Primer pair and kit for identifying and diagnosing benign and malignant pulmonary nodules
By detecting tRF3-17-GlyTCC and 3'tiRNA-43-GlyGCC-4 molecules in plasma exosomes, combined with PCR reactions, the low detection rate and high radiation problems of lung cancer screening in the prior art were solved, and an efficient diagnosis of early and low trauma was achieved.
Patent Information
- Application Number
- CN202510826784.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-08-08
AI Technical Summary
Existing lung cancer screening methods such as imaging examinations have low detection rate, high cost and radiation risks, and the sensitivity and specificity of commonly used blood markers are not high, making it difficult to achieve early, sensitive and low-traumatic lung cancer diagnosis.
The primer pairs that detect plasma exosomes tRF3-17-GlyTCC and/or 3'tiRNA-43-GlyGCC-4 were used to perform PCR reactions in combination with SYBR Green mixed solution for differential diagnosis of benign and malignant lung nodules, and improve diagnostic accuracy and sensitivity.
By detecting specific molecules in plasma exosomes, the sensitivity of malignant lung nodules is achieved at 61.4% and the specificity is 74.7%, which significantly improves diagnostic efficacy in early lung cancer diagnosis and reduces detection costs and radiation risks.
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Figure CN120442784A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a primer pair and a kit for differential diagnosis of benign and malignant pulmonary nodules. Background Art
[0002] Lung cancer is the leading cause of cancer death in my country, and its incidence rate also ranks high. Early-stage patients often have no obvious symptoms, and clinically, most patients are already in the advanced stages of the disease by the time they present symptoms and seek medical attention, which is unfavorable for prognosis and reduces the survival rate of lung cancer patients. By screening for benign and malignant lung nodules, lung cancer can be diagnosed early, reducing the risk of death. Therefore, there is an urgent need to find a tumor diagnostic marker with high sensitivity and specificity that can screen and diagnose lung cancer at an earlier stage, thereby improving the diagnosis and treatment of lung cancer and patient prognosis.
[0003] Current clinical screening methods, such as chest X-rays, CT scans, and ultrasound, require combined pathological examinations for diagnosis, but also suffer from low detection rates, high costs, and exposure to radiation. Elevated levels of hematological markers can precede the onset of clinical symptoms, aiding in early differential diagnosis and prediction of lung cancer pathological types, as well as dynamic monitoring of treatment efficacy and prognosis. However, commonly used tumor markers lack high sensitivity and specificity. Exploring new blood markers for testing aims to minimize invasiveness, accelerate detection, and improve specificity and sensitivity.
[0004] Exosomes are lipid bilayer vesicles secreted by various cells, including tumor cells. Exosomes can be secreted at any stage of disease progression and can be stably present in the blood. Cancer-derived exosomes carry cancer cell-associated signaling molecules. Detecting these molecules can be used to monitor tumor development, treatment monitoring, and prognosis. Summary of the Invention
[0005] In response to the problems existing in the prior art, the present invention provides a primer pair and a kit for differential diagnosis of benign and malignant lung nodules. The kit is used to diagnose lung nodules, thereby improving the accuracy and sensitivity of the diagnostic results and having good application value in the early diagnosis of lung cancer.
[0006] The present invention is achieved through the following technical solutions:
[0007] The present invention provides a primer pair for differential diagnosis of benign and malignant pulmonary nodules, including a primer pair for detecting plasma exosome tRF3-17-GlyTCC and / or a primer pair for detecting plasma exosome 3'tiRNA-43-GlyGCC-4;
[0008] The primer pair for detecting plasma exosome tRF3-17-GlyTCC includes a forward primer for detecting tRF3-17-GlyTCC and a reverse primer for detecting tRF3-17-GlyTCC;
[0009] The primer pair for 3'tiRNA-43-GlyGCC-4 includes a forward primer for detecting 3'tiRNA-43-GlyGCC-4 and a reverse primer for detecting 3'tiRNA-43-GlyGCC-4;
[0010] The tRF3-17-GlyTCC sequence is shown in SEQ ID NO.1, the forward primer for detecting tRF3-17-GlyTCC is shown in SEQ ID NO.2, and the reverse primer for detecting tRF3-17-GlyTCC is shown in SEQ ID NO.3;
[0011] The sequence of the 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.4, the forward primer for detecting 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.5, and the reverse primer for detecting 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.6.
