A method for detecting exosome-associated microRNA molecules

Abstract

The application discloses a method for detecting exosome-related microRNA molecules, and develops a two-step method detection reagent kit based on a PCR platform miRNA, which has a prominent advantage in the auxiliary diagnosis of lung cancer, and the minimum sensitivity can reach 1 copy / muL, and the detection accuracy is greatly improved.

Description

[0001] The present application is a divisional application of CN107365852A. The original application patent has an application number of 2017106930281, an application date of August 14, 2017, and an invention name of "Application of lung cancer related microRNA molecular marker in serum exosome and detection kit thereof". TECHNICAL FIELD

[0002] The present application belongs to the technical field of medical molecular biology, and particularly relates to a method for detecting exosome related microRNA molecules. BACKGROUND

[0003] Exosome is a kind of membrane vesicle widely existing and distributed in various body fluids, which can be secreted by various cells. The diameter is generally between 30-120 nm, and contains cell-specific proteins, lipids and nucleic acids. In addition to carrying and transmitting important signal molecules, forming a new intercellular information transmission system to change the function of other cells, it also plays an important role in many physiological and pathological processes. Studies have shown that the molecular characteristics of tumor exosome reflect the phenotype of the tumor of origin, and the tumor-specific microRNA and antigens carried can be used as tumor diagnostic markers. In addition, exosome can selectively remove certain cell proteins and transmit various types of molecules between cells, which can induce and enhance the immune response of the body, and has important functions in immune surveillance, inflammatory response and cancer development. In various tumor clinical cases including bladder cancer, brain tumor, colorectal cancer and melanoma, exosome can be isolated from the body fluids such as serum or urine of patients for early clinical diagnosis, and can also be used for clinical risk or efficacy evaluation of tumor, and prognosis determination.

[0004] Exosome contains a large amount of mRNA and microRNA, which not only protects the existence of RNA from degradation in vitro, but also can be used as an effective carrier to transport RNA to specific target cells to play an important regulatory role. More than 120 microRNAs carried by exosome have various functions. For example, miR-1, miR-17, miR-18, miR-181 and miR-375 are related to angiogenesis, hematopoiesis, extracellular secretion and tumor occurrence.

[0005] microRNA (also known as miRNA or miR) - as the first of the top ten scientific breakthroughs in 2002 Science - is one of the major discoveries of life science research in the 21st century, which plays a very important role in the development of biological timing control and disease occurrence. miRNA regulates the expression of oncogenes and tumor suppressor genes, regulates cell differentiation, proliferation and apoptosis, thereby promoting or inhibiting the occurrence of tumors, with complex regulatory mechanisms, forming a regulatory network, and promoting or inhibiting the occurrence of tumors. Methylation, biological defects, variations, transcription abnormalities, and genomic loss or amplification all lead to abnormal miRNA in human tumors. Many miRNAs directly exhibit the effects of a proto-oncogene or a tumor suppressor gene, and oncogenic and tumor suppressor miRNAs directly regulate tumor cell proliferation, differentiation and apoptosis by positively or negatively regulating tumor suppressor genes, cancer genes or genes that control cell cycle progression, differentiation or apoptosis, and participate in tumor generation, development and even invasion and metastasis. Numerous studies have shown that miRNA has characteristic expression profile changes in tumor cells, cancer tissues, adjacent tissues, and normal tissues, and has characteristic expression level changes in the blood and urine of tumor patients, which provides a new way for tumor diagnosis and suggests that miRNA can become an important molecular biology marker for tumor diagnosis.

[0006] Peripheral blood has always been the main source of clinical disease marker detection because of its small trauma, easy access, repeatability, and many detectable indicators. In recent years, research has found that there are endogenous circulating miRNAs in peripheral blood, and because of their high stability and specificity, they are expected to become biological markers for various diseases such as tumors. Some researchers have suggested that circulating miRNAs mainly exist in exosomes and may be a good source for detecting serum miRNAs. Therefore, if combined with the characteristics of exo-miRNA, if a corresponding high specificity lung cancer diagnostic kit can be developed to be applied to the field of auxiliary differential diagnosis of benign and malignant lung nodules and scientific research, it will greatly promote lung cancer screening research and the transformation of scientific research achievements, and will play a huge role in the differentiation, diagnosis and treatment of benign and malignant lung tumors.

