CN116574780B - Design method of primer probe for simultaneously detecting two miRNAs through single fluorescent channel, kit and quantitative detection method - Google Patents
Design method of primer probe for simultaneously detecting two miRNAs through single fluorescent channel, kit and quantitative detection method Download PDFInfo
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Abstract
The invention discloses a design method, a kit and a quantitative detection method of primer probes for simultaneously detecting two miRNAs by a single fluorescent channel, wherein the design method comprises the following steps: designing a reverse transcription primer 1, wherein N bases at the 3' end of the sequence of the reverse transcription primer are reversely complementary with N bases at the 13 ' end of the miRNA, and the 5' end of the sequence of the reverse transcription primer is a random sequence containing M bases; designing a reverse transcription primer 2, wherein N bases at the 3' end of the sequence of the reverse transcription primer are reversely complementary with N bases at the 2 3' end of the miRNA, and the 5' end of the sequence of the reverse transcription primer is a random sequence containing M bases; designing a bridge segment, wherein the sequence of the bridge segment is composed of three regions from a 5 'end to a 3' end, namely a region 1, a region 2 and a region 3; the probe was designed to have a sequence reverse complementary to region 2 of the bridge fragment, with a fluorescent group at the 5 'end and a quenching group at the 3' end. The invention realizes simultaneous detection of two miRNAs by one fluorescent channel, reduces detection cost, is simple and convenient to operate, and ensures detection sensitivity and specificity.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a design method, a kit and a quantitative detection method of a primer probe for simultaneously detecting two miRNAs through a single fluorescent channel.
Background
miRNA (microRNA) is an endogenous single-stranded non-coding small RNA, generally consisting of 19-24 nucleotides, with an average length of 22bp, which is widely present in eukaryotes and serves as an endogenous regulator to regulate various biological processes including growth, signal transduction, immunomodulation, apoptosis, proliferation, tumorigenesis, and the like. Current studies indicate that mirnas are closely related to diseases, and that their abnormal expression levels are considered biomarkers for a variety of diseases, including diabetes, cardiovascular disease, kidney disease, neurodegenerative disease, liver disease, immune dysfunction, and the like. mirnas are involved in the development and metastasis of a variety of tumors, have specific expression profiles in cancer cells and tissues, and can enter the circulation of body fluids. mirnas are widely present in tissue cells and in body fluids such as serum, plasma, etc., and the kinds and amounts thereof may vary with physiological conditions or diseases; the mature miRNA is very stable in body fluid, and in addition, compared with protein, the detection of miRNA does not need to prepare antibodies, and is convenient for accurate quantification; compared with cfDNA, the miRNA has higher content in the circulation, and is convenient for detection; this makes mirnas promising as good quality diagnostic markers for diseases, especially as good quality non-invasive diagnostic markers for diseases. Currently, the gold standard for detecting miRNA is still RT-qPCR, and the common methods for RT-qPCR are a neck ring reverse transcription method and a poly (A) tailing method.
miRNA is a small fragment RNA, and the average length is only 22bp; the fluorescent quantitative PCR usually uses a pair of primers with the length of about 20bp to amplify the template, so that the fluorescent quantitative PCR has certain requirements on the length of the template, particularly the fluorescent quantitative PCR by a probe method, the minimum template length is longer, the sequence length of miRNA (micro ribonucleic acid) cannot meet the requirements on the sequence length of the template by direct fluorescent quantitative PCR, and thus, many researchers extend miRNA through specific reverse transcription primers, so that the length of the miRNA reaches the requirements on fluorescent quantitative PCR detection. The neck ring reverse transcription method uses a specific reverse transcription primer containing a neck ring sequence, the 3 'end of the reverse transcription primer contains 6-7 bases reversely complementary with the 3' end of the target miRNA, the target miRNA is subjected to specific reverse transcription by reverse transcription reaction to obtain cDNA, and then fluorescent quantitative PCR detection of the cDNA is completed by the specific forward primer and the universal reverse primer. The neck ring method uses specific reverse transcription primer to raise the specificity of reverse transcription and thus detection, but the primer has only 6-7 bases to the complementary region of target miRNA, so that the specificity is limited and the reverse transcription efficiency is low. According to the method, only one miRNA can be detected by one reverse transcription, when a plurality of miRNAs are required to be detected for the same sample, reverse transcription and fluorescence quantitative PCR are required to be carried out for a plurality of times, and the cost of multi-target detection is increased. Poly (A) tailing method first adds a section of Poly (A) tail to the 3 'end of miRNA by using Poly (A) polymerase (PAP), then carries out reverse transcription by using a nonspecific primer with an oligo (dT) sequence at the 5' end, and finally carries out fluorescence quantitative PCR detection by using a specific forward primer and a universal reverse primer. However, it is analyzed that the 3' -end sequence of miRNA is easy to generate diversification in the reverse transcription process, and the reverse primer is a universal primer, so that the degree of distinction between the precursor and the mature body is not high enough, and the credibility of the result is reduced.