[0012] The present invention provides a kit for differential diagnosis of benign and malignant pulmonary nodules, comprising the primer pair for detecting plasma exosome tRF3-17-GlyTCC and / or the primer pair for detecting plasma exosome 3'tiRNA-43-GlyGCC-4 as described above.
[0013] Furthermore, the kit further comprises SYBR Green mixed solution and water.
[0014] Furthermore, the SYBR Green mixture includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ .
[0015] Furthermore, the plasma exosomes were detected using the kit for differential diagnosis of benign and malignant pulmonary nodules.
[0016] Furthermore, the kit preferably includes the sequences of the internal reference U6 forward primer as shown in SEQ ID NO.7 and the internal reference U6 reverse primer as shown in SEQ ID NO.8.
[0017] Beneficial effects
[0018] The kit for differential diagnosis of benign and malignant pulmonary nodules provided by the present invention can detect plasma exosomes tRF3-17-GlyTCC and 3'tiRNA-43-GlyGCC-4 in the blood; compared with the benign pulmonary nodule group, the expression level of tRF3-17-GlyTCC in the plasma exosomes of the malignant pulmonary nodule group was significantly reduced, with an AUC of 0.703, a sensitivity of 61.4%, and a specificity of 74.7%; compared with patients with benign pulmonary nodules, the expression level of 3'tiRNA-43-GlyGCC-4 in the plasma exosomes of patients with malignant pulmonary nodules was significantly reduced, and ROC curve analysis showed an AUC of 0.671, a sensitivity of 59.1%, and a specificity of 76.4%. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The expression level of plasma exosome tRF3-17-GlyTCC in patients with malignant and benign pulmonary nodules is shown in Figure 2.
[0020] Figure 2 ROC curve for tRF3-17-GlyTCC in the diagnosis of patients with malignant pulmonary nodules;
[0021] Figure 3 The expression level of tRF3-17-GlyTCC in patients with early lung cancer at different stages.
[0022] Figure 4 The expression level of plasma exosomal 3'tiRNA-43-GlyGCC-4 in patients with malignant and benign pulmonary nodules is shown in the figure.
[0023] Figure 5 ROC curve for 3'tiRNA-43-GlyGCC-4 in the diagnosis of patients with malignant pulmonary nodules;
[0024] Figure 6 The expression level of 3'tiRNA-43-GlyGCC-4 in patients with early lung cancer at different stages. DETAILED DESCRIPTION
[0025] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.
[0026] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents and raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents and raw materials used in the present invention are used in accordance with conventional methods in the art or in accordance with the product instructions.
