CN113981047B - Reverse transcription-strand displacement amplification method for miRNA detection and application thereof - Google Patents
Reverse transcription-strand displacement amplification method for miRNA detection and application thereof Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a reverse transcription-strand displacement amplification method for miRNA detection, which combines reverse transcription reaction and strand displacement reaction, takes cDNA synthesized by adding tail to reverse transcription of miRNA as a template, can realize the exponential amplification of a target product and the exponential growth of a signal through one-step reaction under the condition of constant temperature, and improves the detection efficiency; the upstream primer, the downstream primer or the downstream probe have no sequence complementary relation, so that nonspecific amplification generated by hybridization between the primers or between the primers and the probes can be effectively reduced. The invention also provides a reverse transcription-strand displacement amplification dye method kit and a probe method kit for miRNA detection. When any one miRNA target molecule is detected, quantitative detection of any one miRNA target molecule can be realized only by designing and synthesizing different upstream primers according to different miRNA molecule sequences, and repeated design and synthesis of different downstream primers or downstream probes are not needed, so that the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a reverse transcription-strand displacement amplification method for miRNA detection and application thereof.
Background
MicroRNA (miRNA) is a non-coding single-stranded RNA of about 22 nucleotides in length encoded by an endogenous gene, which has been shown to be involved in the regulation of various vital activities. In recent years, research has found that circulating miRNA molecules with rich types exist in body fluids such as serum, plasma and the like, and early screening and prognosis monitoring of cancers can be realized by quantitatively detecting the expression level of the circulating miRNA molecules of a subject (see, for example, shan Baoen and the like, microRNA: new targets for diagnosis and treatment of cancers, journal of biological treatment of Chinese tumors, 2014, volume 21, 6: 603-609).
RT-qPCR is one of the common methods for quantitative detection of miRNA (see Kramer M F, curr. Protoc. Mol. Biol.2011,95 (1): chapter 15: unit 15.10); an RT-PCR method for quantitatively detecting miRNA is disclosed in patent CN 105177132A. However, PCR is a temperature-variable amplification technique, requiring continuous temperature variation and precise temperature control to achieve efficient amplification of target molecules, which places high demands on the performance of the detection device. With the rapid development of isothermal amplification technology, this technical solution, which can react efficiently only at a constant temperature, is considered as a trend of development of molecular detection technology in the future. Several new methods of isothermal amplification for rapid detection of mirnas have been reported (see Sun Y, chem. Com., 2017,53 (80): 11040-11043), most of which are protocols employing direct detection of miRNA target molecules. Because miRNA belongs to RNA molecules, untreated miRNA is easily degraded by the action of RNAase, so that the miRNA cannot be stored for a long time, and the quantitative accuracy of the miRNA expression level in a sample can be influenced. Currently, the reverse transcription (Reverse transcription) pretreatment process is considered to be the most effective technical means for preventing degradation of miRNA and prolonging the preservation period of miRNA samples. However, only one RT-LAMP study developed based on loop-mediated isothermal amplification (LAMP) for miRNA detection has been reported (see Al-Maskri a AA, analytica Chimica Acta,2020, 1126:1-6). Because the LAMP method requires a large number of primers or probes and has a long sequence, false positive results are easy to generate, and the detection specificity cannot be ensured. Therefore, the development of a novel reverse transcription-isothermal amplification method for quantitative detection of miRNA has important significance.
Disclosure of Invention
The technical problems to be solved by the invention are to solve the problems that the existing LAMP method for detecting miRNA requires more primers or probes, the sequence is longer, the detection efficiency is low, and the pretreatment process of the miRNA tail-added reverse transcription cannot be suitable for isothermal amplification, namely the novel molecular amplification detection technology.
