Kit for screening and diagnosing colorectal cancer

Abstract

The invention discloses a kit for screening and diagnosing colorectal cancer, and belongs to the technical field of medical detection. The kit for screening and diagnosing the colorectal cancer comprises a forward primer and a reverse primer, wherein the forward primer and the reverse primer are used for detecting tsRNA-Gly-5-0007; the sequence of the forward primer is as shown in SEQ ID NO.2, and the sequence of the reverse primer is as shown in SEQ ID NO.3. Compared with healthy volunteers, the tsRNA-Gly-5-0007 expression level in serum exosomes of colorectal cancer patients is remarkably reduced for the first time, ROC curve analysis shows that AUC is 0.795, sensitivity is 72.7%, and specificity is 75%; compared with healthy volunteers, the expression level of tsRNA-Gly-5-0007 in serum exosomes of patients with early colorectal cancer is remarkably reduced; rOC curve analysis shows that AUC is 0.788, the sensitivity is 70%, and the specificity is 78.7%.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and in particular relates to a kit for screening and diagnosing colorectal cancer. Background Art

[0002] Colorectal cancer, one of the leading causes of cancer-related death, is caused by a complex interaction of many genetic and environmental factors. Despite significant advances in the diagnosis and treatment of colorectal cancer, the incidence of early-onset colorectal cancer (≤50 years old) has increased significantly. Blood-based testing is expected to improve screening compliance, detect colorectal cancer earlier, and reduce colorectal cancer-related deaths.

[0003] Liquid biopsy is used for early detection and treatment of cancer, as well as the research and application of residual lesions and recurrence monitoring. Exosomes have become one of the research directions with potential wide applications. Exosomes secreted by tumor cells contain rich tumor-derived substances such as RNA, DNA, proteins and metabolites, and are tissue-specific. Exosomes can participate in intercellular communication as messenger molecules and promote the growth of tumor cells. Exosomes can exist stably in the blood and can also be isolated for clinical evaluation in the early stages of the disease.

[0004] Compared with the existing colorectal cancer screening methods in clinical practice, hematological examinations have obvious advantages. Hematological examinations are non-invasive and low-risk; they are easy to operate and can be sampled repeatedly; they are suitable for real-time dynamic monitoring during the development of the disease; they can effectively solve the problem of tumor heterogeneity and provide more comprehensive tumor information. Summary of the invention

[0005] In view of the problems that existing colorectal cancer screening and diagnosis methods have low sensitivity, poor specificity, strong professional operation and high difficulty, the present invention provides a hematological marker and kit for early screening and diagnosis of colorectal cancer, which are quick to operate, improve the sensitivity and accuracy of detection, and have good application value in early screening and diagnosis of colorectal cancer.

[0006] The present invention is achieved through the following technical solutions: A kit for screening and diagnosing colorectal cancer, comprising a forward primer and a reverse primer for detecting tsRNA-Gly-5-0007; The sequence of the forward primer is shown in SEQ ID NO.2; The sequence of the reverse primer is shown in SEQ ID NO.3.

[0007] Furthermore, the kit for screening and diagnosing colorectal cancer also includes a reverse transcription reaction system and an internal reference system; The internal reference system includes a U6 forward primer and a U6 reverse primer; The sequence of the U6 forward primer is shown in SEQ ID NO.4; The sequence of the U6 reverse primer is shown in SEQ ID NO.5.

[0008] Furthermore, the reverse transcription reaction system includes polyadenylic acid polymerase, reverse transcriptase mixture, reverse transcription buffer and enzyme-free water.

[0009] Furthermore, the kit for screening and diagnosing colorectal cancer also includes SYBR Green mixed solution and enzyme-free water.

[0010] Furthermore, the SYBR Green mixture includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ .

