CN112430643A - MiRNA multi-site joint detection method based on isothermal amplification - Google Patents
MiRNA multi-site joint detection method based on isothermal amplification Download PDFInfo
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Abstract
The invention provides a miRNA multi-site joint detection method based on isothermal amplification, which mainly comprises the following steps: 1) designing and synthesizing a specific single-stranded DNA probe aiming at each target miRNA sequence, and hybridizing the padlock probes with corresponding miRNA fragments to be detected to form a plurality of hybrid double strands; 2) connecting two ends of the padlock probe hybridized in the step 1); 3) performing rolling-circle isothermal amplification on the connection product obtained in the step 2); 4) hybridizing the product fragments obtained in the step 3) by using specific magnetic beads; 5) carrying out fluorescent labeling on the product obtained in the step 4) by using a streptavidin-labeled fluorescent dye; 6) detecting the fluorescent signal of the product in the step 5). The invention does not need reverse transcription, saves cost and reduces loss; the specificity is good, and single base can be distinguished; performing multiplex detection; the method can be applied to quantitative detection of multi-site miRNA in various fields, including but not limited to cancer screening, disease diagnosis, scientific research and the like.
Description
Technical Field
The invention belongs to the technical field of miRNA detection, and relates to an miRNA multi-site joint detection method based on isothermal amplification.
Background
microRNA (miRNA for short) is a small endogenous single-stranded RNA only containing 19-23 basic groups and widely exists in various eukaryotic cells. The abnormal expression of miRNA, as a highly conserved small molecule non-coding RNA, is involved in the process of various diseases, including multiple processes of cancer occurrence, development, metastasis, recurrence and the like, and the miRNA plays the role of carcinogenic factors or cancer suppressor factors according to different regulated genes, and the change of the expression level can be used as a marker for disease diagnosis and prognosis.
mirnas are very stable, quantifiable in a variety of bodily fluids, and their levels and composition in bodily fluids also have relevance to the situation of disease progression. In the field of non-invasive liquid biopsy, mirnas may serve as ideal candidate biomarkers for almost all human diseases, including cancer. In fact, miRNA has been added as a hematological molecular marker for liver cancer in primary liver cancer diagnosis and treatment specifications (2019). The standard indicates that the liver cancer detection kit based on the circulating miRNA model is verified by multiple hospitals at present, and three types of medical instrument registration certificates of the national drug supervision and administration are obtained, so that the liver cancer detection kit enters clinical application.
Currently, common miRNA detection methods are classified into two categories, direct detection and indirect detection. The direct detection method is mainly based on methods such as fluorescence, colorimetry, electrochemistry and the like, can directly detect the level and expression of miRNA, but has low sensitivity and is difficult to distinguish homologous miRNA with similar sequences. The indirect detection method mainly comprises Northern Blot, microarray chip, real-time fluorescent quantitative PCR (qRT-PCR) and other detection methods. Among them, Northern Blot and microarray chip methods are semi-quantitative methods, and have poor sensitivity and large demand for initial miRNA amount. In contrast, qRT-PCR is currently the most sensitive and efficient method for quantitatively detecting RNA.
The real-time qRT-PCR detection method based on the TaqMan probe is applied to the detection of miRNA, and the current market dominates the real-time fluorescent quantitative PCR method (TaqMan microRNA detection method) developed by Invitrogen (original ABI). According to the method, miRNA is subjected to reverse transcription through a stem-loop primer designed aiming at specific miRNA, and then fluorescence quantification is performed by using a TaqMan probe and a specific primer. Although the method overcomes the defects of most miRNA detection methods to a certain extent, the method has the following problems. First, although the detection sensitivity of the TaqMan probe is high, the design of each miRNA is not only cost-prohibitive, but also technically challenging. Secondly, the method cannot perform multiple detection of miRNA in the same reaction hole, and the detection of single miRNA locus has relatively low sensitivity and specificity for disease diagnosis. Together with the high detection cost of the method, the defects limit the wide application and clinical transformation of the method.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a miRNA multi-site combined detection method based on isothermal amplification.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a miRNA multi-site joint detection method based on isothermal amplification, which comprises the following steps:
1) designing and synthesizing an upstream universal primer and a downstream universal primer with biotin labels at the 5' ends; designing and synthesizing a specific single-stranded DNA probe aiming at each target miRNA sequence, and hybridizing the padlock probes with corresponding miRNA fragments to be detected to form a plurality of hybrid double strands;
the probes are as follows from the 5' end: phosphorylation marker-8-14 base reverse complementary sequence of 5 'end of miRNA-specific tag sequence-downstream universal primer reverse complementary sequence-upstream universal primer sequence-residual base reverse complementary sequence of 3' end of miRNA;
2) ligating two ends of the padlock probe hybridized successfully in the step 1) by using ligase;
3) performing rolling-circle isothermal amplification on the ligation product in the step 2) by using an upstream universal primer and a downstream universal primer;
4) hybridizing the product fragments obtained in the step 3) by using specific magnetic beads;
5) carrying out fluorescent labeling on the product obtained in the step 4) by using a streptavidin-labeled fluorescent dye;
6) detecting the fluorescent signal of the product in the step 5).