[0007] Currently, the existing microRNA as a lung cancer molecular marker for detection has the defects of low accuracy and specificity or the need for simultaneous detection of several markers, high detection cost, and no suitable commercial production kit.

[0008] In addition, the two-step method detection system of miRNA based on PCR platform mainly includes probe method miRNA quantitative detection technology and dye method detection technology, 1), the quantitative detection technology combined with the probe, including Stem-loop RT-PCR probe method, key-like method and enzyme ligation method (Ligation Assay). The three methods need to use miRNA specific probe, and the outstanding advantages of this method are strong specificity, and different variants of the same miRNA family can often be distinguished. But there is a phenomenon that the Stem-loop RT is not firmly combined with miRNA, and the mismatch of the stem-loop primer with the non-target miRNA. 2), the quantitative detection technology based on PCR and fluorescent dye such as SYBR Green, including poly(A) polymerase tailing method, Stem-loop dye method, primer extension method, multiplexed RT, etc. The use of SYBR Green technology is mostly high in sensitivity and generally low in cost, but the specificity is low. Relatively, the method of adding PolyA tail and stem loop structure lengthens the paired sequence of miRNA, and then normal reverse transcription and subsequent PCR detection are carried out. The stem loop method is only for mature miRNA, and the specificity is relatively high; the tailing method can detect mature miRNA and pre-miRNA, and the specificity and sensitivity are poor, but the operation and primer design are simple. SUMMARY

[0009] A lung cancer auxiliary diagnosis detection kit, the kit is a two-step method detection kit based on PCR platform miRNA, all the two-step detection systems described in the specification are the theoretical basis for constructing the kit. Contain: microRNA molecular marker specific stem loop structure reverse transcription primer, PCR upstream primer, PCR universal downstream primer, specific probe for detecting microRNA molecular marker, wherein the microRNA molecular marker is at least two, one of which is selected from the up-regulated marker miR-21, miR-486-5p, miR-205 or miR-126; the other is selected from the down-regulated marker miR-152, Let-7a or miR-148a.

[0010] Preferably, the loop part of the neck of the specific stem loop structure reverse transcription primer is designed to form a key-like structure with non-continuous complementary base pairs TGCG and CGCA, and the short arm is connected with the microRNA molecule through the ligase in the reverse transcription reaction.

[0011] More preferably, the molecular marker miR-21 reverse transcription primer sequence is as shown in SEQ ID NO. 1:

[0012] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCATCAACAT-3';

[0013] miR-21 PCR upstream primer sequence as SEQ ID NO. 2:

[0014] 5'-CTCCGTCAGGGTAGCTTATCAGACTG-3';

[0015] miR-21 PCR universal downstream primer sequence as SEQ ID NO. 3:

[0016] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0017] miR-21 specific probe sequence as SEQ ID NO. 4:

[0018] 5'-FAM-TTTCCTCATCATCAACAT-MGB-3'

[0019] The reverse transcription primer sequence of the molecular marker miR-486-5p is as SEQ ID NO. 5:

[0020] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCACTCGGGG-3';

[0021] miR-486-5p PCR upstream primer sequence as SEQ ID NO. 6:

[0022] 5'-CTCCGTCAGGGTCCTGTACTGAGCTG-3';

[0023] miR-486-5p PCR universal downstream primer sequence as SEQ ID NO. 3:

[0024] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0025] miR-486-5p specific probe sequence as SEQ ID NO. 7:

[0026] 5'-FAM-TTTCCTCATCACTCGGGG-MGB-3'

[0027] The reverse transcription primer sequence of the molecular marker miR-205 is as SEQ ID NO. 8:

[0028] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCACAGACTC-3';

[0029] miR-205 PCR upstream primer sequence as SEQ ID NO. 9:

[0030] 5'-CTCCGTCAGGGTCCTTCATTCCACCG-3';

