A fluorescence co-detection method for tumor marker miRNA and mRNA based on strand displacement reaction and its application in the diagnosis of gastric cancer

Abstract

The present invention provides a tumor marker fluorescence co-detection method based on strand displacement reaction and its application in gastric cancer diagnosis, and fluorescent probes for tumor markers miRNA and mRNA, including a group of double-stranded probes P1T1 for miR-5585-5p detection, a group of double-stranded probes P2T2 for PLS3 detection, and a group of double-stranded probes A1A2 based on strand displacement reaction for amplifying fluorescence signals. The linear range of the present invention for detecting miR-5585-5p in the serum background is 1 fM-10 nM, and the regression equation is fitted as follows: Y=221.3*X+770.3( R 2 =0.9782), the calculated minimum detection limit was 1.19 fM. The linear range of PLS3 detection was 1 fM-10 nM, and the regression equation was fitted as follows: Y=234.2*X+4169 ( R 2 =0.9717), the calculated minimum detection limit was 16.37 fM. And it has high detection specificity and accuracy. The gastric cancer diagnosis model established based on the detection method for miR-5585-5p and PLS3 has an area under the curve (AUC) of 0.884, a standard error of 0.044, a 95% confidence interval of 0.785-0.948, a diagnostic sensitivity of 0.913, and a specificity of 0.75 for gastric cancer diagnosis, which is significantly better than the traditional tumor marker CA 72-4.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a method for fluorescent co-detection of tumor markers miRNA and mRNA based on strand displacement reaction and its application in gastric cancer diagnosis. Background Art

[0002] Gastric cancer (GC) is the fifth most common type of cancer and the third leading cause of cancer death worldwide. It is estimated that about half of the world's GC deaths occur in China. Due to the insidious onset of GC, most patients are diagnosed in the middle and late stages with a poor prognosis, suggesting that early diagnosis is crucial for the prevention and treatment of GC and improving survival. However, commonly used clinical examination methods such as gastroscopy and CT have a low detection rate for early tumor microlesions. Pathological biopsy is limited by its invasiveness and is not suitable for population screening. Traditional serological tumor biomarkers such as carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 and CA72-4 are not sufficient for GC detection due to their poor sensitivity and specificity. Therefore, it is imperative to develop clinically operable, sensitive, and non-invasive screening methods to screen effective biomarkers for the early diagnosis of GC.

[0003] Tumor circulating nucleic acids, as specific fragments released by physical cells into the blood circulation system, retain the unique genetic characteristics of tumors, effectively get rid of the dependence on primary tissues, and provide a non-invasive, rapid and accurate method for the early diagnosis of tumors. However, its widespread application in clinical practice still faces huge challenges. On the one hand, due to the heterogeneity, diversity and complexity of malignant tumors, a single nucleic acid molecule marker is difficult to meet the needs of clinical precision diagnosis; on the other hand, traditional quantitative methods commonly used in clinical practice, including RT-qPCR, still rely on enzymes, expensive equipment and strict temperature control. Summary of the invention

[0004] Aiming at the problems existing in the traditional technology, the present invention proposes a tumor marker fluorescence co-detection method based on strand displacement reaction and its application in gastric cancer diagnosis

[0005] In the first aspect, the present invention provides a fluorescent probe for tumor marker miRNA and mRNA based on strand displacement reaction, including a group of double-stranded probes P1T1 for detecting miR-5585-5p, a group of double-stranded probes P2T2 for detecting PLS3, and a group of double-stranded probes A1A2 for amplifying fluorescence signals based on strand displacement reaction; the double-stranded probe P1T1 for detecting miR-5585-5pmiRNA, the miR-5585-5p sequence is as shown in SEQ ID NO.1, the P1 modified Cy5 fluorescent molecule sequence is 5'-GGTTGAGC / iCy5dT / CTCTCGAGTAGCTGGTACTTCA-3', and the T1 modified BHQ-3 molecule sequence is 5'-CCAGCCCAGCTACTCGAGAG(BHQ-3)-3'; the double-stranded probe P2T2 for detecting PLS3, the PLS3 sequence is as shown in SEQ ID NO. As shown in IDNO.2, the sequence of the P2-modified FAM fluorescent molecule is 5'-CAGCCTCAACCAGCCCGAGTTT / i6FAMdT / CCAAATGA-3', and the sequence of the T2-modified BHQ-1 molecule is 5'-(BHQ-1)GAAAACTCGGGCTGGGCTGGTT-3'.