Preferably, in step 1), the hybridization conditions are: slowly cooling to room temperature at 80-90 deg.C for 2-5min, and maintaining for 2-4 h.
Preferably, in step 2), the ligase is selected from T4 DNA ligase or T4 RNA ligase or splntr DNA ligase.
Preferably, in step 2), the connection conditions are: 16-37 deg.C, 15-60 min.
Preferably, in step 3), the amplification reaction may be any commercially available isothermal amplification reaction kit.
Preferably, in step 3), the isothermal amplification reaction conditions are 20-40 ℃ for 2-12 h.
Preferably, in step 4), the specific magnetic beads are coupled with specific nucleic acid sequences, and each microsphere is provided with a different label capable of binding to a specific PCR product.
Preferably, in step 4), the hybridization reaction conditions are: 85-100 ℃ for 60-120 s; 25-40 deg.C, 10-20 min.
Preferably, in step 5), the reaction conditions for carrying out the fluorescent labeling are: 25-37 deg.C, 10-30 min.
Compared with the prior art, the invention has the following advantages:
1. reverse transcription is not needed, so that the cost is saved, and the loss is reduced;
2. the specificity is good, and single bases can be distinguished (see example 1);
3. multiplex assays (see example 2);
4. the method provided by the invention can be applied to quantitative detection of the multi-site miRNA in various fields, including but not limited to cancer screening, disease diagnosis, scientific research and the like.
Drawings
FIG. 1 is a schematic flow chart of the combined detection method of multiple miRNA sites.
FIG. 2 shows the results of the specific detection of hsa-let-7c-5p in example 1 of the present invention.
FIG. 3 shows the result of the multiplex detection of breast cancer samples in example 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples of the present invention,
ligase and 10 Xligase buffer with Thermo ScientificTM T4 DNA Ligase，EL0011；
The formula of the hybridization solution is as follows: 0.4M NaCl, 0.2M Tris, 0.016% Triton X-100, pH 8.0, stored at 4 ℃;
the magnetic beads adopt MagPlex-TAG Microspheres,
the formula of the color reaction liquid is as follows: add 1. mu.l of SAPE to every 80. mu.l of hybridization solution, manufacturer: moss SAPE-001G75, Life
S-886。
Unless otherwise specified, each step in the examples can be realized by using materials and conventional technical means in the field.
Example 1 assay for detection specificity
First, primer and probe design principle
1. Designing two universal primers (a primer F and a primer R), wherein the two universal primers are non-human sequences and are not communicated with known human DNA and RNA sequences or are similar to more than 4 continuous bases through sequence comparison; the 5' end of the universal primer F is provided with a biotin label.
2. The length of the universal primer sequence is 18-25bp, and the Tm value is 55-65 ℃.
3. According to a miRNA sequence to be detected, 1 specific single-stranded DNA probe is designed according to the following principle:
1) the length is 80-100 nt;
2) the nucleic acid sequence is a reverse complementary sequence of 8-14 bases from the 5 ' end to the 3 ' end of the miRNA, a specific tag sequence C, a reverse complementary sequence of a universal primer R, a sequence of a universal primer F and a sequence reverse complementary to all the remaining bases at the 3 ' end of the miRNA in sequence from the 5 ' end to the 3 ' end;
3) the 5' end of the probe is modified by phosphorylation.