Detection of mirnas generally involves three steps: extraction of miRNA, reverse transcription and fluorescent quantitative PCR detection. In order to detect abnormal expression of disease-related mirnas by fluorescent quantitative PCR, it is usually necessary to detect both internal and external references at the time of detection, the external reference is usually an artificially synthesized non-human miRNA, such as cel-miR-39-3p, added to the sample before miRNA extraction to control miRNA extraction; whereas the reference is typically an endogenous stably expressed miRNA, e.g., hsa-miR-16-5p, by comparing the change in the delta Ct value of the disease-associated miRNA to the reference, it is detected whether the disease-associated miRNA is aberrantly expressed. miRNA is increasingly paid attention to as a potential noninvasive disease diagnosis marker, and various miRNA detection-based kits are developed on the market at present and are used for auxiliary diagnosis of diseases or screening of cancers. The single miRNA is used as a detection target, and the detection sensitivity and the specificity are insufficient, so that the kit mostly adopts a method for combining and detecting a plurality of miRNAs so as to achieve higher detection sensitivity and specificity. However, in the neck ring reverse transcription method or the poly (A) tailing method, when a certain miRNA is detected, a reverse transcription primer, a forward primer, a reverse primer and a probe are required to be synthesized at the same time, and when a plurality of miRNAs are required to be detected, a plurality of primers and probes are required to be synthesized, so that the detection cost is increased; in addition, in the probe method, fluorescent quantitative PCR (polymerase chain reaction) can only detect one miRNA (micro ribonucleic acid), because of the limitation of fluorescent detection channels of a fluorescent quantitative PCR instrument, 4 fluorescent channels can be detected at most in a tube reaction system, and an internal reference and an external reference respectively occupy one fluorescent channel, which means that 2 target miRNAs can be detected at most in the tube reaction system, and when multiple target miRNAs are required to be detected simultaneously, a multi-tube reaction system is required, so that the detection cost is increased, and the detection flux is limited. Therefore, if one fluorescence channel is used for simultaneously detecting 2 miRNAs, the cost can be reduced, and the detection flux can be improved.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a primer probe design method, a kit and a quantitative detection method for simultaneously detecting two miRNAs by using a single fluorescent channel, so that the aim of simultaneously detecting the two miRNAs by using one fluorescent channel is fulfilled, the detection cost is reduced, the operation is simple and convenient, and the detection sensitivity and specificity are ensured.
The invention provides the following technical scheme:
in a first aspect, a method for designing a primer probe for simultaneously detecting two mirnas through a single fluorescent channel is provided, including:
designing a reverse transcription primer 1, wherein N bases at the 3' end of the sequence of the reverse transcription primer are reversely complementary with N bases at the 13 ' end of the miRNA to be detected, and the 5' end of the sequence of the reverse transcription primer is a random sequence containing M bases;
designing a reverse transcription primer 2, wherein N bases at the 3' end of the sequence of the reverse transcription primer are reversely complementary with N bases at the 2 3' end of the miRNA to be detected, and the 5' end of the sequence of the reverse transcription primer is a random sequence containing M bases;
designing a bridge segment, wherein the sequence of the bridge segment is composed of three regions from a 5 'end to a 3' end, namely a region 1, a region 2 and a region 3;
the probe was designed to have a sequence reverse complementary to region 2 of the bridge fragment, with a fluorescent group at the 5 'end and a quenching group at the 3' end.