[0027] In a specific embodiment of the present invention, a kit for differential diagnosis of benign and malignant pulmonary nodules includes a primer pair for detecting plasma exosome tRF3-17-GlyTCC and / or a primer pair for detecting plasma exosome 3'tiRNA-43-GlyGCC-4. The primer pair for detecting plasma exosome tRF3-17-GlyTCC includes a forward primer for detecting tRF3-17-GlyTCC and a reverse primer for detecting tRF3-17-GlyTCC; the primer pair for 3'tiRNA-43-GlyGCC-4 includes a forward primer for detecting 3'tiRNA-43-GlyGCC-4 and a reverse primer for detecting 3'tiRNA-43-GlyGCC-4;
[0028] As shown in Table 1, the tRF3-17-GlyTCC sequence is shown in SEQ ID NO.1, the forward primer for detecting tRF3-17-GlyTCC is shown in SEQ ID NO.2, and the reverse primer for detecting tRF3-17-GlyTCC is shown in SEQ ID NO.3; the sequence of 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.4, the forward primer for detecting 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.5, and the reverse primer for detecting 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.6. Table 1 Sequences related to the kit for differential diagnosis of benign and malignant pulmonary nodules
[0029]
[0030] In a specific embodiment of the present invention, the kit further comprises a SYBR Green mixture and water. The SYBR Green mixture comprises a PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ ; Also includes a reverse transcription reaction system and an internal reference system; the internal reference system includes a forward primer of internal reference U6 and a reverse primer of internal reference U6, the sequence of the forward primer of internal reference U6 is shown in SEQ ID NO.7 in Table 1, and the sequence of the reverse primer of internal reference U6 is shown in SEQ ID NO.8 in Table 1; the reverse transcription reaction system includes polyadenylic acid polymerase, a reverse transcriptase mixture, a reverse transcription buffer and water.
[0031] The experimental samples in the embodiments of the present invention are 178 patients with malignant pulmonary nodules and 88 patients with benign pulmonary nodules admitted to the Affiliated Tumor Hospital of Shandong First Medical University from June 2022 to January 2023. The enrolled patients were classified as benign and malignant and TNM by pathological methods. The clinical data included in this study mainly include age, gender, tumor stage, smoking history, drinking history and nodule size. All patients with malignant pulmonary nodules were diagnosed by a combined clinical, pathological and radiological diagnostic method, and the tumor stage was determined according to the eighth edition of the TNM staging standard for lung cancer formulated by the International Association for the Study of Lung Cancer (IASLC). About 3 ml of peripheral blood sample was collected from the patient before surgery, chemotherapy and radiotherapy and placed in a vacuum blood collection tube containing a coagulant.
[0032] Example 1
[0033] (1) Determination of tRF3-17-GlyTCC levels in plasma exosomes
[0034] The expression level of tRF3-17-GlyTCC in plasma exosomes of patients with early-stage lung cancer and benign lung nodules was analyzed using qPCR. The specific steps are as follows:
[0035] 1) Extraction of plasma exosomes: Peripheral blood was collected from patients and centrifuged at 3000 g for 10 minutes at room temperature. The separated plasma was centrifuged at 12,000 g for 30 minutes at 4°C. The supernatant was collected and centrifuged at 100,000 g for 2 hours at 4°C. The supernatant was discarded, and 500 μL of Trizol reagent was added to the exosome pellet. The exosomes were lysed at 4°C for 30 minutes, then transferred to a clean enzyme-free EP tube and frozen at -80°C until further use.
[0036] 2) Extraction of total RNA from plasma exosomes
[0037] The procedure was performed in a biosafety cabinet. The pretreated frozen sample was thawed at room temperature. 100 μL of chloroform was added to 500 μL of plasma exosomes, and the mixture was shaken vigorously for 30 seconds. After standing for 3 minutes, the mixture was centrifuged at 12,000 g for 15 minutes at 4°C. (The mixture separated into a lower pink phenol-chloroform liquid phase, a middle white flocculent layer, and an upper colorless aqueous phase, in which RNA was distributed). 250 μL of the upper aqueous phase was transferred to a clean, enzyme-free EP tube, and an equal volume of isopropanol was added. The tube was shaken for 30 seconds, stood for 10 minutes, and centrifuged at 12,000 g for 10 minutes at 4°C. The liquid was discarded. 750 μL of 75% ethanol solution was added, gently inverted to mix, and centrifuged at 7,500 g for 5 minutes at 4°C. The supernatant was discarded as much as possible, retaining the white RNA precipitate at the bottom of the tube. The EP tube was opened and inverted on new filter paper to air dry for 10 to 15 minutes. Add 20 μL of RNase-free water and mix thoroughly using a pipette to completely dissolve the RNA. The dissolved RNA sample can be stored at -80°C until needed. Check RNA concentration and purity using a Nanodrop spectrophotometer.