Second, Hsa-let-7c-5p specificity analysis
1. And (3) synthesizing a standard substance, and designing and synthesizing a primer and a probe according to the method in the step one.
1) The universal primers were synthesized with the following sequences:
a universal primer F: biotin-CCAGATAAGGGTGTTATG (SEQ ID NO: 1), wherein biotin represents a biotin modification;
a universal primer R: GAATGGGCGGATTGCCT (SEQ ID NO: 2).
2) Synthesizing hsa-let-7a-5p, hsa-let-7b-5p and hsa-let-7c-5p standard products, wherein the sequences are as follows:
hsa-let-7a-5p:UGAGGUAGUAGGUUGUAUAGUU(SEQ ID NO：3)
hsa-let-7b-5p:UGAGGUAGUAGGUUGUGUGGUU(SEQ ID NO：4)
hsa-let-7c-5p:UGAGGUAGUAGGUUGUAUGGUU(SEQ ID NO：5)。
3) the probe of hsa-let-7c-5p is synthesized, and the sequence is as follows:
hsa-let-7c-5p-L：
P-CTACTACCTCACAATTTACATTTCACTTTCTTATCAGGCAATCCGCCCATTCagcgagtcaaCCAGATAAGGGTGTTATGAACCATACAAC (SEQ ID NO: 6), wherein P represents a phosphorylation modification.
MiRNA detection
Adding the dry powder of the standard substance of hsa-let-7a-5p, hsa-let-7b-5p and hsa-let-7c-5p into the non-enzyme water respectively for dissolving, and diluting to 1015Copy/ml, 10-fold gradient dilution standard, 10 of each10Copies/ml were used as standard templates and three samples were tested separately using hsa-let-7c-5 p-L.
The detection is carried out according to the following steps:
1) 1. mu.l of the above template was taken, and added to the reaction: 1 ul of probe hsa-let-7c-5p-L, 1 ul of 10 Xligase buffer, and supplementing water to 10 ul;
2) the reaction system carries out hybridization reaction in a PCR instrument, and the procedure is as follows: slowly cooling to 22 deg.C for 2min at 85 deg.C, and maintaining for 2 hr;
3) taking 2 ul of the product, adding 1 ul of ligase and 2 ul of 10 Xligase buffer, and replenishing water to 20 ul;
4) the reaction system carries out a connection reaction in a PCR instrument, and the procedure is as follows: 30min at 25 ℃ and 10min at 65 ℃;
5) taking 20 mul of product, adding 0.5 mul of isothermal amplification enzyme, 5 mul of 10 multiplied by isothermal amplification enzyme buffer, 2 mul of universal primer F and 2 mul of universal primer R, and supplementing water to 50 mul;
6) the reaction system carries out isothermal amplification reaction in a PCR instrument, and the procedure is as follows: 6h at 37 ℃ and 10min at 65 ℃;
7) add 10. mu.l of product, 30. mu.l of hybridization solution, 3. mu.l of magnetic beads to 96-well plate, add water to 50. mu.l, seal membrane, and perform reaction in PCR instrument with the following procedure: at 90 ℃ for 80 s; at 37 ℃ for 20 min;
8) after the hybridization reaction is finished, the 96-well plate is placed on a magnetic frame for 2min, the supernatant is removed by suction, 25 mul of chromogenic reaction liquid is added, the membrane is sealed, the reaction is carried out in a PCR instrument, and the procedure is as follows: at 37 ℃ for 10 min;
9) after the color reaction is finished, the PCR reaction product is taken down from the PCR instrument and is immediately placed on a magnetic plate to stand for 60s, and the supernatant is discarded. Adding 50 μ l of hybridization solution (the same hybridization solution as in step 7), shaking and mixing for 20-30s, standing on magnetic plate for 60s, discarding supernatant, and repeating for 1 time. Adding 75 μ l of the hybridization solution, shaking and mixing for 30 s;
10) the luminex 200 was used for on-machine testing.