[0006] Furthermore, the double-stranded probe A1A2 used to amplify the fluorescence signal, the A1 sequence is shown in SEQ ID NO.3, and the A2 sequence is shown in SEQ ID NO.4. A1 and A2 are respectively involved in the reaction between miR-5585-5p and P1T1, and PLS3 and P2T2, and the fluorescence signal is continuously amplified through the chain displacement reaction to achieve high-sensitivity detection.

[0007] In a second aspect, the present invention provides a method for fluorescent co-detection of tumor markers based on strand displacement reaction, comprising the following steps:

[0008] (1) Double-stranded probe self-assembly: Use ultrapure water to dissolve the probe into a 50 μM solution, and take A1A2, P1T1, and P2T2 to cool down the solution in a gradient manner to allow the probe to self-assemble into a double-stranded probe solution with a concentration of 10 μM;

[0009] (2) Fluorescence detection process: A1A2, P1T1, and P2T2 were diluted with 1× PBS buffer to form a probe solution with a concentration of 100 nM. The A1A2, P1T1, and P2T2 probe solutions were added to 1× PBS buffer, and the sample to be tested was added dropwise and mixed by pipetting. The solution was placed at room temperature for reaction. After the reaction was completed, the fluorescence signal was detected using a Hitachi F7100 fluorescence spectrophotometer.

[0010] (3) Methodological evaluation: Methodological evaluation of the sensitivity, specificity, accuracy and co-detection feasibility of the detection method;

[0011] (4) Serum detection capability verification: RNase A was added to the serum of healthy people to digest free RNA, and different concentrations of target molecules were added after treatment. After optimizing the reaction time, the detection capability of the detection method in the serum background was evaluated using the above samples;

[0012] (5) Establishment of a combined diagnostic model for gastric cancer based on miR-5585-5p and PLS3: The fluorescence signals of miR-5585-5p and PLS3 in the sera of 46 gastric cancer patients and 24 healthy subjects were collected. A Logistics regression diagnostic model was established based on the fluorescence intensity using MedCalc software to evaluate the diagnostic efficacy of the model.

[0013] Furthermore, the gradient annealing process in step (1) is 95°C 5min-65°C 30min-50°C 30min-37°C 30min-22°C 30min-4°C.

[0014] Furthermore, the reaction volume in step (2) is 200 μL, including 20 μL of the sample to be tested, 20 μL of A1A2, 20 μL of P1T1, 40 μL of P2T2 and 100 μL of 1×PBS buffer, and the reaction time is 50 min.

[0015] Furthermore, the detection process in step (2) is divided into two steps, using a spectral scanning method. The first step uses a 640nm wavelength for excitation, scans the emission spectrum in the wavelength range of 660-760nm, and takes the fluorescence intensity at 665nm as the fluorescence signal of miR-5585-5p; the second step uses a 495nm wavelength for excitation, scans the emission spectrum in the wavelength range of 515-620nm, and takes the fluorescence intensity at 520nm as the fluorescence signal of PLS3.

[0016] Furthermore, the serum detection process in step (4) is divided into two steps, using a spectral scanning method. The first step uses a 635nm wavelength excitation, scans the emission spectrum in the wavelength range of 655-760nm, and takes the fluorescence intensity at 665nm as the fluorescence signal of miR-5585-5p; the second step uses a 490nm wavelength excitation, scans the emission spectrum in the wavelength range of 510-620nm, and takes the fluorescence intensity at 520nm as the fluorescence signal of PLS3.

[0017] In the third aspect, the present invention also provides a gastric cancer diagnosis model established by detecting miR-5585-5p and PLS3 using a fluorescent biosensor. The area under the curve for diagnosing gastric cancer is 0.884, the standard error is 0.044, the 95% confidence interval is 0.785-0.948, the diagnostic sensitivity is 0.913, and the specificity is 0.75.

