阅读说明：本技术 通用逆转录引物及miRNA的cDNA合成方法、合成试剂盒和miRNA检测方法 (Universal reverse transcription primer, cDNA (complementary deoxyribonucleic acid) synthesis method of miRNA (micro ribonucleic acid), synthesis kit and miRNA detection method ) 是由 赵俊 朱灵 杨柯 朱灿灿 汪磊 花昌义 邓国庆 刘勇 于 2021-07-29 设计创作，主要内容包括：本发明公开一种通用逆转录引物及miRNA的cDNA合成方法、合成试剂盒和miRNA检测方法。提供的通用逆转录引物的5’端为适用于SDA等温扩增和PCR变温扩增的通用扩增引物结合序列,因此利用该通用逆转录引物由miRNA合成的cDNA不仅适用于SDA等温检测miRNA的体系中,还可同时适用于PCR变温检测miRNA的体系中,因此可以实现SDA等温和PCR变温同时检测同一miRNA的目的,有效提高miRNA定量结果的准确性。提供的cDNA合成方法可在同一反应体系和同一反应条件下同步实现miRNA加poly(A)尾和逆转录反应,整个反应时间较短,可以实现快速合成cDNA的目的。(The invention discloses a universal reverse transcription primer, a cDNA synthesis method of miRNA, a synthesis kit and an miRNA detection method. The 5' end of the universal reverse transcription primer is a universal amplification primer combination sequence suitable for SDA isothermal amplification and PCR temperature-variable amplification, so that cDNA synthesized by miRNA by utilizing the universal reverse transcription primer is not only suitable for a system for SDA isothermal detection of miRNA, but also suitable for a system for PCR temperature-variable detection of miRNA, thereby realizing the purpose of SDA isothermal and PCR temperature-variable simultaneous detection of the same miRNA and effectively improving the accuracy of miRNA quantitative results. The cDNA synthesis method provided can synchronously realize miRNA poly (A) tail addition and reverse transcription reaction in the same reaction system and under the same reaction condition, has short whole reaction time, and can realize the purpose of quickly synthesizing cDNA.)

1. A universal reverse transcription primer whose nucleotide sequence is represented by the following general formula (I):

R-(dT)_n-VN (I)

wherein, R is the 5' end sequence of the universal reverse transcription primer, and the nucleotide sequence is shown as SEQ ID NO. 1; VN is the 3' terminal sequence of the universal reverse transcription primer, wherein V represents adenine residue, guanine residue or cytosine residue, and N represents adenine residue, guanine residue, cytosine residue or thymine residue; (dT)_nIs the middle part of the universal reverse transcription primer and represents n continuous thymine residues, wherein n is any integer from 8 to 20.

2. The universal reverse transcription primer of claim 1, having a nucleotide sequence shown in SEQ ID NO 2.

3. A cDNA synthesis method of miRNA comprises the following reaction steps carried out in the same reaction system and under the same reaction condition:

s1: miRNA plus poly (a) tail reaction: taking miRNA as a target, taking ATP as a reaction raw material, and carrying out poly (A) tail addition reaction under the catalysis of poly (A) polymerase;

s2: reverse transcription synthesis of cDNA reaction: synthesizing cDNA using miRNA with poly (A) tail obtained in step S1 as target under the catalysis of reverse transcriptase by using the universal reverse transcription primer mentioned in claim 1 or 2.

4. The cDNA synthesis method according to claim 3, wherein the reaction system comprises: 10-100mM Tris-HCl pH 8.3, 20-100mM NaCl, 10-100mM KCl, 1-20mM MgCl₂5-50mM DTT, 1-20nM of the universal reverse transcription primer mentioned in claim 1 or 2, 0.1-1mM ATP, 0.1-5U/. mu.L Poly (A) polymerase, 5-50U/. mu.L reverse transcriptase, 0.1-1.0mM dNTP;

the reaction conditions are as follows: preserving heat for 20-60min at 37-42 ℃, and then preserving heat for 5-20min at 65-90 ℃.