[0031] miR-205 PCR universal downstream primer sequence as SEQ ID NO. 3:

[0032] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0033] miR-205 specific probe sequence as SEQ ID NO. 10:

[0034] 5'-FAM-TTTCCTCATCACAGACTC-MGB-3';

[0035] The molecular marker miR-126 reverse transcription primer sequence as SEQ ID NO. 11:

[0036] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCACGCATTA-3';

[0037] miR-126 PCR upstream primer sequence as SEQ ID NO. 12:

[0038] 5'-CTCCGTCAGGGTCGTACCGTGAGTAA-3';

[0039] miR-126 PCR universal downstream primer sequence as SEQ ID NO. 3:

[0040] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0041] miR-126 specific probe sequence as SEQ ID NO. 13:

[0042] 5'-FAM-TTTCCTCATCACGCATTA-MGB-3'

[0043] The molecular marker let-7a reverse transcription primer sequence as SEQ ID NO. 14:

[0044] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCAACTATAC-3';

[0045] let-7a PCR upper primer sequence as SEQ ID NO. 15:

[0046] 5'-CTCCGTCAGGGTGAGGTAGTAGGTT-3';

[0047] let-7a PCR universal lower primer sequence as SEQ ID NO. 3:

[0048] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0049] let-7a specific probe sequence as SEQ ID NO. 16:

[0050] 5'-FAM-TTTCCTCATCAACTATAC-MGB-3'

[0051] The reverse transcription primer sequence of the molecular marker miR-152 is as shown in SEQ ID NO. 17:

[0052] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCAAGTCGGAG-3';

[0053] miR-152 PCR upper primer sequence as SEQ ID NO. 18:

[0054] 5'-CTCCGTCAGGGAGGTTCTGTGATACA-3';

[0055] miR-152 PCR universal lower primer sequence as SEQ ID NO. 3:

[0056] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0057] miR-152 specific probe sequence as SEQ ID NO. 19:

[0058] 5'-FAM-TTTCCTCATCAAGTCGGAG-MGB-3';

[0059] The reverse transcription primer sequence of the molecular marker miR-148a is as shown in SEQ NO. 20:

[0060] 5'-GATGAGGAGTGTCGTGGAGTCGGCAATTTCCTCATCAAGTCGGAG-3';

[0061] miR-148a PCR upstream primer sequence is as shown in SEQ ID NO. 21:

[0062] 5'-CTCCGTCAGGGAAAGTTCTGAGACA-3';

[0063] miR-148a PCR universal downstream primer sequence is as shown in SEQ ID NO. 3:

[0064] 5'-CTCAAGTGTCGTGGAGTCGGC-3';

[0065] miR-148a specific probe sequence is as shown in SEQ ID NO. 22:

[0066] 5'-FAM-TTTCCTCATCAAGTCGGAG-MGB-3';

[0067] More preferably, the miRNA molecular marker calibrators also include: the miR-21 molecular marker standard is miR-21, after dilution, the concentration is 10 13 copy / μL; the miR-486-5p molecular marker standard is miR-486-5p, after dilution, the concentration is 10 13 copy / μL; the miR-205 molecular marker standard is miR-205, after dilution, the concentration is 10 13 copy / μL; the miR-126 molecular marker standard is miR-126, after dilution, the concentration is 10 13 copy / μL; the let-7a molecular marker standard is let-7a, after dilution, the concentration is 10 13 copy / μL; the miR-152 molecular marker standard is miR-152, after dilution, the concentration is 10 13 copy / μL; the miR-148a molecular marker standard is miR-148a, after dilution, the concentration is 10 13 copy / μL.

[0068] The kit also includes microRNA molecule specific amplification templates, Vent (exo-) DNA polymerase, nicking enzyme, double-stranded specific nuclease and molecular hybridization probes.