Further, the N is 8 to 14.
Further, the Tm value of the reverse transcription primer 1 and the reverse transcription primer 2 is 50-65 ℃, and the Tm values of the two primers differ by +/-2 ℃; the Tm value of said probe is 60 to 90 ℃.
Further, the Tm value of the probe is 10℃higher than the Tm values of the reverse transcription primer 1 and the reverse transcription primer 2.
Further, the sequence of the bridge fragment region 1 is reversely complementary to the rest of the sequence of the miRNA1 except for N bases complementary to the 3' -end and the reverse transcription primer 1.
Furthermore, the sequence of the bridge segment region 2 comprises a middle region reversely complementary to the probe and 1-3 bases arranged at two sides of the middle region, and the design of 1-3 bases at two sides of the middle region can prevent the influence of steric effect on the binding of the probe.
Further, the sequence of the bridge fragment region 3 is the rest sequence of the miRNA2 except N bases complementary to the 3' end and the reverse transcription primer 2, and U in the sequence is replaced by T; the 3' end of the bridge fragment region 3 contains a blocking modification, which may be, but is not limited to, a phosphorylation modification, so that it cannot be extended.
Further, the number of complementary paired bases of the reverse transcription primer 1, the reverse transcription primer 2 and the probe is less than or equal to 5, otherwise, random sequences containing M bases at the 5' end of the sequences of the reverse transcription primer 1 and the reverse transcription primer 2 are required to be modified, or the probe sequences are required to be modified, so that mutual interference between the reverse transcription primer 1 and the reverse transcription primer 2 and the probe is prevented, and detection is influenced.
Further, the first base of the probe sequence cannot be G.
Further, the fluorescent group of the probe is selected from one of FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY or JOE, and the quenching group is selected from one of MGB, BHQ1 or BHQ2.
In a second aspect, a composition for simultaneously detecting two miRNAs in a single fluorescent channel is provided, which comprises a reverse transcription primer 1, a reverse transcription primer 2, a bridge fragment and a probe designed by the method of the first aspect.
In a third aspect, a kit for simultaneous detection of two mirnas in a single fluorescent channel is provided, comprising reverse transcription primer 1, reverse transcription primer 2, bridge fragment and probe designed according to the method of the first aspect or the composition of the second aspect.
Further, the kit also comprises a reverse transcription system and a fluorescent quantitative PCR amplification system;
the reverse transcription system comprises a reverse transcription primer 1, a reverse transcription primer 2, a 5 x reverse transcription buffer and a reverse transcriptase;
the fluorescent quantitative PCR amplification system comprises a reverse transcription primer 1 and a reverse transcription primerObject 2, bridge fragment, 5 XPCR buffer, mg 2+ Dntps and hot start DNA polymerase.
In a fourth aspect, a quantitative detection method for simultaneously detecting two mirnas by using a single fluorescent channel is provided, and the composition according to the second aspect or the kit according to the third aspect is adopted, and the specific steps include:
reverse transcription is carried out on the miRNA1 to be detected by using a reverse transcription primer 1, reverse transcription is carried out on the miRNA2 to be detected by using a reverse transcription primer 2, and reverse transcription products cDNA1 and cDNA2 are respectively obtained;
splicing the reverse transcription products cDNA1 and cDNA2 by using a bridge segment to obtain a spliced product;
the splice products were amplified by fluorescent quantitative PCR using reverse transcription primer 1, reverse transcription primer 2 and probe.
Further, the concentrations of the reverse transcription primer 1 and the reverse transcription primer 2 used for reverse transcription are 0.05 to 0.1. Mu.M, the concentration of the bridge fragment used for splicing the cDNA1 and the cDNA2 which are the reverse transcription products is 1 to 1000pM, and the concentrations of the reverse transcription primer 1, the reverse transcription primer 2 and the probe used for fluorescent quantitative PCR amplification are 0.1 to 0.6. Mu.M.
