in samples using techniques such as RT-qPCR, imprint hybridization, in situ hybridization, array hybridization, gene chip, or next-generation sequencing. Based on SNORA58 expression levels, patients with esophageal cancer can be diagnosed and screened, and their prognosis can be predicted. This kit provides stable and accurate test results, and the experimental process and analysis are relatively simple, with a short experimental cycle and low operator requirements, making it highly scalable.

[0016] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0017] Preferably, in the above kit for diagnosing esophageal cancer, the primers for SNORA58 include a forward primer and a reverse primer, the sequence of the forward primer for SNORA58 is shown as SEQ ID No: 1, and the sequence of the reverse primer for SNORA58 is shown as SEQ ID No: 2.

[0018] SEQ ID No: 1

[0019] 5'-TACTCGTAGACCTTGCCTGACT-3'

[0020] SEQ ID No: 2

[0021] 5'-TGATGAAGCTGGTTAGAGCTGG-3'

[0022] The kit involved in this plan contains forward primers and reverse primers for SNORA58. These two primers and Real time-qPCR technology can be used to detect the expression level of SNORA58 in samples, which is beneficial for the early diagnosis and screening of esophageal cancer patients.

[0023] Preferably, the above kit further comprises primers and / or probes targeting GAPDH or 5s rRNA.

[0024] The kit provided in this protocol also contains primers and / or probes targeting GAPDH or 5s rRNA, with GAPDH serving as an internal reference gene for tissue specimens and 5s rRNA serving as an internal reference gene for blood specimens. During the processing of test samples, various factors may affect the test samples, which may affect the accuracy and precision of the test. The inclusion of primers and / or probes targeting GAPDH or 5s rRNA as internal references in the kit is intended to normalize the starting amounts of different samples during the analysis process, ensuring consistent expression of GAPDH or 5s rRNA in each test sample. This facilitates the analysis and evaluation of different test samples and, to a certain extent, improves the reliability of the test results.

[0025] Preferably, the primers for GAPDH include a forward primer and a reverse primer. The sequence of the forward primer for GAPDH is shown as SEQ ID No: 3, and the sequence of the reverse primer for GAPDH is shown as SEQ ID No: 4.

[0026] SEQ ID No: 3

[0027] 5'-GTCTCCTCTGACTTCAACAGCG-3'

[0028] SEQ ID No:4

[0029] 5'-ACCACCCTGTTGCTGTAGCCAA-3'

[0030] Preferably, the primers for 5s rRNA include a forward primer and a reverse primer, the sequence of the forward primer for 5s rRNA is shown as SEQ ID No: 5, and the sequence of the reverse primer for 5s rRNA is shown as SEQ ID No: 6.

[0031] SEQ ID No:5

[0032] 5'-CATACCACCCTGAACGCG-3'

[0033] SEQ ID No:6

[0034] 5'-CTACAGCACCCGGTATTCCC-3'

[0035] According to a third aspect of the present invention, there is provided use of a SNORA58 inhibitor in preparing a pharmaceutical composition for treating esophageal cancer.

[0036] This approach uses SNORA58 as a target for the treatment of esophageal cancer and applies SNORA58 inhibitors to the preparation of pharmaceutical compositions for the treatment of esophageal cancer. SNORA58 inhibitors can effectively inhibit the expression level or functional activity of SNORA58, thereby increasing the sensitivity of esophageal cancer patients to radiotherapy, which is beneficial for improving subsequent treatment effects.

[0037] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0038] According to a fourth aspect of the present invention, a pharmaceutical composition for treating esophageal cancer is provided, wherein the pharmaceutical composition comprises a SNORA58 inhibitor.

[0039] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0040] The pharmaceutical composition for treating esophageal cancer involved in this scheme includes a SNORA58 inhibitor, which can inhibit the expression level or functional activity of SNORA58, reduce the content of SNORA58 in the blood of esophageal cancer patients, increase the sensitivity of esophageal cancer patients to radiotherapy, thereby improving the subsequent treatment efficacy of esophageal cancer patients and prolonging the survival of esophageal cancer patients.

[0041] The beneficial effects of the present invention are as follows: Through research, the present invention discovered that the expression level of SNORA58 is associated with esophageal cancer. SNORA58 is abnormally highly expressed in esophageal cancer patients. Therefore, SNORA58 can be used as a biomarker for the diagnosis of esophageal cancer. By detecting the expression level of SNORA58 in a subject's sample, it is possible to determine whether the subject has esophageal cancer and the prognosis of esophageal cancer patients, which is beneficial for the early diagnosis and screening of esophageal cancer. Using SNORA58 as a target molecule for the treatment of esophageal cancer, silencing SNORA58 can increase the sensitivity of esophageal cancer patients to radiotherapy, improve the efficacy of subsequent treatment for esophageal cancer patients, and thus prolong the survival of esophageal cancer patients. This is of great significance in the clinical treatment of esophageal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram showing the chip detection results of esophageal squamous cell carcinoma tissue and adjacent cancer tissue provided by the present invention.