[0069] Two-step method detection kit for miRNA based on PCR platform

[0070] Reverse transcription primer: the specific reverse transcription primer of the present application combines the design advantages of Stem-loop RT-PCR method and key-like method: 1. Stem-loop RT reverse transcription primer (Stem-loop RT-PCR) Figure 1 ) The neck Stem base pairs are extended, and 4 pairs of non-continuous complementary base pairs are designed in the loop to enhance the ability to form a key-like structure, so that the RT primer can better maintain the stem-loop structure during the entire reverse transcription process, not only eliminating the mispairing of stem-loop primers with non-target miRNAs to improve specificity, but also increasing the number of reverse transcription products, which is more conducive to subsequent PCR detection. 2. Stem-loop RT has 5 pairs of completely complementary bases with miRNA, and an enzyme ligation step is added before reverse transcription ( Figure 1 ), which makes the miRNA bind more firmly with the Stem-loop RT, enhancing the efficiency of reverse transcription. 3. The present application uses Stem-loop RT primer for miRNA reverse transcription product, which can also be used for fluorescent dye PCR detection.

[0071] PCR upstream and downstream primers: specific upstream primers are added with Tag labels to extend the amplification template and increase the amplification efficiency, and the downstream primers are adjusted so that the Tm values of the upstream and downstream primers are basically the same, so that after PCR pre-denaturation, the upstream and downstream primers can bind to the template and perform amplification at the same temperature.

[0072] Hydrolysis probe: the present application uses the design method of TaqMan technology to design a specific hydrolysis probe ( Figure 1 ) complementary to the template, which enhances the specificity of detection.

[0073] For quantitative detection of a miRNA marker, the miRNA is selected as an internal control gene of the miRNA marker, and the relative expression fold change of the marker is calculated using the relative quantitative formula (2 -ΔΔCp ) according to the CP value, and the score of the miRNA is calculated. The Pearson correlation coefficient is used to analyze the correlation between the relative expression of the miRNA marker and the patients with demographic characteristics, benign lesions and healthy individuals. The clinical pathological diagnosis is used as a reference standard to determine the sensitivity and specificity of the miRNA marker. The clinical pathological diagnosis is used as a reference standard to determine the sensitivity and specificity of the miRNA marker. ROC characteristic curve and AUC analysis are used to determine the accuracy of the miRNA combined detection, and the sample results are interpreted with the cut off value.

[0074] For the combined detection of two or more miRNA markers, select 1) up-regulation of at least one of miR-21, miR-486-5p, miR-205 or miR-126; 2) down-regulation of at least one of miR-152, Let-7a or miR-148a; 3) combined use of up-regulation molecular markers and down-regulation molecular markers. According to the CP value, the relative expression amount is calculated using the relative quantitative formula 2 -ΔΔCp , and the score of each miRNA is calculated. The Pearson correlation coefficient is used to analyze the correlation between the relative expression score of each miRNA marker and the patients with demographic characteristics, benign lesions and healthy individuals. The clinical pathological diagnosis is used as a reference standard to determine the sensitivity and specificity of each miRNA marker. The clinical pathological diagnosis is used as a reference standard to determine the sensitivity and specificity of the miRNA marker. Then, the binary logistic regression equation is obtained by using the logistic regression model, and the best diagnostic combination of the miRNA marker is selected. The accuracy of the combined detection of miRNA is determined by using the ROC characteristic curve and AUC analysis, and the sample results are interpreted by the cutoff value.

[0075] The beneficial effects of the present application are:

[0076] (1) The present application optimizes and improves the existing detection method, and develops a two-step detection kit for miRNA based on PCR platform. The corresponding detection and analysis method can be selected according to the purpose of detection and the requirement of experimental conditions. Due to the effectiveness of miRNA itself and the correlation with lung cancer tumor, the kit can be used for early lung nodule benign and malignant differentiation, and can also be used for prognosis, preoperative and postoperative, treatment, efficacy, etc. Real-time monitoring. The two-step detection system contains independently designed specific stem-loop structure RT primers, PCR upstream and downstream primers and Taqman probe primers. The specific primers can distinguish single base difference in miRNA detection specificity, and the sensitivity can reach 1 copy / μL as the lowest detection limit, which greatly improves the detection efficiency and accuracy of miRNA. In addition, the PCR thermal cycling conditions of different markers are the same, which not only can detect multiple markers in the same batch and on the same plate, improves the accuracy and detection efficiency of combined marker detection, but also reduces time and cost.