Further, the reverse transcription primer 1 carries out reverse transcription on the miRNA1 to be detected and the reverse transcription primer 2 is used for carrying out reverse transcription on the miRNA2 to be detected in the same reaction tube, and the reaction procedure is as follows: 16 ℃,15 minutes, 30-50 ℃,40 minutes, 85 ℃,5 minutes and 1 cycle.
Furthermore, the splicing and fluorescent quantitative PCR amplification of the reverse transcription products cDNA1 and cDNA2 are completed in the same reaction system through a section of PCR procedure, and the reaction procedure is as follows: 95 ℃,15 minutes, 1 cycle; 95 ℃,15 seconds, 37-50 ℃,5 minutes, 56-70 ℃,30 seconds, 2 cycles; 95 ℃,15 seconds, 50-65 ℃,30 seconds, 72 ℃,10 seconds and 45 cycles.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention uses two reverse transcription primers to carry out reverse transcription on two miRNAs to be detected, then uses bridge fragments to splice two reverse transcription products, and finally uses two reverse transcription primers and probes to carry out fluorescence quantitative PCR detection, wherein the two reverse transcription primers are used as reverse transcription primers of miRNAs and also used as forward primers and reverse primers of fluorescence quantitative PCR amplification, and the detection of the two miRNAs can be realized by using 2 primers, 1 bridge fragment sequence and 1 probe in total, so that the detection cost can be reduced.
(2) Compared with the traditional method, the quantitative detection method provided by the invention realizes the purpose of simultaneously detecting two miRNAs by using one fluorescent channel, and lays a foundation for detecting more miRNAs in a one-tube reaction system.
(3) The design method and the quantitative detection method of the primer probe for simultaneously detecting two miRNAs by the single fluorescent channel provided by the invention are simple and convenient to operate, and can ensure the sensitivity and the specificity of detection.
Drawings
FIG. 1 is a schematic diagram of a quantitative detection method of miRNA in an embodiment of the present invention;
FIG. 2 is a fluorescence quantitative PCR amplification curve of miRNA (cel-miR-39-3 p and hsa-miR-16-5p coexist) in an embodiment of the invention;
FIG. 3 is a fluorescent quantitative PCR amplification curve of miRNA (cel-miR-39-3 p and hsa-miR-16-5p are not present at the same time) in an embodiment of the invention;
FIG. 4 shows a fluorescence quantitative PCR amplification curve of miRNA (cel-miR-39-3 p and hsa-miR-16-5p coexist and are subjected to concentration gradient detection) in the embodiment of the invention;
FIG. 5 shows fluorescence quantitative PCR amplification curves of miRNAs in examples of the present invention (cel-miR-39-3 p and hsa-miR-16-5p are present at the same time, but at different concentrations);
FIG. 6 shows fluorescence quantitative PCR amplification curves of miRNAs in examples of the present invention (cel-miR-39-3 p and hsa-miR-16-5p are present at the same time, but at different concentrations);
FIG. 7 is a diagram showing agarose gel electrophoresis analysis in the example of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Example 1
The embodiment provides a design method of a primer probe for simultaneously detecting two miRNAs by a single fluorescent channel, which comprises the following steps:
step 1, designing a reverse transcription primer 1, wherein N bases at the 3' end of the sequence are reversely complementary with N bases at the 13 ' end of the miRNA to be detected, and the 5' end of the sequence is a random sequence containing M bases;
step 2, designing a reverse transcription primer 2, wherein N bases at the 3' end of the sequence are reversely complementary with N bases at the 2 3' end of the miRNA to be detected, and the 5' end of the sequence is a random sequence containing M bases;
step 3, designing a bridge segment, wherein the sequence of the bridge segment from the 5 'end to the 3' end consists of three areas, namely an area 1, an area 2 and an area 3;
and 4, designing a probe, wherein the sequence of the probe is reversely complementary with the region 2 of the bridge fragment, the 5 'end of the probe contains a fluorescent group, and the 3' end of the probe contains a quenching group.