The invention solves the technical problems by the following technical means:
a reverse transcription-strand displacement amplification (RT-SDA) method for miRNA detection, comprising the steps of:
s1, performing poly (A) tailing reaction on a miRNA single chain to obtain a C1 chain;
s2, reverse transcription reaction: under the action of a reverse transcription primer P1, the C1 chain carries out reverse transcription reaction to generate a C2 chain reversely complementary with the C1 chain, and the C2 chain is used as a cDNA template for detecting miRNA target molecules by SDA reaction;
s3, the upstream primer P2 of the SDA reaction uses a C2 chain as a template, and chain hybridization and extension reactions are sequentially carried out to generate a C3 chain;
the nucleotide sequence of the upstream primer P2 is shown as a general formula (I):
P-F(I)
wherein P is the 5' end sequence of the primer P2 on the upstream of the RT-SDA reaction, and comprises an enzyme cutting site recognition sequence of the nicking endonuclease and a protecting base thereof; f is the 3 'end sequence of the upstream primer P2 of the RT-SDA reaction, the nucleotide sequence is the same as the 8-14 base sequence of the 5' end of any miRNA target molecule, except that uracil residues are replaced by thymine residues;
s4, after the complementary DNA double strand is formed between the C3 strand and the C2 strand, enzyme cutting reaction is carried out under the catalysis of nicking endonuclease, and a nick is cut off on the C3 strand;
s5, continuously extending the sequence at the 5 'end of the C3 chain incision, and replacing the sequence at the 3' end of the C3 chain incision to generate a C4 chain; simultaneously, a new C3-C2 DNA double-chain structure is formed, and the reaction of the steps S4 and S5 is continuously repeated to generate a large number of C4 chains;
s6, carrying out chain hybridization and extension reaction sequentially by using a C4 chain as a template of a downstream primer P3 of SDA reaction or a downstream probe P4 of SDA reaction to generate a C5 chain;
the nucleotide sequence of the downstream primer P3 is shown as a general formula (II):
P-R(II)
wherein P is the 5' end sequence of the primer P3 at the downstream of the RT-SDA reaction, and comprises an enzyme cutting site recognition sequence of a cutting endonuclease and a protecting base thereof, R is the 3' end sequence of the primer P3 at the downstream of the RT-SDA reaction, and the nucleotide sequence is the same as 8-14 base sequences at the 5' end of the reverse transcription primer P1;
the nucleotide sequence of the downstream probe P4 is shown as a general formula (III):
P-B1-B2-R(III)
wherein, P-B1-B2 is the 5' end sequence of the probe P4 at the downstream of the RT-SDA reaction, and comprises an enzyme cutting site recognition sequence of the incision endonuclease and a protecting base thereof; bases B1 and B2 are a labeled fluorophore and a quencher, respectively; r is the 3 'end sequence of a downstream probe P4 of the RT-SDA reaction, and the nucleotide sequence of the R is the same as the 8-14 base sequence of the 5' end of the reverse transcription primer P1;
s7, after the complementary DNA double strand is formed between the C5 strand and the C4 strand, performing enzyme digestion reaction under the action of a nicking endonuclease, and cutting a nick on the C5 strand;
s8, continuously extending the sequence at the 5 'end of the C5 chain incision, and replacing the sequence at the 3' end of the C5 chain incision to generate a C6 chain; simultaneously, a new C5-C4 DNA double-chain structure is formed, and the reaction of the steps S7 and S8 is continuously repeated to generate a large number of C6 chains;
s9, the C6 chain and the C2 chain are completely the same in sequence, and the reaction of the steps S4-S5 can be continuously carried out to generate a large number of C4 chains, and then the reaction of the steps S7-S8 is continuously carried out, and a large number of C6 chains are generated again; the amplification reaction may be cycled back and forth continuously such that the amplification product and fluorescent signal increase exponentially.
Preferably, the miRNA comprises one or more of humoral circulating miRNA, fresh or paraffin tissue miRNA, total RNA, synthetic miRNA molecules.
Preferably, the nicking enzyme comprises any one of nb.bvci, nb.bsmi, nb.bsrdi, nb.btsi, nt.alwi, nt.bbvci, nt.bsmai, nt.bspqi, nt.bstnbi, nb.bssi, nt.cvipii.
The upstream primer, the downstream primer or the downstream probe provided by the invention have no sequence complementary relationship, so that the non-specific amplification generated by the mutual hybridization between the primers or between the primers and the probe can be effectively reduced, the sequences of the used primers and the probe are shorter, the double-chain binding efficiency is not reduced due to the formation of a dimer structure under the constant temperature condition, and the amplification reaction efficiency is not influenced.
The invention also provides a reverse transcription-strand displacement amplification dye method kit for miRNA detection, which comprises the reverse transcription primer P1, an upstream primer P2 and a downstream primer P3.