[0018] Compared with the prior art, the reaction principle of the present invention is as follows: in the present sensing system, the double-stranded probe P1T1 for miR-5585-5p recognition is composed of the recognition probe P1 containing the fluorophore Cy5 label and the probe T1 labeled with BHQ-3, the P2T2 complex for PLS3 recognition is composed of the FAM-labeled P2 and the BHQ-1-labeled T2, and the double-stranded probe A1A2 can participate in the reaction of P1T1 and P2T2 respectively for signal amplification. Due to their respective partial complementary pairing, each double-stranded probe can coexist in a metastable state in the absence of the Target sequence, and the fluorescence intensity is weak. In the presence of miR-5585-5p, P1 preferentially binds to miR-5585-5p because the binding force between P1 and miR-5585-5p (free energy ΔG = -37.51 kcal / mol) is stronger than the binding force between the P1-T1 complex (-26.79 kcal / mol), which causes the single-stranded T1 to be released from the P1-T1 complex. The ΔG of T1-A2 is designed to be -33.28 kcal / mol, which is lower than the -31 kcal / mol of A1-A2, so as to form a hybrid between T1 and A2 through base pairing and release A1. Since the ΔG of A1-P1 (-29.33 kcal / mol) is lower than that of P1-T1, it tends to form A1-P1 in the subsequent reaction, resulting in the release of T1 and the recovery of Cy5 fluorescence. Further, T1 continues to open the A1-A2 complex through base pairing and releases A1, achieving the recycling of A1 and continuous cascade fluorescence generation. In the same way, PLS3 detection is achieved through a similar principle. In this way, the sensor system achieves ultra-sensitive detection of miR-5585-5p and PLS3 in a non-enzymatic and isothermal form. At the same time, this system realizes the conversion of target detection from RNA to DNA for the first time, reducing the interference caused by RNA degradation and improving the stability of the biosensor.

[0019] Compared with the prior art, the present invention is beneficial in that the linear range of the present invention for detecting miR-5585-5p in the serum background is 1 fM-10 nM, and the regression equation is fitted as follows: Y = 221.3*X + 770.3 (R 2 =0.9782), the calculated minimum detection limit was 1.19 fM. The linear range of PLS3 detection was 1 fM-10 nM, and the regression equation was fitted as follows: Y = 234.2*X + 4169 (R 2=0.9717), and the calculated minimum detection limit was 16.37fM. The results of blank spike recovery experiment and specificity experiment showed that this method has high detection specificity and accuracy. The gastric cancer diagnosis model established based on the detection method for miR-5585-5p and PLS3 had an area under the curve (AUC) of 0.884, a standard error of 0.044, a 95% confidence interval of 0.785-0.948, a diagnostic sensitivity of 0.913, and a specificity of 0.75, which is significantly better than the traditional tumor marker CA 72-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings. The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 This is a detection schematic diagram of the present detection method;

[0022] Figure 2 For the sensitivity evaluation of the present invention;

[0023] Figure 3 To evaluate the specificity of the present invention;

[0024] Figure 4 To evaluate the feasibility of co-detection of mRNA and miRNA of the present invention;

[0025] Figure 5 To optimize the serum detection time of the present invention;

[0026] Figure 6 To evaluate the sensitivity of the detection method in the serum background;

[0027] Figure 7 To evaluate the diagnostic efficacy of the gastric cancer diagnostic model established based on this detection method. DETAILED DESCRIPTION

[0028] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the present invention is not limited to the limitations of the specific embodiments of the following disclosure.

[0029] Example 1

[0030] This embodiment includes a group of double-stranded probes P1T1 for detecting miR-5585-5p, a group of double-stranded probes P2T2 for detecting PLS3, and a group of double-stranded probes A1A2 for amplifying fluorescent signals based on strand displacement reaction. The reaction principle is as follows: Figure 1 shown.