N in the step 1 and the step 2 is 8-14; the Tm values of the reverse transcription primer 1 and the reverse transcription primer 2 are 50-65 ℃, and the Tm values of the two primers are 2 ℃ different; in the step 4, the Tm value of the probe is 60 to 90℃and the Tm value of the probe is about 10℃higher than those of the reverse transcription primer 1 and the reverse transcription primer 2.
The sequence of the bridge fragment region 1 in step 3 is reversely complementary to the rest of the sequence of miRNA1 except for the N bases complementary to the 3' -end and the reverse transcription primer 1. The sequence of the bridge segment region 2 comprises a middle region reversely complementary to the probe and 1-3 bases arranged on two sides of the middle region, and the 1-3 bases are designed on two sides of the middle region, so that the combination of the probe can be prevented from being influenced by steric effect. The sequence of the bridge fragment region 3 is the rest sequence of the miRNA2 except N bases complementary to the 3' end and the reverse transcription primer 2, and U in the sequence is replaced by T; the 3' end of the bridge-fragment region 3 contains a blocking modification, such as, but not limited to, a phosphorylation modification, such that it cannot be extended.
In some other embodiments, the number of complementary paired bases of reverse transcription primer 1, reverse transcription primer 2 and probe is less than or equal to 5, otherwise, it is necessary to modify the random sequences containing M bases at the 5' end of the sequence of reverse transcription primer 1 and reverse transcription primer 2, or modify the probe sequences, to prevent interference between them, and to affect detection.
In some other embodiments, the first base of the probe sequence cannot be G.
In some other embodiments, the fluorophore of the probe is selected from FAM, HEX, ROX, VIC, CY, 5-TAMRA, TET, CY3, or JOE and the quencher is selected from MGB, BHQ1, or BHQ2.
Example 2
As shown in fig. 1, the present embodiment provides a quantitative detection method for simultaneously detecting two mirnas by using a single fluorescent channel, which comprises the following steps:
s1, carrying out reverse transcription on miRNA1 to be detected by using a reverse transcription primer 1, and carrying out reverse transcription on miRNA2 to be detected by using a reverse transcription primer 2 to obtain reverse transcription products cDNA1 and cDNA2 respectively, wherein the concentration of the reverse transcription primer 1 and the reverse transcription primer 2 is 0.05-0.1 mu M;
step S2, splicing the reverse transcription products cDNA1 and cDNA2 by using a bridge segment to obtain a spliced product, wherein the concentration of the bridge segment is 1-1000 pM;
and S3, performing fluorescent quantitative PCR amplification on the spliced product by using the reverse transcription primer 1, the reverse transcription primer 2 and the probe, wherein the concentration of the reverse transcription primer 1, the reverse transcription primer 2 and the probe is 0.1-0.6 mu M.
The reverse transcription in step S1 is performed simultaneously in the same reaction tube, and the reaction procedure is as follows: 16 ℃,15 minutes, 30-50 ℃,40 minutes, 85 ℃,5 minutes and 1 cycle.
Step S2 and step S3 are completed in the same reaction system through a section of PCR procedure, and the reaction procedure is as follows: 95 ℃,15 minutes, 1 cycle; 95 ℃,15 seconds, 37-50 ℃,5 minutes, 56-70 ℃,30 seconds, 2 cycles; 95 ℃,15 seconds, 50-65 ℃,30 seconds, 72 ℃,10 seconds and 45 cycles.
Example 3
In the embodiment, cel-miR-39-3p and hsa-miR-16-5p are used as miRNA1 and miRNA2 to be detected. miRNA mimics were synthesized from sequences reported in the miRNA database (http:// www.mirbase.org /) (see Table 1) and primers, probes and bridge fragments were designed as described in example 1, as shown in Table 2.
Table 1miRNA mimetic information
TABLE 2 primers, probes and bridge fragments
In this example, the 5 'end of the probe contains a FAM fluorophore and the 3' end contains a BHQ1 quencher; the 3' -end of the bridge fragment contains a phosphorylation modification.