5. A cDNA synthesis kit for miRNA comprising the universal reverse transcription primer of claim 1 or 2;

preferably, the cDNA synthesis kit further comprises components for performing a miRNA synthesis cDNA reaction: ATP, Poly (A) polymerase, reverse transcriptase, dNTPs and a reaction buffer, wherein the reaction buffer comprises Tris-HCl pH 8.3, NaCl, KCl, MgCl₂And DTT;

further preferably, the concentrations of the components in the cDNA synthesis reaction system when the cDNA synthesis kit is used are respectively: 10-100mM Tris-HCl pH 8.3, 20-100mM NaCl, 10-100mM KCl, 1-20mM MgCl₂5-50mM DTT, 1-20nM of the universal reverse transcription primer mentioned in claim 1 or 2, 0.1-1mM ATP, 0.1-5U/. mu.LPoly (A) polymerase, 5-50U/. mu.L reverse transcriptase, 0.1-1.0mM dNTP.

6. A universal amplification reverse primer, the nucleotide sequence of which is represented by the following general formula (II):

P-R (II)

wherein, P is the 5' end sequence of the general amplification reverse primer, and represents the restriction enzyme cutting site of the restriction endonuclease and the protective base thereof, wherein the restriction enzyme cutting site of the restriction endonuclease is selected from any one of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; r is the 3' end sequence of the universal amplification reverse primer, and the nucleotide sequence of the R is shown as SEQ ID NO. 1;

preferably, the nucleotide sequence of the universal amplification reverse primer is shown in any one of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10.

7. A polynucleotide, the nucleotide sequence of which is shown in SEQ ID NO. 1.

8. A method for quantitatively detecting miRNA, which is based on the cDNA synthesis method as claimed in claim 3 or 4, and further comprises:

s3: performing RT-SDA and/or RT-PCR quantitative detection by taking the cDNA reverse-transcribed and synthesized in the step S2 as a template; wherein the reverse primer used for amplifying the cDNA template is the universal amplification reverse primer mentioned in claim 6.

9. A miRNA detection kit comprising the universal reverse transcription primer of claim 1 or 2 and the universal amplification reverse primer of claim 6.

10. The universal reverse transcription primer according to claim 1 or 2, the cDNA synthesis method of claim 3 or 4, the cDNA synthesis kit of claim 5, the universal amplification reverse primer of claim 6, the method of quantitatively detecting miRNA of claim 8, or the miRNA detection kit of claim 9, wherein the miRNA is selected from one or more of: circulating mirnas, tissue mirnas, total RNAs, and synthetic miRNA molecules of eukaryotes.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a universal reverse transcription primer, a cDNA synthesis method, a synthesis kit and a miRNA detection method of miRNA, in particular to a universal reverse transcription primer for synthesizing cDNA by miRNA, a cDNA synthesis method, a cDNA synthesis kit and an miRNA detection method, which are simultaneously suitable for SDA (strand displacement isothermal amplification) and PCR detection of miRNA systems.

Background

miRNA is a non-coding single-stranded RNA composed of 18-24 nucleotides, and is widely involved in regulation and control of eukaryotic genes of animals, plants and the like. Multiple studies have shown that circulating mirnas are a class of molecular markers closely related to tumorigenesis, and that early screening and prognostic monitoring of cancer can be achieved by quantitatively detecting the expression level of circulating mirnas in serum or plasma of a patient (see, e.g., anecdotal et al, circulating micrornas and tumor diagnosis, chinese science, 2009, vol.39, stage 1: 64-68).

At present, RT-qPCR is one of the commonly used technical means for real-time quantitative detection of miRNA, and after cDNA is synthesized by tailing and reverse transcription, miRNA molecules can be detected, see, for example, patent documents CN105177132A and CN 110804656A. However, the PCR method requires continuous temperature change to achieve effective amplification of miRNA targets, which puts high demands on the performance of detection equipment, and the cDNA synthesis method suitable for PCR detection of miRNA is poor in compatibility and only suitable for the PCR detection principle. In recent years, with the rapid development of isothermal amplification techniques, attention has been paid to a new technique for efficiently amplifying nucleic acid molecules at a constant temperature. Researches find that isothermal amplification technology, such as strand displacement isothermal amplification (SDA), is particularly suitable for detecting short single-stranded RNA (ribonucleic acid) of miRNA (micro ribonucleic acid), and can amplify miRNA targets by 10 within 30-60 minutes⁹More than twice. However, at present, there is no report of a cDNA synthesis method for detecting miRNA by SDA, and even no cDNA synthesis method for detecting miRNA by both SDA isothermal amplification and PCR temperature-variable amplification is available, which limits the application of the SDA isothermal amplification technology in miRNA detection to a certain extent.