[0043] Figure 2 This is a diagram showing the expression of SNORA58 in esophageal squamous cell carcinoma tissue and adjacent cancer tissues provided by the present invention.

[0044] Figure 3 This is a diagram of the expression of SNORA58 in the plasma of healthy people and esophageal squamous cell carcinoma patients provided by the present invention.

[0045] Figure 4This is a correlation diagram between high expression of SNORA58 and the prognosis of patients with esophageal squamous cell carcinoma provided by the present invention.

[0046] Figure 5 This is a diagram showing the effect of silencing SNORA58 on the radiotherapy sensitivity of esophageal squamous cell carcinoma cells provided by the present invention. DETAILED DESCRIPTION

[0047] The following is a further clear and complete description of the technical features of the technical solution provided by the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present invention.

[0048] Example 1 Screening and identification of SNORA58

[0049] The snoRNA PCR chip detection technology was used to find the differentially expressed snoRNAs in esophageal squamous cell carcinoma tissues and their adjacent tissues. Based on the chip detection results of three pairs of esophageal squamous cell carcinoma tissues and adjacent tissues, the following were obtained: Figure 1 As shown. Figure 1 It can be seen that a total of 19 snoRNAs are upregulated in esophageal squamous cell carcinoma and its adjacent tissues, including SNORA58. In addition to SNORA58, the 18 snoRNAs upregulated in the microarray results have been reported in existing literature to varying degrees. Currently, SNORA58 has not been studied in human diseases, and according to TCGA big data analysis results, the other 18 snoRNAs do not show significant differences in esophageal squamous cell carcinoma and its adjacent tissues, while SNORA58 shows significant differences in esophageal squamous cell carcinoma and its adjacent tissues, and its high expression is significantly correlated with poor prognosis in esophageal cancer patients. Based on the above, the present invention has screened SNORA58 as a biomarker for the diagnosis of esophageal cancer.

[0050] Example 2 Collection, Processing, and RNA Extraction of Esophageal Squamous Cell Carcinoma Tissues and Para-Cancerous Tissues

[0051] This example mainly involves collecting, processing, and extracting RNA from esophageal squamous cell carcinoma tissue and adjacent tissue. The specific steps are as follows:

[0052] 1. Collection and processing of esophageal squamous cell carcinoma tissue and adjacent tissue

[0053] Tumor tissue and adjacent tissue were collected from surgical resection specimens of 3 patients with esophageal squamous cell carcinoma. After the tissues were removed from the body, they were cut into mung bean-sized pieces and placed in EP tubes. The tubes were quickly placed in liquid nitrogen and stored in a -80°C refrigerator.

[0054] 2. Extraction of RNA from Esophageal Squamous Cell Carcinoma Tissue and Paracancerous Tissue

[0055] (1) Take out the esophageal squamous cell carcinoma tissue and adjacent tissue frozen at -80°C, cut about 100 mg of each into a mortar, add an appropriate amount of liquid nitrogen, and grind the tissue into powder;

[0056] (2) Add 1 mL of TRIzol reagent (purchased from Invitrogen, USA) to the ground tissue powder and use an electric homogenizer to thoroughly homogenize the tissue;

[0057] (3) The homogenized tissue was placed at room temperature for 5 minutes, and 200 μL of chloroform was added to it. The EP tube was tightly capped and shaken vigorously for 15 seconds. The tube was placed at room temperature for 2 minutes, and centrifuged at 4°C and 12,000 g for 15 minutes.

[0058] (4) Prepare a new 1.5 mL EP tube, mark it, and transfer the upper aqueous phase from the centrifuged EP tube to a new EP tube. Add 500 μL of isopropanol, incubate at room temperature for 10 minutes, and centrifuge at 4°C and 12,000 g for 10 minutes.

[0059] (5) Use a pipette to carefully remove the supernatant, take 1 mL of 75% ethanol and add it to the EP tube with RNA precipitate to wash the RNA. Centrifuge at 8000g for 5 minutes at 4°C and remove as much remaining supernatant as possible with a pipette.

[0060] (6) Place the EP tube with RNA precipitate in a fume hood for 10 minutes. After the ethanol has completely evaporated, add an appropriate volume of RNase-free water and place at room temperature for 10 minutes.