Example 4
4.1 reverse transcription
Reverse transcription was performed in the same manner as in example 2 using the primers designed in example 3, and was designated as reverse transcription system 1, as shown in Table 3.
TABLE 3 reverse transcription System 1
In order to verify the specificity of the detection according to the method of the present invention, three additional sets of reverse transcription systems were set as control sets, each:
reverse transcription system 2: a reverse transcription system containing only one miRNA template to be detected (cel-miR-39-3 p), as shown in table 4;
reverse transcription system 3: a reverse transcription system containing only one miRNA template to be detected (hsa-miR-16-5 p), as shown in table 5;
reverse transcription system 4: reverse transcription systems without miRNA templates to be detected are shown in table 6.
TABLE 4 reverse transcription System 2
TABLE 5 reverse transcription System 3
| Reagent(s) | Usage amount | Final concentration |
| 5 Xreverse transcription buffer | 4ul | 1× |
| Reverse transcriptase (200U/ul) | 1ul | 10U/ul |
| hsa-miR-16-5p(100pM/ul) | 1ul | 5pM/ul |
| Reverse transcription primer 1 (1 uM/ul) | 1ul | 0.05uM/ul |
| Reverse transcription primer 2 (1 uM/ul) | 1ul | 0.05uM/ul |
| DEPC water | To 20ul |
TABLE 6 reverse transcription System 4
| Reagent(s) | Usage amount | Final concentration |
| 5 Xreverse transcription buffer | 4ul | 1× |
| Reverse transcriptase (200U/ul) | 1ul | 10U/ul |
| Reverse transcription primer 1 (1 uM/ul) | 1ul | 0.05uM/ul |
| Reverse transcription primer 2 (1 uM/ul) | 1ul | 0.05uM/ul |
| DEPC water | To 20ul |
In order to verify the detection sensitivity of the method, four groups of reverse transcription systems are additionally arranged, and the reverse transcription systems simultaneously contain two miRNAs to be detected, but the concentrations or relative concentrations of the two miRNA templates in the same reverse transcription system are different and are respectively as follows:
reverse transcription system 5 and reverse transcription system 6: the concentrations of the two miRNAs are the same in the same system, and the two miRNAs are in concentration gradient change between different systems, as shown in tables 7 and 8;
reverse transcription system 7 and reverse transcription system 8: the concentrations of the two mirnas in the same system were different, as shown in tables 9 and 10.
TABLE 7 reverse transcription System 5
| Reagent(s) | Usage amount | Final concentration |
| 5 Xreverse transcription buffer | 4ul | 1× |
| Reverse transcriptase (200U/ul) | 1ul | 10U/ul |
| hsa-miR-16-5p(100pM/ul) | 1ul | 0.5pM/ul |
| cel-miR-39-3p(100pM/ul) | 1ul | 0.5pM/ul |
| Reverse transcription primer 1 (1 uM/ul) | 1ul | 0.05uM/ul |
| Reverse transcription primer 2 (1 uM/ul) | 1ul | 0.05uM/ul |
| DEPC water | To 20ul |
TABLE 8 reverse transcription System 6
TABLE 9 reverse transcription System 7
| Reagent(s) | Usage amount | Final concentration |
| 5 Xreverse transcription buffer | 4ul | 1× |
| Reverse transcriptase (200U/ul) | 1ul | 10U/ul |
| hsa-miR-16-5p(100pM/ul) | 1ul | 5pM/ul |
| cel-miR-39-3p(100pM/ul) | 1ul | 0.5pM/ul |
| Reverse transcription primer 1 (1 uM/ul) | 1ul | 0.05uM/ul |
| Reverse transcription primer 2 (1 uM/ul) | 1ul | 0.05uM/ul |
| DEPC water | To 20ul |
TABLE 10 reverse transcription System 8
The reverse transcription system was subjected to reverse transcription, and the procedure for reverse transcription is shown in Table 11.