[0077] (2) Serum exo-miRNA as a marker for joint detection is better than plasma exo-miRNA or serum-miRNA, plasma-miRNA direct detection. Exo-miRNA has good stability, serum stored at 4℃ for 20 days can still extract exo-miRNA effectively, and the extracted exo-miRNA can be stored at -20℃ for 50 days and at -80℃ for a long time. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 miRNA two-step method PCR detection amplification principle diagram;

[0079] Figure 2 miRNA marker PCR standard curve and detection sensitivity (A: Let-7a PCR minimum detection limit; B: Let-7a PCR standard curve; C: miR-21 PCR minimum detection limit; D: miR-21 PCR standard curve; E: miR-486-5p PCR minimum detection limit; F: miR-486-5p PCR standard curve; G: miR-205 PCR minimum detection limit; H: miR-205 PCR standard curve; I: miR-126 PCR minimum detection limit; J: miR-126 PCR standard curve; K: miR-152 PCR minimum detection limit; L: miR-152 PCR standard curve; M: miR-148a PCR minimum detection limit; N: miR-148a PCR standard curve).

[0080] Figure 3 Two-step miRNA detection system for clinical sample detection stability results (A: batch difference CV value; B: batch difference CV value). DETAILED DESCRIPTION

[0081] The preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings. The experimental methods not specified in the preferred embodiments are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0082] The technical solutions in the embodiments of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0083] Example 1, two-step detection system kit based on PCR platform miRNA

[0084] 1) The instruments used in this embodiment are as follows:

[0085] 4°C low-temperature centrifuge (Thermo Fisher Fresco17), LightCycler 480 real-time fluorescent quantitative PCR instrument (Roche), super-clean workbench (SW-CJ-1D, Longyang Scientific Instruments), conventional PCR instrument (A100, Hangzhou Langji Scientific Instruments Co., Ltd.).

[0086] 2) RNA reverse transcription reaction system:

[0087] Reagents: The reagents used to prepare the reverse transcription reaction system include reverse transcription primers (RT-Primer, Shanghai Yingwei Jieji Synthesis), miRNA standard powder (Shanghai Yingwei Jieji Synthesis), T4 DNA ligase (T4 DNA Ligase, supplier: NEB, product number: M0202S, containing 10×T4 DNA Ligase Buffer), RNAase inhibitor (RNAase inhibitor, supplier: Fermentas, product number: K1622), transcriptase (supplier: Shanghai Yingwei Jieji Biotechnology Co., Ltd., product number: K1622, containing RNAase inhibitor, dNTPs, nuclease-free water), T4 polynucleotide kinase (T4 Polynucleotide Kinase, supplier: NEB, product number: M0201S), and nuclease-free water (nuclease-free water, supplier: Shanghai Yingwei Jieji Biotechnology Co., Ltd., product number: K1622). The reagents used to prepare the reverse transcription reaction system are packaged in bottles, and the reverse transcription system is prepared in a certain proportion when used. The reverse transcription reaction system is 20 μL / time, and the volume of the divided portion is 50 times the amount, as shown in Table 1.

[0088] Table 1 Components of the general reverse transcription reaction system

[0089] Component Final concentration Volume / μL Reverse transcription RT-Primer 5 μmol / L 5 T4 DNA Ligase Buffer 10× 2 dNTPs (with dUTP) 1 mmol / L 2 RNase inhibitor 20 UμL -1 ]] 1 Transcriptase 200 UμL -1 ]]> 1 T4 DNA Ligase 10 UμL -1 ]] 0.5 T4 Polynucleotide Kinase 2.5 U μL -1 ]] 0.25 Template 5 nuclease-free water Add water to 20 μL

[0090] Reverse transcription was performed according to the conditions in Table 2.

[0091] Table 2 General RNA reverse transcription conditions

[0092]

[0093] The cDNA was diluted 10 times and stored at 4°C for subsequent PCR amplification.