[0031] The double-stranded probe P1T1 used for miR-5585-5p miRNA detection, the miR-5585-5p sequence is shown in SEQ ID NO.1, the P1 modified Cy5 fluorescent molecule sequence is 5'-GGTTGAGC / iCy5dT / CTCTCGAGTAGCTGGTACTTCA-3', and the T1 modified BHQ-3 molecule sequence is 5'-CCAGCCCAGCTACTCGAGAG(BHQ-3)-3'. The double-stranded probe P2T2 used for PLS3 detection, the PLS3 sequence is shown in SEQ ID NO.2, the P2 modified FAM fluorescent molecule sequence is 5'-CAGCCTCAACCAGCCCGAGTTT / i6FAMdT / CCAAATGA-3', and the T2 modified BHQ-1 molecule sequence is 5'-(BHQ-1)GAAAACTCGGGCTGGGCTGGTT-3'. The double-stranded probe A1A2 used to amplify the fluorescence signal, the A1 sequence is shown in SEQ ID NO.3, and the A2 sequence is shown in SEQ ID NO.4. A1 and A2 are respectively involved in the reaction between miR-5585-5p and P1T1, and PLS3 and P2T2, and the fluorescence signal is continuously amplified through the chain displacement reaction to achieve high-sensitivity detection.

[0032] Double-stranded probe self-assembly: Use ultrapure water to dissolve the probe into a 50 μM solution, and gradient cooling to allow the probe to self-assemble into a 10 μM double-stranded probe solution. The gradient annealing process is 95°C 5min-65°C 30min-50°C 30min-37°C 30min-22°C 30min-4°C.

[0033] Example 2

[0034] 1. Optimization of reaction conditions: In order to obtain better detection performance, the fluorescence intensity of the target molecule of 10nM was used to optimize the reaction concentrations of A1A2, P1T1, and P2T2 as well as the reaction time. The results showed that when the reaction concentration of P1T1 was 10nM, the reaction concentration of P2T2 was 20nM, the reaction concentration of A1A2 was 10nM, and the reaction time was 50min, the fluorescence intensity of the target molecule was the highest.

[0035] 2. Fluorescence detection process: A1A2, P1T1, and P2T2 were diluted with 1×PBS buffer to form a probe solution with a concentration of 100nM. 20μL of the sample to be tested, 20μL P1T1, 40μL P2T2, 20μL A1A2, and 100μL 1×PBS buffer were taken to form a 200μL reaction system and mixed by pipetting. The solution was placed at room temperature for 20 minutes. After the reaction was completed, the fluorescence signal was detected using a Hitachi F7100 fluorescence spectrophotometer. The detection process is divided into two steps, using a spectral scanning method. The first step is to use 640nm wavelength excitation, scan the emission spectrum in the wavelength range of 660-760nm, and take the fluorescence intensity at 665nm as the fluorescence signal of miR-5585-5p. The second step is to use 495nm wavelength excitation, scan the emission spectrum in the wavelength range of 515-620nm, and take the fluorescence intensity at 520nm as the fluorescence signal of PLS3.

[0036] Example 3

[0037] 1. Sensitivity evaluation: Use DNA simulation standards of different concentrations of the target molecule to evaluate the sensitivity of the detection method, such as Figure 2 As shown, R -5585-5p in the range of 10fM to 5.5nM, the fluorescence intensity gradually increased with the increase of concentration. The regression equation was fitted as follows: Y = 74.05*X + 707.3 (R 2 =0.9239), the calculated minimum detection limit was 2.38fM. In the range of 1pM to 5.5nM, the fluorescence intensity of PLS3 gradually increased with the increase of concentration. The regression equation was fitted as follows: Y = 149.5*X + 2153 (R 2 =0.9493), and the calculated minimum detection limit was 3.52 pM.