[0011] Beneficial Effects (1) The present invention proposes for the first time that the expression level of tsRNA-Gly-5-0007 in serum exosomes of colorectal cancer patients is significantly lower than that of healthy volunteers. ROC curve analysis shows that the AUC is 0.795, the sensitivity is 72.7%, and the specificity is 75%; (2) Compared with healthy volunteers, the expression level of tsRNA-Gly-5-0007 in serum exosomes of patients with early colorectal cancer was significantly reduced; ROC curve analysis showed that the AUC was 0.788, the sensitivity was 70%, and the specificity was 78.7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the expression level of tsRNA-Gly-5-0007 in serum exosomes of colorectal cancer patients and healthy volunteers; Figure 2 This is the expression level diagram of tsRNA-Gly-5-0007 in early colorectal cancer; Figure 3 ROC curve for tsRNA-Gly-5-0007 in diagnosing colorectal cancer patients; Figure 4 ROC curve for tsRNA-Gly-5-0007 in diagnosing early colorectal cancer patients; DETAILED DESCRIPTION The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the implementation cases are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0013] Through a large number of studies, it was found that compared with healthy volunteers, the expression level of tsRNA-Gly-5-0007 in serum exosomes of patients with colorectal cancer or early colorectal cancer was significantly reduced, and it was speculated that it could be used as a blood marker for screening and diagnosis of colorectal cancer or early colorectal cancer; the sequence of serum exosome tsRNA-Gly-5-0007 is shown in SEQ ID NO.1 in Table 1.

[0014] Example 1 A kit for early screening and diagnosis of colorectal cancer, the kit is used to detect the above-mentioned hematological markers, the kit for early screening and diagnosis of colorectal cancer comprises a forward primer for tsRNA-Gly-5-0007 and a reverse primer for tsRNA-Gly-5-0007, a SYBR Green mixed solution (PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ ) and enzyme-free water to form a PCR reaction system, the sequence of the forward primer for tsRNA-Gly-5-0007 is shown in SEQ ID NO.2 in Table 1, and the sequence of the reverse primer for tsRNA-Gly-5-0007 is shown in SEQ ID NO.3 in Table 1; The kit for early screening and diagnosis of colorectal cancer also includes an internal reference system, which includes a U6 forward primer and a U6 reverse primer. The sequence of the U6 forward primer is shown in SEQ ID NO.4 in Table 1, and the sequence of the U6 reverse primer is shown in SEQ ID NO.5 in Table 1. The kit for early screening and diagnosis of colorectal cancer also includes a reverse transcription reaction system, which includes polyadenylic acid polymerase, reverse transcriptase mixture, reverse transcription buffer and enzyme-free water.

[0015] Table 1 Sequences related to the kit for early diagnosis or early warning of progression of colorectal cancer .

[0016] Example 2 Application of a kit for early screening and diagnosis of colorectal cancer in diagnosing early colorectal cancer (1) Experimental design Serum samples were collected from patients with primary colorectal cancer and healthy volunteers in the Affiliated Tumor Hospital of Shandong First Medical University, and exosomes were separated and enriched, and exosomal RNA was extracted. The expression of tsRNA-Gly-5-0007 was detected by qPCR, and the expression difference between colorectal cancer patients and healthy volunteers was statistically analyzed, and its clinical diagnostic efficacy was analyzed.

[0017] (2) Patient samples involved in the experiment From July to December 2021, 172 colorectal cancer patients and 164 healthy volunteers were admitted to the Affiliated Tumor Hospital of Shandong First Medical University. Among them, 80 patients with early colorectal cancer were included. The enrolled patients were classified by TNM by pathological methods. The clinical data included in this study mainly included age, gender, UICC tumor stage (eighth edition), smoking and drinking history, hypertension and diabetes history. Before surgery, chemotherapy and radiotherapy, about 3 ml of peripheral blood samples were collected from patients and placed in a vacuum blood collection tube containing a coagulant. After standing for blood coagulation, the blood was centrifuged at 3000g for 10 minutes at room temperature. The separated serum was centrifuged at 12,000g for 30 minutes at 4°C, the supernatant was taken and then centrifuged at 100,000g for 2 hours at 4°C, the supernatant was discarded, 500 μL of Trizol reagent was added to the exosome precipitate, lysed at 4°C for 30 minutes, and then transferred to a clean enzyme-free EP tube and frozen at -80°C for later use.