Disclosure of Invention

In view of one or more problems of the prior art, an aspect of the present invention provides a universal reverse transcription primer for cDNA synthesis of miRNA, the nucleotide sequence of the universal reverse transcription primer is represented by the following general formula (I):

R-(dT)_n-VN (I)

wherein, R is the 5' end sequence of the universal reverse transcription primer, and the nucleotide sequence is shown as SEQ ID NO. 1; VN is the 3' terminal sequence of the universal reverse transcription primer, wherein V represents adenine residue, guanine residue or cytosine residue, and N represents adenine residue, guanine residue, cytosine residue or thymine residue; (dT)_nIs the middle part of the universal reverse transcription primer and represents n continuous thymine residues, wherein n is any integer from 8 to 20.

In a preferred embodiment, the nucleotide sequence of the universal reverse transcription primer is shown as SEQ ID NO. 2.

In another aspect, the present invention provides a method for synthesizing cDNA of miRNA, comprising the following steps performed in the same reaction system and under the same reaction conditions:

s1: miRNA plus poly (a) tail reaction: taking miRNA as a target, taking ATP as a reaction raw material, and carrying out poly (A) tail addition reaction under the catalysis of poly (A) polymerase;

s2: reverse transcription synthesis of cDNA reaction: using the miRNA with the poly (a) tail obtained in step S1 as a target, synthesizing cDNA using the universal reverse transcription primer under the catalysis of reverse transcriptase.

In the above cDNA synthesis method, the reaction system comprises: 10-100mM Tris-HCl pH 8.3, 20-100mM NaCl, 10-100mM KCl, 1-20mM MgCl₂5-50mM DTT, 1-20nM of the universal reverse transcription primer, 0.1-1mM ATP, 0.1-5U/. mu.L Poly (A) polymerase, 5-50U/. mu.L reverse transcriptase, 0.1-1.0mM dNTP; preferably: 20-60mM Tris-HCl pH 8.3, 30-60mM NaCl, 20-60mM KCl, 5-15mM MgCl₂10-30mM DTT, 5-10nM of the universal reverse transcription primer, 0.3-0.7mM ATP, 0.25-2.5U/. mu.L poly (A) polymerase, 5-20U/. mu.L AMV reverse transcriptase, 0.4-0.8mM dNTP; the reaction conditions are as follows: keeping the temperature at 37-42 ℃ for 20-60min, and keeping the temperature at 65-90 ℃ for 5-20 min.

In another aspect, the invention further provides a cDNA synthesis kit for miRNA, which comprises the above universal reverse transcription primer.

The above-mentioned cDNA synthesis kit further comprises components for performing miRNA synthesis cDNA reaction: ATP, Poly (A) polymerase, reverse transcriptase, dNTPs and a reaction buffer, wherein the reaction buffer comprises Tris-HCl pH 8.3, NaCl, KCl, MgCl₂And DTT; preferably, the concentrations of the components in the cDNA synthesis reaction system when the cDNA synthesis kit is used are respectively as follows: 10-100mM Tris-HCl pH 8.3, 20-100mM NaCl, 10-100mM KCl, 1-20mM MgCl₂5-50mM DTT, 1-20nM of the universal reverse transcription primer, 0.1-1mM ATP, 0.1-5U/. mu.L Poly (A) polymerase, 5-50U/. mu.L reverse transcriptase, 0.1-1.0mM dNTP.

In still another aspect of the present invention, there is provided a universal amplification reverse primer, the nucleotide sequence of which is represented by the following general formula (II):

P-R (II)

wherein, P is the 5' end sequence of the general amplification reverse primer, and represents the restriction enzyme cutting site of the restriction endonuclease and the protective base thereof, wherein the restriction enzyme cutting site of the restriction endonuclease is selected from any one of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; r is the 3' end sequence of the universal amplification reverse primer, and the nucleotide sequence of the R is shown as SEQ ID NO. 1; preferably, the nucleotide sequence of the universal amplification reverse primer is shown in any one of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10. The universal amplification reverse primer is used for amplification of cDNA in an SDA isothermal amplification or PCR temperature-variable amplification reaction system with the cDNA as a template, wherein the cDNA is obtained by synthesis of miRNA by using the universal reverse transcription primer.