Further, the dye method kit further comprises components for a miRNA tail-biting reverse transcription reaction: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer; the reaction buffer comprises Tris-HCl pH 8.3, naCl, KCl, mgCl 2 、DTT；
The dye method kit also comprises components for performing a strand displacement isothermal amplification reaction: DNA polymerase, nicking endonuclease, dNTPs, fluorescent dye and reaction buffer; the reaction buffer solution comprises Tris-HCl pH 7.9, naCl and MgCl 2 、BSA。
Further, the concentrations of each component used in the dye method kit in the reaction system when the miRNA tail-added reverse transcription is performed are respectively as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl, 1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs;
the dye method kit is characterized in that the concentrations of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification are respectively as follows: 10-100mM Tris-HCl pH 7.9, 50-200mM NaCl, 5-50mM MgCl 2 50-200. Mu.g/mL BSA, 0.05-0.5U/. Mu.L DNA polymerase, 0.05-0.5U/. Mu.L nicking endonuclease, 0.025-0.5mM dNTPs, 0.025-0.5. Mu.M upstream primer, 0.025-0.5. Mu.M downstream primer, 1 Xfluorescent dye.
Further, the dye methodThe concentration of each component used in the kit when the miRNA tail-added reverse transcription is carried out in a reaction system is as follows: 20-80mM Tris-HCl pH 8.3, 20-80mM NaCl, 20-80mM KCl, 5-30mM MgCl 2 5-50mM DTT, 2-20nM reverse transcription primer, 0.2-1.0mM ATP, 0.2-5.0U/. Mu.L Poly (A) polymerase, 2-20U/. Mu.L reverse transcriptase, 0.2-1.0mM dNTPs;
the dye method kit is characterized in that the concentrations of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification are respectively as follows: 20-80mM Tris-HCl pH 7.9, 80-150mM NaCl, 10-30mM MgCl 2 80-150. Mu.g/mL BSA, 0.1-0.3U/. Mu.L DNA polymerase, 0.1-0.3U/. Mu.L nicking endonuclease, 0.05-0.4mM dNTPs, 0.05-0.3. Mu.M upstream primer, 0.05-0.3. Mu.M downstream primer, 1 Xfluorescent dye.
Further, the reaction conditions of the dye method kit when the miRNA tail-added reverse transcription is carried out are as follows: firstly, preserving the temperature at 37-42 ℃ for 15-60min, and then heating at 65-95 ℃ for 5-20min;
the reaction conditions of the dye method kit during the strand displacement isothermal amplification are as follows: the temperature is kept at 37-65 ℃ for 15-60min, and then the mixture is heated at 70-95 ℃ for 5-20min.
The invention also provides a reverse transcription-strand displacement amplification probe method kit for miRNA detection, which comprises the reverse transcription primer P1, an upstream primer P2 and a downstream probe P4.
Still further, the probe method kit further comprises components for a miRNA tail-biting reverse transcription reaction: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer; the probe method kit further comprises components for the reverse transcription reaction of miRNA tailing: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer;
the probe method kit further comprises components for performing a strand displacement isothermal amplification reaction: DNA polymerase, nicking endonucleases, dNTPs and reaction buffer; the reaction buffer solution comprises Tris-HCl pH 7.9, naCl and MgCl 2 、BSA。
Furthermore, the concentration of each component used in the probe method kit when the miRNA tail-added reverse transcription is carried out in a reaction system is as follows: 10-100mM Tris-HClpH 8.3、10-100mM NaCl、10-100mM KCl、1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs;
the probe method kit is characterized in that the concentration of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification is respectively as follows: 10-100mM Tris-HCl pH 7.9, 50-200mM NaCl, 5-50mM MgCl 2 50-200. Mu.g/mL BSA, 0.05-0.5U/. Mu.L DNA polymerase, 0.05-0.5U/. Mu.L nicking endonuclease, 0.025-0.5mM dNTPs, 0.025-0.5. Mu.M upstream primer, 0.025-0.5. Mu.M downstream probe.
Furthermore, the concentration of each component used in the probe method kit when the miRNA tail-added reverse transcription is carried out in a reaction system is as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl, 1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs;
the probe method kit is characterized in that the concentration of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification is respectively as follows: 20-80mM Tris-HCl pH 7.9, 80-150mM NaCl, 10-30mM MgCl 2 80-150. Mu.g/mL BSA, 0.1-0.3U/. Mu.L DNA polymerase, 0.1-0.3U/. Mu.L nicking endonuclease, 0.05-0.4mM dNTPs, 0.05-0.3. Mu.M upstream primer, 0.05-0.3. Mu.M downstream probe.
Furthermore, the reaction conditions of the probe method kit when the miRNA tail-added reverse transcription is carried out are as follows: firstly, preserving the temperature at 37-42 ℃ for 15-60min, and then heating at 65-95 ℃ for 5-20min;
the reaction conditions of the probe method kit during the strand displacement isothermal amplification are as follows: the temperature is kept at 37-65 ℃ for 15-60min, and then the mixture is heated at 70-95 ℃ for 5-20min.