TABLE 11 reverse transcription reaction procedure
| Temperature (temperature) | Time | Cycle number |
| 16℃ | 15 minutes | 1 |
| 42℃ | 50 minutes | 1 |
| 85℃ | For 5 minutes | 1 |
| 4℃ | For 1 minute | 1 |
4.2 splicing and fluorescent quantitative PCR detection
The reverse transcription products obtained from the 8 reverse transcription systems in 4.1 were subjected to splicing and fluorescent quantitative PCR detection by using the method steps S2 and S3 described in example 2, respectively, and the splicing and fluorescent quantitative PCR detection were completed in the same reaction system by using a single PCR procedure, the reaction system is shown in Table 12, and the PCR procedure is shown in Table 13.
Table 12 fluorescent quantitative PCR reaction System
TABLE 13 fluorescent quantitative PCR reaction procedure
4.3 analysis of results
When two miRNAs to be detected (cel-miR-39-3 p and hsa-miR-16-5 p) exist simultaneously, a normal S-shaped amplification curve can be obtained through fluorescent quantitative PCR detection, and the Ct value of the fluorescent quantitative PCR is shown in a table 14 as shown in FIG. 2; when only one miRNA to be detected (cel-miR-39-3 p or hsa-miR-16-5 p) is present or no miRNA to be detected is present, there is no amplification curve, as shown in FIG. 3.
When the concentrations of the two miRNAs are the same in the same reverse transcription system and the concentration gradients change between different systems, a normal S-shaped amplification curve (shown in figure 4) can be obtained, and Ct values between different concentration gradients change in a gradient manner (shown in table 14); when the concentrations of the two mirnas in the same reverse transcription system are different, the Ct value of the fluorescent quantitative PCR depends on the mirnas with lower template concentration, the fluorescent quantitative PCR amplification curves are shown in fig. 5 and 6, and the Ct value of the fluorescent quantitative PCR is shown in table 14.
TABLE 14 fluorescent quantitative PCR Ct values
Agarose gel electrophoresis analysis of the fluorescent quantitative PCR product showed that when two miRNAs to be detected (cel-miR-39-3 p and hsa-miR-16-5 p) were present at the same time, an amplified band (as shown in well 1 and well 2 of FIG. 7) as expected in size (94 bp) could be obtained, and when only one miRNA to be detected (cel-miR-39-3 p or hsa-miR-16-5 p) was present or no miRNA to be detected was present, no amplified band (as shown in well 3-7 of FIG. 7) was obtained.
In summary, the method of the invention achieves the purpose of simultaneously detecting two miRNAs by using one fluorescent channel, and when two miRNAs exist simultaneously, an amplification curve can be obtained, the Ct value of fluorescent quantitative PCR depends on the miRNA with lower template concentration, and when only one miRNA to be detected exists or both miRNAs to be detected do not exist, no amplification curve exists; in addition, by using the method provided by the invention, the detection of two miRNAs can be realized by synthesizing fewer primers, and the detection cost can be reduced.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Claims (10)
1. A design method of a primer probe for simultaneously detecting two miRNAs by a single fluorescent channel is characterized by comprising the following steps:
designing a reverse transcription primer 1, wherein N bases at the 3' end of the sequence of the reverse transcription primer are reversely complementary with N bases at the 13 ' end of the miRNA to be detected, and the 5' end of the sequence of the reverse transcription primer is a random sequence containing M bases;
designing a reverse transcription primer 2, wherein N bases at the 3' end of the sequence of the reverse transcription primer are reversely complementary with N bases at the 2 3' end of the miRNA to be detected, and the 5' end of the sequence of the reverse transcription primer is a random sequence containing M bases;
designing a bridge segment, wherein the sequence of the bridge segment is composed of three regions from a 5 'end to a 3' end, namely a region 1, a region 2 and a region 3;
designing a probe, wherein the sequence of the probe is reversely complementary with the region 2 of the bridge fragment, the 5 'end of the probe contains a fluorescent group, and the 3' end of the probe contains a quenching group;
the N is 8-14; the Tm values of the reverse transcription primer 1 and the reverse transcription primer 2 differ by + -2 ℃;
the sequence of the bridge fragment region 1 is reversely complementary with the rest sequence of the miRNA1 except N bases complementary with the 3' -end and the reverse transcription primer 1; the sequence of the bridge segment region 2 comprises a middle region reversely complemented with the probe and 1-3 bases arranged on two sides of the middle region; the sequence of the bridge segment region 3 is the rest sequence of the miRNA2 except N bases complementary to the 3' end and the reverse transcription primer 2, and U in the sequence is replaced by T.