[0061] (7) Use NanoDrop 2000 ultra-micro spectrophotometer (ThermoFisher Scientific) to detect the purity and concentration of RNA, keep records, and aliquot the extracted total RNA and store it in a -80°C refrigerator.

[0062] Example 3 Collection, processing and RNA extraction of blood samples from patients with esophageal squamous cell carcinoma and healthy subjects

[0063] This example mainly collects, processes, and extracts RNA from blood samples of esophageal squamous cell carcinoma patients and healthy subjects. The specific steps are as follows:

[0064] 1. Collection of Blood Samples

[0065] 4-5 mL of peripheral blood was collected from healthy subjects and esophageal squamous cell carcinoma patients before surgery using EDTA anticoagulant tubes and allowed to stand for 1-2 hours.

[0066] 2. Blood Sample Processing

[0067] Place the EDTA anticoagulant tube containing the blood sample in a centrifuge and centrifuge at 4°C, 1500g for 15 minutes. Collect the supernatant and divide it into EP tubes and store it in a -80°C refrigerator.

[0068] 3. RNA Extraction

[0069] 100 μL of each blood sample was taken and total RNA was extracted using the miRNeasy Serum / Plasma Kit purchased from Qiagen.

[0070] Example 4 Reverse transcription PCR

[0071] The purpose of this example is to use reverse transcription PCR technology to reverse transcribe the total RNA extracted from esophageal squamous cell carcinoma tissue and adjacent tissue in Example 2 and the total RNA extracted from blood specimens of healthy subjects from esophageal squamous cell carcinoma patients in Example 3 (as templates for reverse transcription PCR) to obtain PCR products (i.e., cDNA).

[0072] The kit used for reverse transcription PCR was the Evoscript Universal cDNA Master Kit purchased from Roche, which is mainly used for reverse transcription of RNA into cDNA.

[0073] The main operating steps of reverse transcription PCR are as follows: various reagents are added to a 0.2 mL PCR tube according to the reaction system described in Table 1, mixed thoroughly, centrifuged briefly, and incubated at room temperature for 5 minutes. Then, 2.0 μL of Enzyme Mix is added, mixed thoroughly, and PCR reaction is carried out according to the conditions shown in Table 2. After the reaction is completed, the PCR product (i.e., cDNA) is obtained and stored in a -30°C refrigerator. Among them, the product obtained by reverse transcription PCR using the total RNA extracted in Example 2 is called PCR product A, and the product obtained by reverse transcription PCR using the total RNA extracted in Example 3 is called PCR product B.

[0074] Table 1 Reverse transcription PCR reaction system

[0075]

[0076] Table 2 Reverse transcription PCR reaction conditions

[0077]

[0078] Example 5 Real-time qPCR detection of SNORA58 expression level

[0079] Table 3 Real-time qPCR reaction system

[0080]

[0081] Table 4 Real-time qPCR reaction conditions

[0082]

[0083] Real-time qPCR experiments were performed using FastStart Universal SYBR Green Master purchased from Roche. The reaction system is shown in Table 3, and a total of 40 cycles of amplification were performed. Specific operating conditions are shown in Table 4. In this example, the real-time qPCR reaction used GAPDH (an internal reference gene for tissue specimens) and 5s rRNA (an internal reference gene for plasma specimens) as internal reference genes.

[0084] Table 5 Primer sequences used in this example

[0085]

[0086] The specific primers for each gene used in this example are shown in Table 5. Prepare the samples for the PCR system as described above. Perform 2 to 3 replicates for each sample, take the average value for analysis, and use the Roche LightCycler 480 II Fluorescence Quantitative PCR instrument for detection. Melting curve and quantitative analysis of the reaction products were performed using the Roche LightCycler® 480 System. -ΔΔCT Methods RT-qPCR data were analyzed to calculate the expression level of SNORA58 in ESCC or plasma. The calculation process was as follows: ΔCT = ΔCT SNORA58 – ΔCT GAPDH (or ΔCT 5srRNA), ΔΔCT = ΔCT Tumor - ΔCT Non-tumor, where Tumor and Non-tumor represent the expression levels of SNORA58 in ESCC tissue (or blood samples from ESCC patients) and para-cancer tissue (or blood samples from healthy controls), respectively. -ΔΔCT The expression level of SNORA58 in esophageal cancer tissue was calculated and expressed as the corresponding fold change. Finally, the above data were normalized. The results are as follows Figure 2 and Figure 3 shown.