The invention has the following beneficial effects:
1. the upstream primer, the downstream primer or the downstream probe provided by the invention have no sequence complementary relationship, so that the non-specific amplification generated by the mutual hybridization between the primers or between the primers and the probe can be effectively reduced, the sequences of the used primers and the probe are shorter, the double-chain binding efficiency is not reduced due to the formation of a dimer structure under the constant temperature condition, and the amplification reaction efficiency is not influenced.
2. According to the dye method or probe method reaction system for detecting miRNA by using RT-SDA, when any miRNA target molecule is detected, different upstream primers are designed and synthesized according to different miRNA molecule sequences, and different downstream primers or downstream probes are not required to be repeatedly designed and synthesized, so that quantitative detection of any miRNA target molecule can be realized, the detection efficiency is improved, and the detection cost is greatly reduced.
Drawings
FIG. 1 is a schematic diagram of the reaction principle of a reverse transcription-strand displacement amplification (RT-SDA) method for miRNA detection according to an embodiment of the present invention;
FIG. 2 is an amplification curve of miRNA detection by using the RT-SDA dye-based method provided by the embodiment of the invention;
FIG. 3 is an amplification curve of miRNA detection by using the RT-SDA probe-based method provided by the embodiment of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
The reverse transcription-strand displacement amplification method for miRNA detection is applicable to any type of miRNA, including natural miRNA molecules of all eukaryotes such as human beings, animals, plants and the like, for example, one or more of body fluid circulating miRNA, fresh tissue or paraffin tissue miRNA and total RNA, and can also include any artificially synthesized miRNA molecules. The method of the invention is described in the following examples by way of an artificially synthesized miR-320 molecular standard (AAAAGCUGGGUUGAGAGGGCGA, SEQ ID NO: 5).
The sequences referred to in the examples below can all be synthesized using the prior art.
Example 1
The dye method detection method for detecting miRNA by RT-SDA comprises the following steps:
in the embodiment, an artificially synthesized miR-320 standard substance is used as a sample, a dye method detection scheme for detecting miRNA by RT-SDA is constructed according to the technical principle shown in FIG. 1, and a 20 mu L reaction system is prepared according to the following requirements:
2.0. Mu.L of reaction buffer (final concentration: 50mM Tris-HCl pH 7.9, 100mM NaCl, 10mM MgCl, respectively) 2 100. Mu.g/mL BSA), 0.125. Mu.M upstream primer P2 (SEQ ID NO: 2), 0.125. Mu.M downstream primer P3 (SEQ ID NO: 3), 0.1U/. Mu.L DNA polymerase, 0.2U/. Mu.L Nt.BstNBI,0.125mM dNTPs,1 XGreen dye, and sequentially diluting cDNA synthesized from 10pM miR-320 molecular standard by tailed reverse transcription into three-concentration mother liquor (10) -1 、10 -2 And 10 -3 ) And taking 1.0 mu L of mother solution as templates of three reactions respectively, adding water to 20 mu L, preserving heat at 55 ℃ for 40min, then heating at 85 ℃ for 5min to terminate the reactions, and collecting fluorescent signals for 40 times after each cycle is finished at 1min intervals, wherein the amplification detection result of RT-SDA is shown in figure 2.
According to the results shown in fig. 2, it can be seen that three test groups for detecting miRNA by using the RT-SDA dye method all have standard S-shaped amplification curves, which fully indicate that the RT-SDA dye method provided by the invention can realize effective detection of miRNA; meanwhile, the linear correlation between amplification curves corresponding to three-time gradient dilution of the synthesized cDNA template is high, which shows that the RT-SDA dye method provided by the invention can efficiently realize the amplification detection of miRNA molecules.
Because the RT-SDA detection miRNA method is characterized in that on the premise of not changing the sequence of an upstream primer and a downstream primer, the obtained probe can be used for a probe method detection test by modifying the base of the downstream primer with a fluorescent group and a quenching group, and further the quantitative detection of miRNA can be realized by using an RT-SDA probe method detection scheme, and the quantitative detection of miRNA can be verified by the following example 2.