[0038] 3) Reverse transcription reaction
[0039] Reverse transcription reaction system: 3.75 μL Total RNA, 1.25 μL RT Enzyme Mix, 5 μL RT Reaction Solution;
[0040] Reverse transcription reaction conditions: 37°C for 60 minutes, 85°C for 15 minutes to obtain cDNA, which was stored at 4°C.
[0041] 4) Preparation before real-time fluorescence PCR amplification
[0042] Prepare primer solution: The primer reagent is a dry powder. Centrifuge at 3000 rpm for 60 seconds. Then slowly open the lid and add an appropriate amount of nuclease-free double-distilled water to a final concentration of 10 μM. Close the lid and shake thoroughly to dissolve and mix.
[0043] 5) Fluorescence PCR amplification reaction
[0044] The fluorescence PCR reaction system is shown in Table 2 below:
[0045] Table 2 Fluorescence PCR reaction system
[0046]
[0047] Add each component of the fluorescent PCR reaction system to the corresponding amplification reaction plate, followed by sample cDNA. To prevent reagent contamination, immediately apply a transparent sealant to the plate after sample addition. (Note: To avoid exposure and contamination during operation, system preparation and sample addition should be performed in a biosafety cabinet.) Centrifuge at 800g for 3 minutes at room temperature to mix thoroughly, then transfer to the instrument and perform the fluorescent PCR reaction using the LightCycler 480 RT-PCR instrument according to the conditions set in Table 3 below. Each sample was replicated, and the average was calculated to reduce error. The experiment was repeated three times.
[0048] Table 3 Fluorescence PCR reaction conditions
[0049]
[0050] 6) Internal reference system amplification reaction
[0051] The U6 gene was used as the internal reference gene. The internal reference system included 10 μL SYBR Green mixture, 0.8 μL internal reference U6 forward primer, 0.8 μL internal reference U6 reverse primer, 6.4 μL enzyme-free water, and 2 μL cDNA. The reaction conditions of the internal reference system were as follows: pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing at 60°C for 20 seconds, and extension at 65°C for 15 seconds, for a total of 40 cycles.
[0052] (2) Data statistical analysis
[0053] qPCR data were analyzed using Prism 9.0. Statistical data from both groups that met normal distribution were expressed as mean ± standard deviation using the t-test. Otherwise, data were expressed as median ± interquartile range using the Mann-Whitney test. Receiver operating characteristic (ROC) curves were calculated using SPSS 26.0 to evaluate the clinical diagnostic performance of tRF3-17-GlyTCC. A P value of < 0.05 was considered statistically significant. All tests were two-tailed.
[0054] (3) Summary of clinicopathological characteristics of patients with benign and malignant pulmonary nodules and their relationship with tRF3-17-GlyTCC expression levels
[0055] Table 4 shows the clinical characteristics of patients with benign and malignant pulmonary nodules, including age, gender, smoking history, and alcohol consumption history. tRF3-17-GlyTCC expression levels were associated with pulmonary nodule size but not with age, gender, smoking, or alcohol consumption.
[0056] Table 4 Summary of clinicopathological characteristics of patients with benign and malignant pulmonary nodules and their relationship with tRF3-17-GlyTCC expression levels
[0057]
[0058] (4) Differences in expression levels of plasma exosomal tRF3-17-GlyTCC in patients with malignant and benign pulmonary nodules
[0059] The qPCR method was used to verify the plasma exosome tRF3-17-GlyTCC levels in patients with malignant pulmonary nodules and benign pulmonary nodules; the expression levels of plasma exosome tRF3-17-GlyTCC in patients with malignant pulmonary nodules and benign pulmonary nodules were as follows: Figure 1 As shown in the figure, compared with patients with benign pulmonary nodules, the expression level of tRF3-17-GlyTCC in patients with malignant pulmonary nodules was significantly reduced, and the difference was statistically significant (P < 0.0001).