[0018] (3) Determination of exosomal tsRNA-Gly-5-0007 levels in serum The expression level of exosomal tsRNA-Gly-5-0007 in the serum of colorectal cancer patients and healthy volunteers was analyzed using the qPCR method. The specific steps are as follows: 1) Extraction of exosomes.

[0019] 2) Extraction of total exosome RNA The operation was carried out in a biosafety cabinet. The frozen exosome samples after pretreatment were thawed at room temperature, 100 μL of chloroform was added, and the mixture was shaken vigorously for 30 seconds. After standing for 3 minutes, it was centrifuged at 12,000 g for 15 minutes at 4°C (the mixture was separated into a lower pink phenol-chloroform liquid phase, a middle layer of white flocs, and an upper layer of colorless aqueous phase, and RNA was distributed in the upper aqueous phase); 250 μL of the upper aqueous phase was pipetted into a clean enzyme-free EP tube, an equal volume of isopropanol was added, and the mixture was shaken for 30 seconds. After standing for 10 minutes, it was centrifuged at 12,000 g for 10 minutes at 4°C, and the liquid was discarded; 750 μL of 75% ethanol solution was added, and the mixture was gently inverted and mixed, and centrifuged at 7,500 g for 5 minutes at 4°C; the supernatant was discarded as much as possible, and the white RNA precipitate at the bottom of the tube was retained. The EP tube was opened and inverted on a new filter paper, and dried naturally for 10 to 15 minutes. Add 10 μL RNase-free water and mix thoroughly with a pipette to completely dissolve the RNA. The dissolved RNA sample can be stored at -80°C for later use. Use Nanodrop ultra-micro spectrophotometer to detect RNA concentration and purity.

[0020] 3) Reverse transcription reaction Reverse transcription reaction system: 3.75μL Total RNA, 1.25μL RT Enzyme Mix, 5μL RT Reaction Solution; Reverse transcription reaction conditions: 37°C for 60 min, 85°C for 15 min, and storage at 4°C.

[0021] 4) Preparation before real-time fluorescence PCR amplification Prepare primer solution: The primer reagent is a dry powder. Centrifuge at 3000 rpm for 60 seconds, then slowly open the lid, add an appropriate amount of nuclease-free double-distilled water to make the final concentration 10 μM, close the lid and shake thoroughly to dissolve and mix; 5) Fluorescence PCR amplification reaction The fluorescence PCR reaction system is shown in Table 2 below: Table 2 Fluorescence PCR reaction system Each component in the fluorescent PCR reaction system was added to the corresponding amplification reaction plate in turn, and the sample cDNA was added. To avoid reagent contamination, a transparent sealing film should be attached immediately after the sample addition. Centrifuge at 800g for 3 minutes at room temperature to mix well and then load onto the machine. Use the Light Cycler 480 RT-PCR instrument to perform the fluorescent PCR reaction conditions set in Table 3 below. Each sample in the reaction was set to one replicate, and the average was taken to reduce the error. The experiment was repeated three times.

[0022] Table 3 Fluorescence PCR reaction conditions 6) Amplification reaction of internal reference system The U6 gene was used as the internal reference gene. The internal reference system included 10 μL SYBR Green mixture, 0.8 μL U6 forward primer, 0.8 μL U6 reverse primer, 6.4 μL enzyme-free water, and 2 μL cDNA. The reaction conditions of the internal reference system included 95°C pre-denaturation for 30 seconds, 95°C denaturation for 5 seconds, 60°C annealing for 20 seconds, and 65°C extension for 15 seconds, for a total of 40 cycles.

[0023] (4) Data statistical analysis Draw the fluorescence amplification curve and melting curve to obtain the Ct value, and use ΔCt to analyze the real-time fluorescence quantitative PCR reaction results. The ΔCt value represents the relative expression of the target RNA. The larger the ΔCt value, the weaker the expression of the target RNA. ΔCt = Ct 目的RNA Value - Ct 内参RNA value.