In still another aspect, the present invention provides a polynucleotide for use in the above-mentioned universal reverse transcription primer or universal amplification reverse primer, wherein the nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 1. The existence of the polynucleotide enables the cDNA synthesized by the universal reverse transcription primer to be used as an amplification template in an SDA isothermal amplification system and a PCR variable temperature amplification system, or enables the universal amplification reverse primer to be used as a reverse primer of the amplification template in the SDA isothermal amplification system and the PCR variable temperature amplification system, and is suitable for the amplification of the cDNA synthesized by any miRNA by utilizing the universal reverse transcription primer.

In another aspect, the present invention further provides a method for quantitatively detecting miRNA, which further includes, on the basis of the above-mentioned cDNA synthesis method:

s3: performing RT-SDA and/or RT-PCR quantitative detection by taking the cDNA reverse-transcribed and synthesized in the step S2 as a template; wherein the reverse primer used for amplifying the cDNA template is the universal amplification reverse primer.

In still another aspect, the invention provides a miRNA detection kit, which includes the above-mentioned universal reverse transcription primer and universal amplification reverse primer.

The miRNA may be any type of miRNA, for example selected from one or more of the following: circulating mirnas, tissue mirnas, total RNAs, and synthetic miRNA molecules of eukaryotes.

The 5 'end of the universal reverse transcription primer provided based on the technical scheme is a universal amplification primer combination sequence suitable for SDA isothermal amplification and PCR variable temperature amplification, the 3' end is a universal base sequence of a recognition site, and a poly (T) base sequence complementary with a poly (A) tail of miRNA is arranged between the universal reverse transcription primer and the recognition site, so that the cDNA synthesized by the cDNA synthesis method and the synthesis kit of miRNA provided based on the universal reverse transcription primer is not only suitable for a system for SDA isothermal detection of miRNA, but also suitable for a system for PCR variable temperature detection of miRNA, the purpose of simultaneously detecting the same miRNA at the same time of SDA and other moderate PCR variable temperature can be realized, the results of the two can be verified mutually, the accurate quantification of miRNA is facilitated, and the accuracy of the miRNA quantification result is effectively improved. On the other hand, the cDNA synthesis method provided by the invention can synchronously realize the reaction of miRNA poly (A) tail addition and reverse transcription in the same reaction system and under the same reaction condition, the whole reaction time is short, the reaction system is simple, the aim of quickly synthesizing cDNA can be realized, and the detection efficiency of miRNA is improved. On the other hand, in the miRNA detection method provided by the present invention, a universal amplification reverse primer designed based on the universal reverse transcription primer provided by the present invention may be used, and the use of the universal amplification reverse primer does not change with the change of the detected miRNA, so that when different mirnas are detected, no additional synthesis of a new reverse primer is required, which may improve the detection efficiency and save the detection cost.

Drawings

FIG. 1 is a schematic diagram of the reaction principle of a cDNA synthesis method suitable for SDA and PCR detection of miRNA in one embodiment of the present invention;

FIG. 2 is a graph showing the amplification curve of cDNA synthesized using one embodiment of the present invention as a template in the SDA and PCR detection methods, in which panel A shows the amplification curve of the synthesized cDNA as a template in the SDA detection method, and panel B shows the amplification curve of the synthesized cDNA as a template in the PCR detection method.

Detailed Description

Aiming at the technical defects that a cDNA synthesis method suitable for SDA (such as RT-SDA) to detect miRNA still lacks in the prior art, and particularly a cDNA synthesis method suitable for SDA and PCR (such as RT-PCR) to detect miRNA simultaneously lacks, the invention aims to provide a cDNA synthesis method suitable for SDA to detect miRNA, particularly a cDNA synthesis method suitable for SDA and PCR to detect miRNA simultaneously, and provides a universal reverse transcription primer and a cDNA synthesis kit for reverse transcribing miRNA into cDNA in the cDNA synthesis method.