Example 2
The probe method for detecting miRNA by RT-SDA comprises the following steps:
in the embodiment, an artificially synthesized miR-320 standard product is used as a sample, a probe method detection scheme for detecting miRNA by RT-SDA is constructed according to the technical principle shown in FIG. 1, and a 20 mu L reaction system is prepared according to the following requirements:
2.0. Mu.L of reaction buffer (final concentration: 50mM Tris-HCl pH 7.9, 100mM NaCl, 10mM MgCl, respectively) 2 100. Mu.g/mL BSA), 0.125. Mu.M upstream primer P2 (SEQ ID NO: 2), 0.125. Mu.M downstream probe P4 (SEQ ID NO: 4), 0.1U/. Mu.L DNA polymerase, 0.2U/. Mu.L Nt.BstNBI,0.125mM dNTPs, and sequentially diluting cDNA synthesized from a 10pM miR-320 molecular standard by reverse transcription primer P1 (SEQ ID NO: 1) tailed reverse transcription into three concentrations of mother liquor (10 -1 、10 -2 And 10 -3 ) And taking 1.0 mu L of mother solution as templates of three reactions respectively, adding water to 20 mu L, preserving heat at 55 ℃ for 40min, then heating at 85 ℃ for 5min to terminate the reactions, and collecting fluorescent signals for 40 times after each cycle is finished at 1min intervals, wherein the amplification detection result of RT-SDA is shown in figure 3.
According to the results shown in fig. 3, it can be seen that three test groups for detecting miRNA by using the RT-SDA probe method have standard S-shaped amplification curves, which fully indicate that the RT-SDA probe method provided by the invention can realize effective detection of miRNA; meanwhile, the linear correlation between amplification curves corresponding to three-time gradient dilution of the synthesized cDNA template is high, which indicates that the RT-SDA probe method provided by the invention can efficiently realize the amplification detection of miRNA molecules.
Example 3
Based on the results of example 1, the present invention also provides a dye-process kit that can be used for RT-SDA detection of mirnas in example 1 above.
The dye method kit for detecting miRNA by RT-SDA comprises a reverse transcription primer P1 and an upstream primer in the embodiment 1The primer P2 and the downstream primer P3 also comprise components for the reverse transcription reaction of miRNA tailing: poly (a) polymerase, reverse transcriptase, ATP, dNTPs and reaction buffer; the reaction buffer comprises Tris-HCl pH 8.3, naCl, KCl, mgCl 2 DTT; the dye method kit is characterized in that the concentrations of each component used in the process of carrying out miRNA tail-added reverse transcription in a reaction system are respectively as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl, 1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs; preferably 20-80mM Tris-HCl pH 8.3, 20-80mM NaCl, 20-80mM KCl, 5-30mM MgCl 2 5-50mM DTT, 2-20nM reverse transcription primer, 0.2-1.0mM ATP, 0.2-5.0U/. Mu.L Poly (A) polymerase, 2-20U/. Mu.L reverse transcriptase, 0.2-1.0mM dNTPs; meanwhile, the method also comprises the components for carrying out the strand displacement isothermal amplification reaction: DNA polymerase, nicking endonuclease, dNTPs, fluorescent dye and reaction buffer; the reaction buffer solution comprises Tris-HCl pH 7.9, naCl and MgCl 2 BSA; the concentrations of each component used in the dye-process kit in the reaction system for performing the strand displacement isothermal amplification are respectively as follows: 10-100mM Tris-HCl pH 7.9, 50-200mM NaCl, 5-50mM MgCl 2 50-200. Mu.g/mL BSA, 0.05-0.5U/. Mu.L DNA polymerase, 0.05-0.5U/. Mu.L nicking endonuclease, 0.025-0.5mM dNTPs, 0.025-0.5. Mu.M upstream primer, 0.025-0.5. Mu.M downstream primer, 1 Xfluorescent dye; preferably 20-80mM Tris-HCl pH 7.9, 80-150mM NaCl, 10-30mM MgCl 2 80-150 mug/mL BSA, 0.1-0.3U/. Mu.L DNA polymerase, 0.1-0.3U/. Mu.L nicking endonuclease, 0.05-0.4mM dNTPs, 0.05-0.3 mu.M upstream primer, 0.05-0.3 mu.M downstream primer, 1 Xfluorescent dye;
the reaction conditions of the dye-process kit in the embodiment when the miRNA is subjected to tail-end reverse transcription are as follows: firstly, preserving the temperature at 37-42 ℃ for 15-60min, and then heating at 65-95 ℃ for 5-20min; the reaction conditions at the time of strand displacement isothermal amplification were then: the temperature is kept at 37-65 ℃ for 15-60min, and then the mixture is heated at 70-95 ℃ for 5-20min.
Example 4
Based on the results of example 2, the present invention also provides a probe method kit that can be used for RT-SDA detection of mirnas in example 2 above.