[0060] (5) Analysis of the diagnostic efficacy of plasma exosome tRF3-17-GlyTCC for malignant pulmonary nodules
[0061] The ROC curve was constructed to evaluate the clinical diagnostic efficacy of tRF3-17-GlyTCC for malignant pulmonary nodules. Figure 2 As shown, the results showed that the AUC was 0.703, the sensitivity was 61.4%, and the specificity was 74.7%.
[0062] (6) Differences in plasma exosomal tRF3-17-GlyTCC levels in patients with early-stage lung cancer and benign pulmonary nodules
[0063] The expression levels of tRF3-17-GlyTCC in patients with early lung cancer at different stages are shown in the figure. Figure 3 As described above, the qPCR test results showed that compared with patients with benign pulmonary nodules, the expression levels of plasma exosome tRF3-17-GlyTCC in patients with stage 0 and stage I lung cancer were significantly reduced, and the differences were statistically significant (both P < 0.0001).
[0064] Example 2
[0065] (1) Determination of 3'tiRNA-43-GlyGCC-4 levels in plasma exosomes
[0066] The expression level of 3'tiRNA-43-GlyGCC-4 in plasma exosomes of patients with early-stage lung cancer and benign lung nodules was analyzed using qPCR. The specific steps are as follows:
[0067] 1) The plasma exosome extraction method, total RNA extraction, and reverse transcription reaction were the same as in Example 1.
[0068] 2) Preparation before real-time fluorescence PCR amplification
[0069] Prepare primer solution: The primer reagent is a dry powder. Centrifuge at 3000 rpm for 60 seconds. Then slowly open the lid and add an appropriate amount of nuclease-free double-distilled water to a final concentration of 10 μM. Close the lid and shake thoroughly to dissolve and mix.
[0070] 3) Fluorescence PCR amplification reaction
[0071] The fluorescence PCR reaction system is shown in Table 5 below:
[0072] Table 5 Fluorescence PCR reaction system
[0073]
[0074] Each component of the fluorescent PCR reaction system was sequentially added to the corresponding amplification reaction plate, and the sample cDNA was added. To avoid reagent contamination, a transparent sealing film should be attached immediately after sample addition. (Note: To avoid exposure contamination during operation, the system preparation and sample addition should be performed in a biosafety cabinet.) After centrifugation at 800g for 3 minutes at room temperature to mix, the plate was loaded onto the instrument and the fluorescent PCR reaction conditions set in Table 3 were set. Each sample in this reaction was set as a replicate, and the average was taken to reduce error. The experiment was repeated three times; the fluorescent PCR reaction conditions were the same as in Example 1.
[0075] 6) The amplification reaction conditions of the internal reference system were the same as those in Example 1.
[0076] (2) Data statistical analysis
[0077] qPCR data were analyzed using Prism 9.0. Data from both groups that met normal distribution were expressed as mean ± standard deviation using the t-test. Data that did not meet normal distribution were expressed as median ± interquartile range using the Mann-Whitney test. Receiver operating characteristic (ROC) curves were calculated using SPSS 26.0 to evaluate the clinical diagnostic efficacy of 3'tiRNA-43-GlyGCC-4. P < 0.05 was considered statistically significant. All tests were two-tailed.
[0078] (3) Summary of clinicopathological characteristics of patients with benign and malignant pulmonary nodules and their relationship with 3'tiRNA-43-GlyGCC-4 expression levels
[0079] The clinical characteristics of patients with benign and malignant pulmonary nodules were analyzed separately, including age, gender, smoking history, and alcohol consumption history. The results showed that the expression level of 3'tiRNA-43-GlyGCC-4 was related to the size of pulmonary nodules, but was not related to age, gender, smoking history, or alcohol consumption.