[0024] Prism 9.0 was used to analyze the qPCR data. tThe test was expressed as mean ± standard deviation. When the test was not satisfied, the Mann-Whitney test was used to express the median ± interquartile range. SPSS26.0 was used to calculate the ROC curve to evaluate the clinical diagnostic efficacy of tsRNA-Gly-5-0007. P The difference was considered statistically significant when p<0.05; all tests were two-tailed.

[0025] (5) Summary of clinical pathological characteristics of colorectal cancer patients and their relationship with tsRNA-Gly-5-0007 expression levels The results in Table 4 analyzed the clinical characteristics of colorectal cancer patients, including age, gender, history of smoking and drinking, history of hypertension, history of diabetes, tumor location, etc. The expression level of tsRNA-Gly-5-0007 was related to tumor location, but not to age, gender, drinking, smoking, diabetes, hypertension, metastasis, and TNM stage.

[0026] Table 4 Summary of clinicopathological characteristics of colorectal cancer patients and their relationship with tsRNA-Gly-5-0007 expression levels * : Did not participate in hypothesis testing.

[0027] (6) Difference in expression levels of serum exosomal tsRNA-Gly-5-0007 between colorectal cancer patients and healthy volunteers The qPCR method was used to verify the level of serum exosome tsRNA-Gly-5-0007 in colorectal cancer patients and healthy volunteers; the expression level of serum exosome tsRNA-Gly-5-0007 in colorectal cancer patients and healthy volunteers is shown in the figure Figure 1 As shown in Figure 2, the expression level of tsRNA-Gly-5-0007 in early colorectal cancer is as follows Figure 2 As shown, compared with healthy volunteers, the expression level of tsRNA-Gly-5-0007 in colorectal cancer patients was significantly reduced, and the difference was statistically significant ( P <0.0001). In addition, the expression levels of serum exosome tsRNA-Gly-5-0007 in patients with early colorectal cancer and healthy volunteers were compared. The results showed that the expression level of tsRNA-Gly-5-0007 was significantly reduced in patients with early colorectal cancer, and the difference was statistically significant ( P <0.0001).

[0028] (7) Analysis of the diagnostic efficacy of serum exosomal tsRNA-Gly-5-0007 for colorectal cancer The ROC curve was constructed to evaluate the clinical diagnostic efficacy of tsRNA-Gly-5-0007 for colorectal cancer. Figure 3 As shown, the results showed that the AUC was 0.795, the sensitivity was 72.7%, and the specificity was 75%.

[0029] (8) Analysis of the diagnostic efficacy of serum exosomal tsRNA-Gly-5-0007 for early colorectal cancer The ROC curve was constructed to evaluate the clinical diagnostic efficacy of tsRNA-Gly-5-0007 for early colorectal cancer. Figure 4 As shown, the results showed that the AUC was 0.788, the sensitivity was 70%, and the specificity was 78.7%.

Claims

1. A kit for screening and diagnosing colorectal cancer, characterized in that: Includes a forward primer and a reverse primer for detecting tsRNA-Gly-5-0007; The sequence of the forward primer is shown in SEQ ID NO.2; The sequence of the reverse primer is shown in SEQ ID NO.

3.

2. The kit for screening and diagnosing colorectal cancer according to claim 1, characterized in that: The kit for screening and diagnosing colorectal cancer also includes a reverse transcription reaction system and an internal reference system; The internal reference system includes a U6 forward primer and a U6 reverse primer; The sequence of the U6 forward primer is shown in SEQ ID NO.4; The sequence of the U6 reverse primer is shown in SEQ ID NO.

5.

3. The kit for screening and diagnosing colorectal cancer according to claim 2, characterized in that: The reverse transcription reaction system comprises polyadenylic acid polymerase, reverse transcriptase mixed solution, reverse transcription buffer and enzyme-free water.

4. The kit for screening and diagnosing colorectal cancer according to claim 1, characterized in that: The kit for screening and diagnosing colorectal cancer also includes SYBR Green mixed solution and enzyme-free water.

5. The kit for screening and diagnosing colorectal cancer according to claim 4, characterized in that: The SYBRGreen mixed solution includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ .

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