3. The experimental results are shown in FIG. 2, NC is a negative control, the result of hsa-let-7c-5p is 3 times greater than that of NC, and the results of hsa-let-7a-5p and hsa-let-7b-5p are both 3 times less than that of NC, thus proving that the probe hsa-let-7c-5p-L can specifically detect hsa-let-7c-5p, but cannot detect fragments with only 1 base difference, and proving that the specificity of the method is better.
Example 2 multiplex assay
1. Extracting miRNA from the plasma sample:
miRNA extraction was performed on 5 breast cancer patients and 5 healthy control plasma (plasma samples of healthy persons and patients from cooperative hospitals, signed with written consent and sequence verified) using a serum/plasma miRNA extraction and isolation kit (DP503) produced by tiangen biochemistry technology (beijing) ltd according to the instruction method.
2. The probe design and synthesis were performed according to the method described in example 1.
Synthesizing hsa-miR-200c-3p, hsa-miR-101-3p, hsa-miR-372-3p and hsa-miR-105-5p standard products, wherein the sequences are as follows:
hsa-miR-200c-3p:UAAUACUGCCGGGUAAUGAUGGA(SEQ ID NO：7)
hsa-miR-101-3p:UACAGUACUGUGAUAACUGAA(SEQ ID NO：8)
hsa-miR-372-3p:AAAGUGCUGCGACAUUUGAGCGU(SEQ ID NO：9)
hsa-miR-105-5p:UAACACUGUCUGGUAAAGAUGG(SEQ ID NO：10)
probes were designed and synthesized with the following sequences:
hsa-miR-200c-3p-L：
P-CCGGCAGTATTAACAAATATCTAACTACTATCACAATCACAATCCGCCCATTCagcgagtcaaCCAGATAAGGGTGTTGCGTCCATCATTAC (SEQ ID NO: 11), wherein P represents a phosphorylation modification;
hsa-miR-101-3p-L：
P-ACAGTACTGTACAATTTACATTTCACTTTCTTATCTCACAATCCGCCCATTCagcgagtcaaCCAGATAAGGGTGTTGCGTTCAGTTATC (SEQ ID NO: 12), wherein P represents a phosphorylation modification;
hsa-miR-372-3p-L：
P-GTCGCAGCACTTTTTAACAACTTATACAAACACAAACTCACAATCCGCCCATTCagcgagtcaaCCAGATAAGGGTGTTGCGACGCTCAAAT (SEQ ID NO: 13), wherein P represents a phosphorylation modification;
hsa-miR-105-5p-L：
P-CTGAGCATTTGAACATCAAATTCTTTCAATATCTTCTCACAATCCGCCCATTCagcgagtcaaCCAGATAAGGGTGTTGCGACCACAGGAGT (SEQ ID NO: 14), wherein P represents a phosphorylation modification.
3. Multiplex assays
Dissolving various standard dry powders in non-enzyme water respectively, and then adding the mixture into the mixture according to the proportion of 1: 1 proportion and diluted to 1014Copy/ml, following the procedure in example 2, a gradient dilution of each standard was prepared separately. The concentration of each component after dilution is 1010Copy/ml standard and extracted miRNA in breast cancer patient/healthy human plasma sampleAs a template, all probes were mixed at the same concentration to serve as a multiplex detection probe, and detection was performed according to the following procedure:
1) taking 10 ul of the template, adding the following components into the reaction system: mu.l probe, 2. mu.l 10 Xligase buffer, and water to 20. mu.l;
2) the reaction system carries out hybridization reaction in a PCR instrument, and the procedure is as follows: slowly cooling to 22 deg.C for 2min at 85 deg.C, and maintaining for 2 hr;
3) taking 10 ul of the product, adding 1 ul of ligase and 2 ul of 10 Xligase buffer, and replenishing water to 20 ul;
4) the reaction system carries out a connection reaction in a PCR instrument, and the procedure is as follows: 30min at 25 ℃ and 10min at 65 ℃;
5) adding 0.5. mu.l of isothermal amplification enzyme, 5. mu.l of 10 Xisothermal amplification enzyme buffer, 3. mu.l of primer F and 3. mu.l of primer R into 20. mu.l of product, and replenishing water to 50. mu.l;
6) the reaction system carries out isothermal amplification reaction in a PCR instrument, and the procedure is as follows: 6h at 37 ℃ and 10min at 65 ℃;
7) add 10. mu.l of product, 30. mu.l of hybridization solution, 3. mu.l of magnetic beads to 96-well plate, add water to 50. mu.l, seal membrane, and perform reaction in PCR instrument with the following procedure: at 90 ℃ for 80 s; at 37 ℃ for 20 min;
8) after the hybridization reaction is finished, the 96-well plate is placed on a magnetic frame for 2min, the supernatant is removed by suction, 25 mul of chromogenic reaction liquid is added, the membrane is sealed, the reaction is carried out in a PCR instrument, and the procedure is as follows: at 37 ℃ for 10 min;
9) after the color reaction is finished, the PCR reaction product is taken down from the PCR instrument and is immediately placed on a magnetic plate to stand for 60s, and the supernatant is discarded. Adding 50 μ l of hybridization solution, shaking and mixing for 20-30s, standing on magnetic plate for 60s, discarding supernatant, and repeating for 1 time. Adding 75 μ l of the hybridization solution, shaking and mixing for 30 s;
10) the luminex 200 was used for on-machine testing.