2. The method for designing primer probes for simultaneously detecting two miRNAs with a single fluorescent channel according to claim 1, wherein the Tm value of the reverse transcription primer 1 and the reverse transcription primer 2 is 50-65 ℃ and the Tm value of the probes is 60-90 ℃.
3. The method for designing a primer probe for simultaneously detecting two miRNAs through a single fluorescent channel according to claim 1, wherein the 3' -end of the bridge fragment region 3 contains a blocking modification.
4. A composition for simultaneously detecting two miRNAs through a single fluorescent channel is characterized by comprising a reverse transcription primer 1, a reverse transcription primer 2, a bridge fragment and a probe which are designed by the method of any one of claims 1-3.
5. A kit for simultaneously detecting two mirnas through a single fluorescent channel, comprising a reverse transcription primer 1, a reverse transcription primer 2, a bridge fragment and a probe designed by the method according to any one of claims 1 to 3 or the composition according to claim 4.
6. The kit for simultaneous detection of two mirnas by a single fluorescent channel according to claim 5, further comprising a reverse transcription system and a fluorescent quantitative PCR amplification system;
the reverse transcription system comprises a reverse transcription primer 1, a reverse transcription primer 2, a 5 x reverse transcription buffer and a reverse transcriptase;
the fluorescent quantitative PCR amplification system comprises a reverse transcription primer 1, a reverse transcription primer 2, a bridge fragment, a 5 XPCR buffer solution and Mg 2 + Dntps and hot start DNA polymerase.
7. A quantitative detection method for simultaneously detecting two mirnas by using a single fluorescent channel, which is characterized by adopting the composition of claim 4 or the kit of any one of claims 5 to 6, and specifically comprising the following steps:
reverse transcription is carried out on the miRNA1 to be detected by using a reverse transcription primer 1, reverse transcription is carried out on the miRNA2 to be detected by using a reverse transcription primer 2, and reverse transcription products cDNA1 and cDNA2 are respectively obtained;
splicing the reverse transcription products cDNA1 and cDNA2 by using a bridge segment to obtain a spliced product;
the splice products were amplified by fluorescent quantitative PCR using reverse transcription primer 1, reverse transcription primer 2 and probe.
8. The quantitative detection method for simultaneously detecting two miRNAs through a single fluorescent channel according to claim 7, wherein the concentration of a reverse transcription primer 1 and a reverse transcription primer 2 used for reverse transcription is 0.05-0.1 mu M, the concentration of a bridge fragment used for splicing cDNA1 and cDNA2 of a reverse transcription product is 1-1000 pM, and the concentration of a reverse transcription primer 1, a reverse transcription primer 2 and a probe used for fluorescent quantitative PCR amplification is 0.1-0.6 mu M.
9. The quantitative detection method for simultaneously detecting two miRNAs through a single fluorescent channel according to claim 7, wherein reverse transcription of the miRNA1 to be detected is performed by the reverse transcription primer 1 and reverse transcription of the miRNA2 to be detected is performed simultaneously in the same reaction tube by using the reverse transcription primer 2, and the reaction procedure is as follows: 16 ℃,15 minutes, 30-50 ℃,40 minutes, 85 ℃,5 minutes and 1 cycle.
10. The quantitative detection method for simultaneously detecting two miRNAs by using a single fluorescent channel according to claim 7, wherein the splicing of the reverse transcription products cDNA1 and cDNA2 and the fluorescent quantitative PCR amplification are completed in the same reaction system by a section of PCR program, and the reaction program is as follows: 95 ℃,15 minutes, 1 cycle; 95 ℃,15 seconds, 37-50 ℃,5 minutes, 56-70 ℃,30 seconds, 2 cycles; 95 ℃,15 seconds, 50-65 ℃,30 seconds, 72 ℃,10 seconds and 45 cycles.
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