[0087] Depend on Figure 2 It can be seen that compared with adjacent adjacent tissues, small nucleolar RNA SNORA58 is abnormally highly expressed in tumor tissues of patients with esophageal squamous cell carcinoma. Figure 3 It can be seen that compared with plasma samples from healthy people, the small nucleolar RNA SNORA58 is abnormally highly expressed in plasma samples from patients with esophageal squamous cell carcinoma. This shows that SNORA58 is abnormally highly expressed in both esophageal squamous cell carcinoma tissue and patient plasma, and can be used as a biomarker for the diagnosis of esophageal squamous cell carcinoma patients. The application of SNORA58 in the preparation of products for the diagnosis of esophageal cancer can diagnose and dynamically detect esophageal cancer with high detection accuracy and sensitivity, which is beneficial for the screening and diagnosis of early-stage esophageal cancer patients, thereby improving the subsequent treatment effects of patients.

[0088] Example 6 KM-plot survival curve analysis

[0089] The clinical data and protein expression information of esophageal squamous cell carcinoma patients in the TCGA and GEO databases were used to analyze the prognosis of esophageal squamous cell carcinoma patients and draw KM-plot survival curves. The relationship between the expression level of SNORA58 and the survival period in esophageal squamous cell carcinoma patients is shown in Figure 3. Figure 4 As shown. Figure 4 It can be seen that high expression of SNORA58 in patients with esophageal squamous cell carcinoma indicates poor prognosis of the patients.

[0090] Example 7 Effect of Silencing SNORA58 on Radiosensitivity in Patients with Esophageal Squamous Cell Carcinoma

[0091] The purpose of this example is to explore the effect of SNORA58 on the radiotherapy effect of esophageal squamous cell carcinoma. This example uses two different esophageal cancer cell lines (KYSE510 and KYSE180) as experimental subjects. Both cells were subjected to SNORA58 knockout, and cells without SNORA58 knockout were used as controls. Wild-type cells that can normally express SNORA58A (WT, i.e. normal KYSE510 cells and KYSE180 cells) and SNORA58 knockout cells (KO, i.e. KYSE510 cells and KYSE180 cells with SNORA58 knocked out) were respectively given different radiation therapy doses (unit: Gy), and then the cells were inoculated and cultured on the wall to observe the proliferation and clone formation of the cells. The results are shown in Figure 2. Figure 5 shown.

[0092] Depend on Figure 5 It was found that after SNORA58 knockout in two esophageal squamous cell carcinoma cell lines, their proliferation was inhibited and cell colony formation was reduced. The inhibition of proliferation and cell colony formation became more significant with increasing radiation dose. These results indicate that silencing SNORA58 can increase the sensitivity of esophageal squamous cell carcinoma (ESCC) cells to radiotherapy.

[0093] In summary, the present invention has discovered through research that the expression level of SNORA58 is associated with esophageal cancer. SNORA58 is abnormally highly expressed in esophageal cancer patients. By using SNORA58 as a biomarker for esophageal cancer diagnosis, by detecting the expression level of SNORA58 in subject samples, it is possible to determine whether the subject has esophageal cancer and the prognosis of esophageal cancer patients, which is beneficial for the early diagnosis and screening of esophageal cancer. Using SNORA58 as a target molecule for esophageal cancer treatment, silencing SNORA58 can increase the sensitivity of esophageal cancer patients to radiotherapy, improve the efficacy of subsequent treatment for esophageal cancer patients, and thus prolong the survival of esophageal cancer patients. This is of great significance in the clinical treatment of esophageal cancer.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention. Sequence Listing <110> Sun Yat-sen University Cancer Center (Sun Yat-sen University Cancer Hospital, Sun Yat-sen University Cancer Research Institute) <120> Application of SNORA58 in the preparation of products for diagnosing esophageal cancer <160> 6 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <400> 1 tactcgtaga ccttgcctga ct 22 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <400> 2 tgatgaagct ggttagagct gg 22 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <400> 3 gtctcctctg acttcaacag cg 22 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <400> 4 accaccctgt tgctgtagcc aa 22 <210> 5 <211> 18 <212> DNA <213> Artificial sequence <400> 5 cataccaccc tgaacgcg 18 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 ctacagcacc cggtattccc 20

Claims

1. Use of primers for detecting the expression level of SNORA58 in the preparation of a product for diagnosing esophageal squamous cell carcinoma, characterized in that: The expression level of SNORA58 is high in patients with esophageal squamous cell carcinoma.

2. Use of the primers for detecting the expression level of SNORA58 according to claim 1 in the preparation of a product for diagnosing esophageal squamous cell carcinoma, characterized in that: The products include preparations, kits and / or chips.

3. Use of the primers for detecting the expression level of SNORA58 according to claim 1 in the preparation of a product for diagnosing esophageal squamous cell carcinoma, characterized in that: The primers for detecting the expression level of SNORA58 include a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID No: 1, and the sequence of the reverse primer is shown in SEQ ID No: 2.

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