The provided probe method kit for detecting miRNA by RT-SDA comprises a reverse transcription primer P1, an upstream primer P2 and a downstream probe P4 in the embodiment 2, and further comprises components for the reverse transcription reaction of miRNA tailing: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer; the reaction buffer comprises Tris-HCl pH 8.3, naCl, KCl, mgCl 2 DTT. The concentration of each component used in the probe method kit during the reverse transcription of miRNA with tail in a reaction system is respectively as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl, 1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs; preferably 20-80mM Tris-HCl pH 8.3, 20-80mM NaCl, 20-80mM KCl, 5-30mM MgCl 2 5-50mM DTT, 2-20nM reverse transcription primer, 0.2-1.0mM ATP, 0.2-5.0U/. Mu.L Poly (A) polymerase, 2-20U/. Mu.L reverse transcriptase, 0.2-1.0mM dNTPs; meanwhile, the method also comprises the components for carrying out the strand displacement isothermal amplification reaction: DNA polymerase, nicking endonucleases, dNTPs and reaction buffer; the reaction buffer solution comprises Tris-HCl pH 7.9, naCl and MgCl 2 BSA; the probe method kit has the following concentrations in a reaction system of each component used for strand displacement isothermal amplification: 10-100mM Tris-HCl pH 7.9, 50-200mM NaCl, 5-50mM MgCl 2 50-200. Mu.g/mL BSA, 0.05-0.5U/. Mu.L DNA polymerase, 0.05-0.5U/. Mu.L nicking endonuclease, 0.025-0.5mM dNTPs, 0.025-0.5. Mu.M upstream primer, 0.025-0.5. Mu.M downstream probe, preferably 20-80mM Tris-HCl pH 7.9, 80-150mM NaCl, 10-30mM MgCl 2 80-150 mug/mL BSA, 0.1-0.3U/. Mu.L DNA polymerase, 0.1-0.3U/. Mu.L nicking endonuclease, 0.05-0.4mM dNTPs, 0.05-0.3 mu.M upstream primer, 0.05-0.3 mu.M downstream probe;
the reaction conditions of the probe method kit in the embodiment when the miRNA tail-added reverse transcription is performed are as follows: firstly, preserving the temperature at 37-42 ℃ for 15-60min, and then heating at 65-95 ℃ for 5-20min; the reaction conditions at the time of strand displacement isothermal amplification were then: the temperature is kept at 37-65 ℃ for 15-60min, and then the mixture is heated at 70-95 ℃ for 5-20min.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (13)
1. A reverse transcription-strand displacement amplification method for miRNA detection, comprising the steps of:
s1, performing poly (A) tailing reaction on a miRNA single chain to obtain a C1 chain;
s2, reverse transcription reaction: under the action of a reverse transcription primer P1, the C1 chain carries out reverse transcription reaction to generate a C2 chain reversely complementary with the C1 chain, and the C2 chain is used as a cDNA template for detecting miRNA target molecules by SDA reaction;
s3, the upstream primer P2 of the SDA reaction uses a C2 chain as a template, and chain hybridization and extension reactions are sequentially carried out to generate a C3 chain;
the nucleotide sequence of the upstream primer P2 is shown as a general formula (I):
P-F (I)
wherein P is the 5' end sequence of the primer P2 on the upstream of the RT-SDA reaction, and comprises an enzyme cutting site recognition sequence of the nicking endonuclease and a protecting base thereof; f is the 3 'end sequence of the upstream primer P2 of the RT-SDA reaction, the nucleotide sequence is the same as the 8-14 base sequence of the 5' end of any miRNA target molecule, except that uracil residues are replaced by thymine residues;
s4, after the complementary DNA double strand is formed between the C3 strand and the C2 strand, enzyme cutting reaction is carried out under the catalysis of nicking endonuclease, and a nick is cut off on the C3 strand;
s5, continuously extending the sequence at the 5 'end of the C3 chain incision, and replacing the sequence at the 3' end of the C3 chain incision to generate a C4 chain; simultaneously, a new C3-C2 DNA double-chain structure is formed, and the reaction of the steps S4 and S5 is continuously repeated to generate a large number of C4 chains;
s6, carrying out chain hybridization and extension reaction sequentially by using a C4 chain as a template of a downstream primer P3 of SDA reaction or a downstream probe P4 of SDA reaction to generate a C5 chain;
the nucleotide sequence of the downstream primer P3 is shown as a general formula (II):