[0080] (4) Differences in expression levels of plasma exosomal 3'tiRNA-43-GlyGCC-4 in patients with malignant and benign pulmonary nodules
[0081] The qPCR method was used to verify the plasma exosome 3'tiRNA-43-GlyGCC-4 levels in patients with malignant pulmonary nodules and benign pulmonary nodules; the expression levels of plasma exosome 3'tiRNA-43-GlyGCC-4 in patients with malignant pulmonary nodules and benign pulmonary nodules were as follows: Figure 4 As shown in the figure, compared with patients with benign pulmonary nodules, the expression level of 3'tiRNA-43-GlyGCC-4 in patients with malignant pulmonary nodules was significantly reduced, and the difference was statistically significant (P < 0.0001).
[0082] (5) Analysis of the diagnostic efficacy of plasma exosomal 3'tiRNA-43-GlyGCC-4 for malignant pulmonary nodules
[0083] The ROC curve was constructed to evaluate the clinical diagnostic efficacy of 3'tiRNA-43-GlyGCC-4 for malignant pulmonary nodules. Figure 5 As shown, the results showed that the AUC was 0.671, the sensitivity was 59.1%, and the specificity was 76.4%.
[0084] (6) Differences in the levels of plasma exosomal 3'tiRNA-43-GlyGCC-4 in patients with early-stage lung cancer and benign pulmonary nodules The expression levels of 3'tiRNA-43-GlyGCC-4 in patients with early-stage lung cancer at different stages are shown in the figure. Figure 6 As shown in the results, the expression levels of plasma exosomal 3'tiRNA-43-GlyGCC-4 were significantly decreased in patients with stage 0 and stage I lung cancer compared with those with benign pulmonary nodules (P = 0.0425, P < 0.0001, respectively).
Claims
1. A primer pair for differential diagnosis of benign and malignant pulmonary nodules, characterized in that: Including a primer pair for detecting plasma exosome tRF3-17-GlyTCC and / or a primer pair for detecting plasma exosome 3'tiRNA-43-GlyGCC-4; the primer pair for detecting plasma exosome tRF3-17-GlyTCC includes a forward primer for detecting tRF3-17-GlyTCC and a reverse primer for detecting tRF3-17-GlyTCC; The primer pair for 3'tiRNA-43-GlyGCC-4 includes a forward primer for detecting 3'tiRNA-43-GlyGCC-4 and a reverse primer for detecting 3'tiRNA-43-GlyGCC-4; The tRF3-17-GlyTCC sequence is shown in SEQ ID NO.1, the forward primer for detecting tRF3-17-GlyTCC is shown in SEQ ID NO.2, and the reverse primer for detecting tRF3-17-GlyTCC is shown in SEQ ID NO.3; The sequence of the 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.4, the forward primer for detecting 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.5, and the reverse primer for detecting 3'tiRNA-43-GlyGCC-4 is shown in SEQ ID NO.
6.
2. A kit for differential diagnosis of benign and malignant pulmonary nodules, characterized in that: The method comprises the primer pair for detecting plasma exosome tRF3-17-GlyTCC and / or the primer pair for detecting plasma exosome 3'tiRNA-43-GlyGCC-4 according to claim 1.
3. The kit for differential diagnosis of benign and malignant pulmonary nodules according to claim 2, characterized in that: The kit also includes SYBR Green mixture and water.
4. The kit for differential diagnosis of benign and malignant pulmonary nodules according to claim 3, characterized in that: The SYBRGreen mixed solution includes PCR reaction buffer, SYBR Green I dye, ROX, dNTPs, Mg 2+ .
5. The kit for differential diagnosis of benign and malignant pulmonary nodules according to claim 2, characterized in that: The plasma exosomes were detected using the kit for differential diagnosis of benign and malignant pulmonary nodules.
6. The kit for differential diagnosis of benign and malignant pulmonary nodules according to claim 2, characterized in that: The kit preferably includes the sequences of the internal reference U6 forward primer as shown in SEQ ID NO.7 and the internal reference U6 reverse primer as shown in SEQ ID NO.8.