[0094] 3) PCR reaction system:

[0095] Reagents: The reagents used to prepare the PCR reaction system include dNTPs (dCTP, dGTP, dATP, dTTP, dUTP (supplier: Thermo Scientific) prepared into dNTPs. F primer liquid (F primer, Shanghai Yingwei Jieji Synthesis), universal R primer liquid (R primer, Shanghai Yingwei Jieji Synthesis), probe (Probe, ABI synthesis), DNA polymerase (HS Taq, supplier: Takara Company, product number: R007A), uracil-DNA glycosylase (UDG, supplier: NEB, product number: M0280S), and pure water (H2O).

[0096] The reagents used to prepare the PCR reaction system were packaged in bottles, and when used, they were prepared into a PCR reaction system in a certain proportion. The PCR reaction system was 20 μL / time, and the volume of the sub-packaging was 50 times the amount of use, as shown in Table 3.

[0097] Table 3: Optimized PCR reaction system

[0098]

[0099]

[0100] Then the amplification reaction was carried out according to the conditions in Table 4.

[0101] Table 4: PCR thermal cycling conditions

[0102]

[0103] 4) miRNA two-step method molecular marker standard preparation:

[0104] The cDNA stock solution of the standard miRNA after reverse transcription was 10 12 copy / μL, 10 μL of the cDNA stock solution was taken and diluted to 10 11 copy / μL with 90 μL of sterile purified water, and then 10 μL of the 10 11 copy / μL dilution was taken and diluted to 10 10 copy / μL with 90 μL of sterile purified water, and then sequentially diluted to a dilution of 1 copy / μL.

[0105] 5) Sensitivity of miRNA two-step detection system:

[0106] The two-step method detection system kit based on the PCR platform miRNA was used to detect the standard of miR-152, Let-7a, miR-148a, miR-21, miR-486-5p, miR-205 or miR-126, and the lower limit of detection and amplification efficiency were obtained. The detection principle is shown in Figure 1 .

[0107] Taking miR-21 as an example, the two-step method molecular marker standard of miR-21 was configured.

[0108] The cDNA stock solution of the standard miR-21 after reverse transcription was 10 12 copy / μL, 10 μL of the cDNA stock solution was added to 90 μL of sterilized purified water to dilute to 10 11 copy / μL, and 10 μL of the 10 11 copy / μL dilution was added to 90 μL of sterilized purified water to dilute to 10 10 copy / μL, and sequentially diluted to 1 copy / μL dilution.

[0109] The two-step method detection system of other miRNA molecular markers was configured by referring to miR-21, and the PCR reaction conditions were the same.

[0110] The detection results of the two-step method detection system miRNA standard are shown in Table 5.

[0111] Table 5 miRNA standard detection results

[0112]

[0113]

[0114] 6) Evaluation of the stability of the two-step method miRNA detection system for clinical samples

[0115] The stability of the detection results of the lung cancer clinical sample serum Exo-miR-21 combined with the miRNA down-regulation marker Exo-Let-7a was evaluated. Four different clinical serum samples were detected in three batches, and each batch had three repeats to verify the stability of the detection evaluation system (including Exo-miRNA extraction and purification, reverse transcription, and PCR machine detection). The results are shown in Figure 3 , the batch difference CV value of the same sample can be within 4%, and the batch difference CV value can be within 8%, indicating that the two-step method miRNA detection evaluation system has good stability.