[0038] 2. Specificity evaluation: The specificity of this method for detecting miR-5585-5p was evaluated by detecting miR-5585-5p (10nM), PLS3 (10nM), piR-28527 (10nM), piR-28190 (10nM), miR-21 (10nM), and miR-92a-3p (10nM). Figure 3As shown, the results showed that only the miR-5585-5p group had a higher fluorescence intensity, confirming that the biosensor has good specificity for the detection of miR-5585-5p. The specificity of the method for detecting PLS3 was evaluated by detecting PLS3 (10nM), miR-5585-5p (10nM), GAPDH (10nM), MDR1 (10nM), Survivin (10nM), and TK1 (10nM). The results showed that only the PLS3 group had a higher fluorescence intensity, confirming that the biosensor also has good specificity for the detection of PLS3.

[0039] The miR-5585-5p sequence is shown in SEQ ID NO.1, the PLS3 sequence is shown in SEQ ID NO.2, the MDR1 sequence is shown in SEQ ID NO.5, the TK1 sequence is shown in SEQ ID NO.6, the Survivin sequence is shown in SEQ ID NO.7, the GAPDH sequence is shown in SEQ ID NO.8, the miR-92a-3p sequence is shown in SEQ ID NO.9, the miR-21 sequence is shown in SEQ ID NO.10, the piR-28190 sequence is shown in SEQ ID NO.11, and the piR-28527 sequence is shown in SEQ ID NO.12.

[0040] 3. Accuracy evaluation: The accuracy of the detection method was evaluated by blank spike recovery experiment. The test results are shown in Tables 1 and 2 below, which prove that the detection method has high accuracy.

[0041] Table 1 Results of blank spike recovery experiment of miR-5585-5p

[0042]

[0043] Table 2 PLS3 blank spike recovery test results

[0044]

[0045] 4. Co-detection feasibility evaluation: Compare the fluorescence intensity of the target molecule with the same concentration in single detection mode and dual detection mode, such as Figure 4 As shown, the results showed that there was no significant difference in the fluorescence intensity of the two molecules in the two detection modes, and they had good co-detection feasibility.

[0046] Example 4

[0047] Verification of serum detection capability: add 10 μL RNase A solution to every 500 μL healthy human serum and incubate at 37°C for 3 hours to digest free RNA. The serum detection process is divided into two steps, using spectral scanning. The first step uses 635nm wavelength excitation, scans the emission spectrum in the wavelength range of 655-760nm, and takes the fluorescence intensity at 665nm as the fluorescence signal of miR-5585-5p; the second step uses 490nm wavelength excitation, scans the emission spectrum in the wavelength range of 510-620nm, and takes the fluorescence intensity at 520nm as the fluorescence signal of PLS3. Figure 5 As shown in the figure, the results show that when the reaction time is 20 min, the fluorescence intensity of the target molecule is the highest, and the fluorescence intensity of miR-5585-5p is in the range of 1 fM to 10 nM, and gradually increases with the increase of concentration. Figure 6 As shown, the regression equation is fitted as follows: Y = 221.3*X + 770.3 (R 2 =0.9782), the calculated minimum detection limit is 1.19 fM. In the range of 1 fM to 10 nM, the fluorescence intensity of PLS3 gradually increases with the increase of concentration. The regression equation is fitted as follows: Y = 234.2*X + 4169 (R 2 =0.9717), and the calculated minimum detection limit was 16.37 fM.

[0048] Example 5

[0049] Establishment of a combined diagnostic model for gastric cancer based on miR-5585-5p and PLS3: The fluorescence signals of miR-5585-5p and PLS3 in the serum of 46 gastric cancer patients and 24 healthy subjects were collected, and a Logistics regression diagnostic model was established based on the fluorescence intensity using MedCalc software. The results are shown in Table 3 below. The ROC curve ( Figure 7 )The analysis results showed that the gastric cancer diagnostic model established based on the detection of miR-5585-5p and PLS3 by fluorescent biosensor had an area under the curve (AUC) of 0.884, a standard error of 0.044, and a 95% confidence interval of 0.785-0.948 for diagnosing gastric cancer, which was significantly better than the traditional tumor marker CA72-4.