4. The experimental results are shown in FIG. 3, NC is negative control, PC is positive control, and the detection results of several sites of the positive control are all more than 3 times of the corresponding value N, which proves that the method can simultaneously detect a plurality of sites; at least 2 sites of all 5 patient samples are positive, and all the healthy controls are positive, the results are less than 2, so that the method is proved to be capable of well distinguishing breast cancer samples.
Sequence listing
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Claims (8)
1. An isothermal amplification-based miRNA multi-site combined detection method comprises the following steps:
1) designing and synthesizing an upstream universal primer and a downstream universal primer with biotin labels at the 5' ends; designing and synthesizing a specific single-stranded DNA probe aiming at each target miRNA sequence, and hybridizing the padlock probes with corresponding miRNA fragments to be detected to form a plurality of hybrid double strands;
the probes are as follows from the 5' end: phosphorylation marker-8-14 base reverse complementary sequence of 5 'end of miRNA-specific tag sequence-downstream universal primer reverse complementary sequence-upstream universal primer sequence-residual base reverse complementary sequence of 3' end of miRNA;
2) ligating two ends of the padlock probe hybridized successfully in the step 1) by using ligase;
3) performing rolling-circle isothermal amplification on the ligation product in the step 2) by using an upstream universal primer and a downstream universal primer;
4) hybridizing the product fragments obtained in the step 3) by using specific magnetic beads;
5) carrying out fluorescent labeling on the product obtained in the step 4) by using a streptavidin-labeled fluorescent dye;
6) detecting the fluorescent signal of the product in the step 5).
2. The miRNA multi-site joint detection method based on isothermal amplification according to claim 1, wherein in the step 1), the hybridization conditions are as follows: slowly cooling to room temperature at 80-90 deg.C for 2-5min, and maintaining for 2-4 h.
3. The method for detecting miRNA multi-site combination based on isothermal amplification of claim 1, wherein in step 2), the ligase is selected from T4 DNA ligase, T4 RNA ligase or SplintRNAligase.
4. The method for detecting miRNA multi-site combination based on isothermal amplification according to claim 1 or 3, wherein in step 2), the connection conditions are as follows: 16-37 deg.C, 15-60 min.
5. The miRNA multi-site joint detection method based on isothermal amplification according to claim 1, wherein in the step 3), the isothermal amplification reaction conditions are 20-40 ℃ and 2-12 h.
6. The method as claimed in claim 1, wherein in step 4), the specific magnetic beads are coupled with specific nucleic acid sequences, and each microsphere has a different label, so that the specific beads can bind to a specific PCR product.
7. The method for detecting miRNA multi-site combination based on isothermal amplification according to claim 1 or 6, wherein in step 4), the hybridization reaction conditions are as follows: 85-100 ℃ for 60-120 s; 25-40 deg.C, 10-20 min.
8. The miRNA multi-site combined detection method based on isothermal amplification according to claim 1, wherein in the step 5), the reaction conditions for fluorescence labeling are as follows: 25-37 deg.C, 10-30 min.
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