[0116] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present application and not to limit the present application. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details without departing from the scope defined by the claims of the present application. SEQUENCE LISTING <110> Jiangsu Fuzhen Biopharmaceutical Technology Co., Ltd. <120> A method for detecting exosome-related microRNA molecules <160> 22 <170> SIPOSequenceListing 1.0 <210> 1 <211> 44 <212> DNA <213> Artificial Sequence <400> 1 gatgaggagt gtcgtggagt cggcaatttc ctcatcatca acat 44 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <400> 2 ctccgtcagg gtagcttatc agactg 26 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 ctcaagtgtc gtggagtcgg c 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 tttcctcatc atcaacat 18 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <400> 5 gatgaggagt gtcgtggagt cggcaatttc ctcatcactc gggg 44 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <400> 6 ctccgtcagg gtcctgtact gagctg 26 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 tttcctcatc actcgggg 18 <210> 8 <211> 44 <212> DNA <213> Artificial Sequence <400> 8 gatgaggagt gtcgtggagt cggcaatttc ctcatcacag actc 44 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <400> 9 ctccgtcagg gtccttcatt ccaccg 26 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <400> 10 tttcctcatc acagactc 18 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <400> 11 gatgaggagt gtcgtggagt cggcaatttc ctcatcacgc atta 44 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 ctccgtcagg gtcgtaccgt gagtaa 26 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <400> 13 tttcctcatc acgcatta 18 <210> 14 <211> 44 <212> DNA <213> Artificial Sequence <400> 14 gatgaggagt gtcgtggagt cggcaatttc ctcatcaact atac 44 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 ctccgtcagg gtgaggtagt aggtt 25 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <400> 16 tttcctcatc aactatac 18 <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <400> 17 gatgaggagt gtcgtggagt cggcaatttc ctcatcaagt cggag 45 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <400> 18 ctccgtcagg gaggttctgt gataca 26 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <400> 19 tttcctcatc aagtcggag 19 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <400> 20 gatgaggagt gtcgtggagt cggcaatttc ctcatcaagt cggag 45 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <400> 21 ctccgtcagg gaaagttctg agaca 25 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <400> 22 tttcctcatc aagtcggag 19

Claims

1. A kit for detecting an exosome-associated microRNA molecule, characterized by: The kit is a two-step method detection kit based on a PCR platform, containing a microRNA molecular marker specific stem-loop structure reverse transcription primer, a PCR upstream primer, a PCR universal downstream primer, a specific probe for detecting a microRNA molecular marker, and a series of gradient dilution concentrations of microRNA standard products; The short arm is connected to the microRNA molecule by a ligase during the reverse transcription reaction; the loop part of the neck of the reverse transcription primer is designed with two pairs of non-continuous complementary base pairs; the microRNA molecule is miR-21, miR-486-5p, miR-205, miR-126 or Let-7a, the reverse transcription primer sequence of the miR-21 is shown as SEQ ID NO. 1, the reverse transcription primer sequence of the miR-486-5p is shown as SEQ ID NO. 5, the reverse transcription primer sequence of the miR-205 is shown as SEQ ID NO. 8, the reverse transcription primer sequence of the miR-126 is shown as SEQ ID NO. 11, and the reverse transcription primer sequence of the let-7a is shown as SEQ ID NO.

14.

2. The kit of claim 1, wherein: The loop part of the neck of the specific stem-loop structure reverse transcription primer is designed with non-continuous complementary base pairs TG and CA to form a key-shaped structure.

3. The kit of claim 1, wherein: The PCR upstream primer of the miR-21 is shown as SEQ ID NO. 2, the PCR universal downstream primer is shown as SEQ ID NO. 3, and the specific probe nucleotide sequence is shown as SEQ ID NO.

4.

4. The kit of claim 1, wherein: The PCR upstream primer of the miR-486-5p is shown as SEQ ID NO. 6, the PCR universal downstream primer is shown as SEQ ID NO. 3, and the specific probe nucleotide sequence is shown as SEQ ID NO.

7.

5. The kit of claim 1, wherein: The PCR upstream primer of the miR-205 is shown as SEQ ID NO. 9, the PCR universal downstream primer is shown as SEQ ID NO. 3, and the specific probe nucleotide sequence is shown as SEQ ID NO.

10.

6. The kit of claim 1, wherein: The PCR upstream primer of the miR-126 is shown as SEQ ID NO. 12, the PCR universal downstream primer is shown as SEQ ID NO. 3, and the specific probe nucleotide sequence is shown as SEQ ID NO.

13.

7. The kit of claim 1, wherein: The PCR upstream primer of the let-7a is shown as SEQ ID NO. 15, the PCR universal downstream primer is shown as SEQ ID NO. 3, and the specific probe nucleotide sequence is shown as SEQ ID NO. 16.

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