[0050] Table 3Logistics regression analysis results

[0051]

[0052] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A fluorescent probe for tumor marker miRNA and mRNA based on strand displacement reaction, characterized in that: The invention comprises a group of double-stranded probes P1T1 for detecting miR-5585-5p, a group of double-stranded probes P2T2 for detecting PLS3, and a group of double-stranded probes A1A2 for amplifying fluorescence signals based on a strand displacement reaction; the double-stranded probe P1T1 for detecting miR-5585-5p miRNA, the miR-5585-5p sequence is as shown in SEQ ID NO.1, the P1 modified Cy5 fluorescent molecule sequence is 5'-GGTTGAGC / iCy5dT / CTCTCGAGTAGCTGGTACTTCA-3', the T1 modified BHQ-3 molecule sequence is 5'-CCAGCCCAGCTACTCGAGAG(BHQ-3)-3'; the double-stranded probe P2T2 for detecting PLS3, the PLS3 sequence is as shown in SEQ ID As shown in NO.2, the sequence of the P2-modified FAM fluorescent molecule is 5'-CAGCCTCAACCAGCCCGAGTTT / i6FAMdT / CCAAATGA-3', and the sequence of the T2-modified BHQ-1 molecule is 5'-(BHQ-1)GAAAACTCGGGCTGGGCTGGTT-3'.

2. The fluorescent probe of tumor marker miRNA and mRNA based on strand displacement reaction according to claim 1, characterized in that: The double-stranded probe A1A2 used to amplify the fluorescence signal, the A1 sequence is shown in SEQ ID NO.3, and the A2 sequence is shown in SEQ ID NO.

4. A1 and A2 are respectively involved in the reaction between miR-5585-5p and P1T1, and PLS3 and P2T2, and the fluorescence signal is continuously amplified through the chain displacement reaction to achieve high-sensitivity detection.

3. A method for constructing a gastric cancer joint diagnosis model based on a strand displacement reaction of tumor marker miRNA and mRNA fluorescent probe according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Double-stranded probe self-assembly: Use ultrapure water to dissolve the probe into a 50 μM solution, and take A1A2, P1T1, and P2T2 to cool down the solution in a gradient manner to allow the probe to self-assemble into a double-stranded probe solution with a concentration of 10 μM; (2) Fluorescence detection process: A1A2, P1T1, and P2T2 were diluted with 1× PBS buffer to form a probe solution with a concentration of 100 nM. The A1A2, P1T1, and P2T2 probe solutions were added to 1× PBS buffer, and the sample to be tested was added dropwise and mixed by pipetting. The solution was placed at room temperature for reaction. After the reaction was completed, the fluorescence signal was detected using a fluorescence spectrophotometer. (3) Methodological evaluation: Methodological evaluation of the sensitivity, specificity, accuracy and co-detection feasibility of the detection method; (4) Serum detection capability verification: RNase A was added to the serum of healthy people to digest free RNA, and different concentrations of target molecules were added after treatment. After optimizing the reaction time, the detection capability of the detection method in the serum background was evaluated; (5) Establish a combined diagnostic model for gastric cancer based on miR-5585-5p and PLS3. Collect the fluorescence signals of miR-5585-5p and PLS3 in the serum of gastric cancer patients and healthy subjects. Use MedCalc software to establish a logistics model based on the fluorescence intensity. Regression diagnostic model.

4. The method according to claim 3, characterized in that: The gradient annealing process in step (1) is 95°C 5min-65°C 30min-50°C 30min-37°C 30min-22°C 30min-4°C.

5. The method according to claim 3, characterized in that: The reaction volume in step (2) is 200 μL, including 20 μL of the sample to be tested, 20 μL of A1A2, 20 μL of P1T1, 40 μL of P2T2 and 100 μL of 1×PBS buffer, and the reaction time is 50 min.

6. The method according to claim 3, characterized in that: In step (4), the detection process is divided into two steps. The first step is to use 640nm wavelength excitation, scan the emission spectrum in the wavelength range of 660-760nm, and take the fluorescence intensity at 665nm as the fluorescence signal of miR-5585-5p; the second step is to use 495nm wavelength excitation, scan the emission spectrum in the wavelength range of 515-620nm, and take the fluorescence intensity at 520nm as the fluorescence signal of PLS3.

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