P-R (II)
wherein P is the 5' end sequence of the primer P3 at the downstream of the RT-SDA reaction, and comprises an enzyme cutting site recognition sequence of a cutting endonuclease and a protecting base thereof, R is the 3' end sequence of the primer P3 at the downstream of the RT-SDA reaction, and the nucleotide sequence is the same as 8-14 base sequences at the 5' end of the reverse transcription primer P1;
the nucleotide sequence of the downstream probe P4 is shown as a general formula (III):
P-B1-B2-R (III)
wherein, P-B1-B2 is the 5' end sequence of the probe P4 at the downstream of the RT-SDA reaction, and comprises an enzyme cutting site recognition sequence of the incision endonuclease and a protecting base thereof; bases B1 and B2 are a labeled fluorophore and a quencher, respectively; r is the 3 'end sequence of a downstream probe P4 of the RT-SDA reaction, and the nucleotide sequence of the R is the same as the 8-14 base sequence of the 5' end of the reverse transcription primer P1;
s7, after the complementary DNA double strand is formed between the C5 strand and the C4 strand, performing enzyme digestion reaction under the action of a nicking endonuclease, and cutting a nick on the C5 strand;
s8, continuously extending the sequence at the 5 'end of the C5 chain incision, and replacing the sequence at the 3' end of the C5 chain incision to generate a C6 chain; simultaneously, a new C5-C4 DNA double-chain structure is formed, and the reaction of the steps S7 and S8 is continuously repeated to generate a large number of C6 chains;
s9, the C6 chain and the C2 chain are completely the same in sequence, and the reaction of the steps S4-S5 can be continuously carried out to generate a large number of C4 chains, and then the reaction of the steps S7-S8 is continuously carried out, and a large number of C6 chains are generated again; the amplification reaction may be cycled back and forth continuously such that the amplification product and fluorescent signal increase exponentially.
2. A reverse transcription-strand displacement amplification method for miRNA detection according to claim 1, characterized in that: the miRNA comprises one or more of humoral cycle miRNA, fresh tissue or paraffin tissue miRNA, total RNA and artificially synthesized miRNA molecules.
3. A reverse transcription-strand displacement amplification method for miRNA detection according to claim 1, characterized in that: the endonuclease comprises any one of Nb.BbvCI, nb.BsmI, nb.BsrDI, nb.BtsI, nt.AlwI, nt.BbvCI, nt.BspI, nt.BspQI, nt.BstNBI, nb.BssSI and Nt.CviPII.
4. The reverse transcription-strand displacement amplification dye method kit for miRNA detection is characterized in that: the dye-process kit comprises a reverse transcription primer P1, an upstream primer P2 and a downstream primer P3 according to any one of claims 1 to 3.
5. The reverse transcription-strand displacement amplification dye method kit for miRNA detection according to claim 4, wherein: the dye method kit further comprises components for a miRNA tailing reverse transcription reaction: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer; the reaction buffer comprises Tris-HCl pH 8.3, naCl, KCl, mgCl 2 、DTT；
The dye method kit also comprises components for performing a strand displacement isothermal amplification reaction: DNA polymerase, nicking endonuclease, dNTPs, fluorescent dye and reaction buffer; the reaction buffer solution comprises Tris-HCl pH 7.9, naCl and MgCl 2 、BSA。
6. The reverse transcription-strand displacement amplification dye method kit for miRNA detection according to claim 4, wherein: the dye method kit is characterized in that the concentrations of each component in a reaction system used in the process of carrying out miRNA tail-added reverse transcription are respectively as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl、1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs;
the dye method kit is characterized in that the concentrations of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification are respectively as follows: 10-100mM Tris-HCl pH 7.9, 50-200mM NaCl, 5-50mM MgCl 2 50-200. Mu.g/mL BSA, 0.05-0.5U/. Mu.L DNA polymerase, 0.05-0.5U/. Mu.L nicking endonuclease, 0.025-0.5mM dNTPs, 0.025-0.5. Mu.M upstream primer, 0.025-0.5. Mu.M downstream primer, 1 Xfluorescent dye.
7. The reverse transcription-strand displacement amplification dye method kit for miRNA detection according to claim 4, wherein: the dye method kit is characterized in that the concentrations of each component in a reaction system used in the process of carrying out miRNA tail-added reverse transcription are respectively as follows: 20-80mM Tris-HCl pH 8.3, 20-80mM NaCl, 20-80mM KCl, 5-30mM MgCl 2 5-50mM DTT, 2-20nM reverse transcription primer, 0.2-1.0mM ATP, 0.2-5.0U/. Mu.L Poly (A) polymerase, 2-20U/. Mu.L reverse transcriptase, 0.2-1.0mM dNTPs;
the dye method kit is characterized in that the concentrations of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification are respectively as follows: 20-80mM Tris-HCl pH 7.9, 80-150mM NaCl, 10-30mM MgCl 2 80-150. Mu.g/mL BSA, 0.1-0.3U/. Mu.L DNA polymerase, 0.1-0.3U/. Mu.L nicking endonuclease, 0.05-0.4mM dNTPs, 0.05-0.3. Mu.M upstream primer, 0.05-0.3. Mu.M downstream primer, 1 Xfluorescent dye.
8. The reverse transcription-strand displacement amplification dye method kit for miRNA detection according to claim 4, wherein: the reaction conditions of the dye method kit when the miRNA tail-added reverse transcription is carried out are as follows: firstly, preserving the temperature at 37-42 ℃ for 15-60min, and then heating at 65-95 ℃ for 5-20min;
the reaction conditions of the dye method kit during the strand displacement isothermal amplification are as follows: the temperature is kept at 37-65 ℃ for 15-60min, and then the mixture is heated at 70-95 ℃ for 5-20min.
9. The reverse transcription-strand displacement amplification probe method kit for miRNA detection is characterized in that: the probe method kit comprises the reverse transcription primer P1, the upstream primer P2 and the downstream probe P4 according to any one of claims 1 to 3.
10. The reverse transcription-strand displacement amplification probe method kit for miRNA detection according to claim 9, wherein: the probe method kit further comprises components for the reverse transcription reaction of miRNA tailing: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer; the probe method kit further comprises components for the reverse transcription reaction of miRNA tailing: poly (a) polymerase, reverse transcriptase, ATP, dNTPs, and reaction buffer;
the probe method kit further comprises components for performing a strand displacement isothermal amplification reaction: DNA polymerase, nicking endonucleases, dNTPs and reaction buffer; the reaction buffer solution comprises Tris-HCl pH 7.9, naCl and MgCl 2 、BSA。
11. The reverse transcription-strand displacement amplification probe method kit for miRNA detection according to claim 9, wherein: the probe method kit is characterized in that the concentration of each component used in the process of carrying out miRNA tail-added reverse transcription in a reaction system is as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl, 1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs;
the probe method kit is characterized in that the concentration of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification is respectively as follows: 10-100mM Tris-HCl pH 7.9, 50-200mM NaCl, 5-50mM MgCl 2 50-200. Mu.g/mL BSA, 0.05-0.5U/. Mu.L DNA polymerase, 0.05-0.5U/. Mu.L nicking endonuclease, 0.025-0.5mM dNTPs, 0.025-0.5. Mu.M upstream primer, 0.025-0.5. Mu.M downstream probe.
12. The reverse transcription-strand displacement amplification probe method kit for miRNA detection according to claim 9, wherein: the saidThe concentration of each component used in the probe method kit when the miRNA tail-added reverse transcription is carried out in a reaction system is as follows: 10-100mM Tris-HCl pH 8.3, 10-100mM NaCl, 10-100mM KCl, 1-50mM MgCl 2 1-100mM DTT, 1-50nM reverse transcription primer, 0.1-2.0mM ATP, 0.1-10U/. Mu.L Poly (A) polymerase, 1-50U/. Mu.L reverse transcriptase, 0.1-2.0mM dNTPs;
the probe method kit is characterized in that the concentration of each component in a reaction system used in the process of carrying out the strand displacement isothermal amplification is respectively as follows: 20-80mM Tris-HCl pH 7.9, 80-150mM NaCl, 10-30mM MgCl 2 80-150. Mu.g/mL BSA, 0.1-0.3U/. Mu.L DNA polymerase, 0.1-0.3U/. Mu.L nicking endonuclease, 0.05-0.4mM dNTPs, 0.05-0.3. Mu.M upstream primer, 0.05-0.3. Mu.M downstream probe.
13. The reverse transcription-strand displacement amplification probe method kit for miRNA detection according to claim 9, wherein: the reaction conditions of the probe method kit when the miRNA tail-added reverse transcription is carried out are as follows: firstly, preserving the temperature at 37-42 ℃ for 15-60min, and then heating at 65-95 ℃ for 5-20min;
the reaction conditions of the probe method kit during the strand displacement isothermal amplification are as follows: the temperature is kept at 37-65 ℃ for 15-60min, and then the mixture is heated at 70-95 ℃ for 5-20min.
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