Specifically, the present invention provides a cDNA synthesis method suitable for SDA detection of miRNA, and more particularly, a cDNA synthesis method suitable for SDA detection and miRNA detection by PCR, the technical principle of which is shown in fig. 1, the cDNA synthesis method includes two reaction steps under the same reaction system and the same reaction conditions: (1) under the catalysis of poly (A) polymerase, the miRNA target takes 5' -Adenosine Triphosphate (ATP) as a reaction raw material to carry out poly (A) tailing reaction; and (2) combining the miRNA sequence with the poly (A) tail with a poly (T) reverse transcription primer (namely a universal reverse transcription primer) (through complementary combination of the poly (A) sequence in the miRNA sequence with the poly (T) sequence in the universal reverse transcription primer), and carrying out reverse transcription to synthesize the cDNA under the catalysis of reverse transcriptase. Wherein the reaction system used in the above cDNA synthesis method comprises the following components: poly (T) reverse transcription primer (i.e., universal reverse transcription primer), adenosine 5' -triphosphate (ATP), Poly (A) polymerase, reverse transcriptase (which may be one or more of AMV reverse transcriptase, M-MuLV reverse transcriptase, ProtoScript II reverse transcriptase), reaction buffer (comprising Tris-HCl (pH 8.3), NaCl, KCl, MgCl₂DTT (dithiothreitol)) and dNTPs. The reaction conditions are as follows: preserving heat for 20-60min at 37-42 ℃, and then preserving heat for 5-20min at 65-90 ℃ to finish the reaction. The miRNA as a target may be subjected to the above-described poly (a) tailing reaction and reverse transcription reaction simultaneously under the above reaction system and reaction conditions, thereby synthesizing cDNA. Wherein the nucleotide sequence of the universal reverse transcription primer used in the reverse transcription reaction is represented by the following general formula (I):

R-(dT)_n-VN (I)

in the general formula (I), R is a 5' end sequence of a universal reverse transcription primer, and the nucleotide sequence of the universal reverse transcription primer is a universal amplification primer combination sequence (CAGTCGTCGCACCCTCC, SEQ ID NO:1) which is simultaneously suitable for SDA isothermal amplification and PCR temperature-variable amplification; the 3' end is composed of nucleotide residues represented by VN and is used as a universal base sequence of a recognition site, wherein V represents adenine residue A, guanine residue G or cytosine residue C, and N represents adenine residue A, guanine residue CA purine residue G, a cytosine residue C, or a thymine residue T; (dT)_nThe middle part of the universal reverse transcription primer represents n consecutive thymine residues, wherein n can be any integer within the value of 8-20 (inclusive), preferably 12, i.e., the nucleotide sequence of the universal reverse transcription primer is preferably CAGTCGTCGCACCCTCCTTTTTTTTTTTTVN (SEQ ID NO: 2).

Since the general reverse transcription primer represented by the general formula (I) comprises the general amplification primer binding sequence (SEQ ID NO:1) which is applicable to both the SDA isothermal amplification and the PCR temperature-variable amplification, according to the technical principle shown in FIG. 1, the first strand cDNA (i.e. the single-stranded DNA obtained according to the first step of the miRNA template) obtained by reverse transcription of the miRNA sequence with the poly (A) tail also comprises the general amplification primer binding sequence (SEQ ID NO:1), the first strand cDNA synthesized by the method can be used as a template in the SDA isothermal amplification system and also can be used as a template in the PCR temperature-variable amplification system, and the complementary strand of the template amplified by the guide of the forward primer in the amplification system comprises a sequence segment complementary to the SEQ ID NO:1, so that one general amplification reverse primer can be used in the SDA isothermal amplification system and the PCR temperature-variable amplification system, the 3' end sequence of the universal amplification reverse primer is designed as SEQ ID NO. 1, so that the universal amplification reverse primer can be complementarily combined with a template complementary strand obtained by amplification of a forward primer in an amplification system, and then reverse amplification is guided. On the other hand, for the continuation of amplification (i.e., to cleave the formed double-stranded cDNA to obtain a single-stranded template), the 5' end of the universal amplification reverse primer is designed to contain the cleavage site of a nicking endonuclease and its protective bases, wherein the cleavage sites that can be selected depending on the nicking endonuclease used include: GAGTC (SEQ ID NO:3, the cleavage site of Nt.BstNBI), GGATC (SEQ ID NO:4, the cleavage site of Nt.AlwI), GTCTC (SEQ ID NO:5, the cleavage site of Nt.BsmAI), and GCTCTTC (SEQ ID NO:6, the cleavage site of Nt.BspQI). Thus, when a cDNA synthesized from the universal reverse transcription primer represented by the above general formula (I) is used as a template in an SDA isothermal amplification system or a PCR variable temperature amplification system, any miRNA can be detected using a nucleotide sequence represented by the following general formula (II) as a universal amplification reverse primer in both amplification systems:

P-R (II)

in the general formula (II), P is the 5' terminal sequence of the general amplification reverse primer, the restriction site of the nicking endonuclease and the protective base thereof, wherein the restriction site of the nicking endonuclease can be any one of SEQ ID NO:3 to SEQ ID NO:6, and the protective base of the restriction site can be selected conventionally according to the type of the restriction site, which is well known to those skilled in the art; r is the 3' end sequence of the universal amplification reverse primer, and the nucleotide sequence of the R is shown as SEQ ID NO. 1. Preferably, the nucleotide sequence of the universal amplification reverse primer represented by the general formula (II) is as shown in any one of SEQ ID NO:7 to SEQ ID NO: 10:

in the above sequences SEQ ID NO 7 to SEQ ID NO 10, the single underlined portion indicates the cleavage site of the nicking endonuclease, the double underlined portion indicates SEQ ID NO 1, and the other portions are protective bases of the cleavage site.

The invention is further illustrated by the following examples. It should be understood that the specific examples are intended to be illustrative of the invention and are not intended to limit the scope of the invention.

The methods used in the following examples are conventional methods unless otherwise specified. The specific steps can be seen in: a Molecular Cloning Laboratory Manual (Molecular Cloning: A Laboratory Manual, Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples. The test materials used in the following examples are all conventional biochemical reagents, and are commercially available, unless otherwise specified.

The method provided by the invention is applicable to any type of miRNA, including natural miRNA molecules of all eukaryotes such as human beings, animals, plants and the like, such as one or more of circulating miRNA, tissue miRNA and total RNA, and can also include any artificially synthesized miRNA molecules. The method of the present invention is described in the following examples using artificially synthesized miR-21 molecular standard (UAGCUUAUCAGACUGAUGUUGA, SEQ ID NO:11) as an example.

The sequences referred to in the examples below were all synthesized using known techniques.

Example 1: cDNA synthesis method of miRNA

In the embodiment, an artificially synthesized miR-21 standard substance is used as a sample, a cDNA synthesis method which is simultaneously suitable for SDA and PCR detection of miRNA is constructed according to the technical principle shown in figure 1, and specifically a 20-microliter reaction system is prepared according to the following requirements: miR-21 molecular standard 50pM, ATP 1.0 μ L (final concentration is 0.5mM), Poly (A) polymerase 5U (final concentration is 0.25U/. mu.L), reaction buffer 2.0 μ L (final concentrations are 50mM Tris-HCl (pH 8.3), 50mM NaCl, 50mM KCl, and 5mM MgCl₂10mM DTT), AMV reverse transcriptase 100U (final concentration of 5U/. mu.L), poly (T) reverse transcription primer (SEQ ID NO:2) 1.0. mu.L (final concentration of 5nM), 0.5mM dNTP, and make up water to 20. mu.L. Under the reaction conditions of incubation at 37 ℃ for 30min and then at 85 ℃ for 5min, the following two reactions occurred simultaneously in this 20. mu.L reaction system:

1.1) miR-21 and poly (A) tail reaction: taking miR-21 molecules as targets, taking ATP as reaction raw materials, and carrying out miR-21 and poly (A) tail reaction under the catalysis of Poly (A) polymerase; and

1.2) reverse transcription synthesis of cDNA reaction: taking the miR-21 molecule added with poly (A) tail obtained in the step 1.1) as a target, taking dNTP as a raw material, and synthesizing cDNA by utilizing poly (T) reverse transcription primer under the catalysis of AMV reverse transcriptase.

Because the poly (T) reverse transcription primer used in the reaction of synthesizing cDNA by reverse transcription contains the general amplification primer binding sequence (SEQ ID NO:1) which is simultaneously suitable for SDA isothermal amplification and PCR temperature-variable amplification, the first strand cDNA obtained by synthesis also contains the general amplification primer binding sequence (SEQ ID NO:1) according to the technical principle shown in FIG. 1, so that the synthesized cDNA can also be simultaneously suitable for an SDA isothermal amplification system and a PCR temperature-variable amplification system as an amplification template, and further can quantitatively detect miR-21 by using an SDA isothermal and PCR temperature-variable detection method, which is verified by the following example 2.

Example 2: RT-SDA isothermal RT-PCR variable temperature detection of miR-21

In this embodiment, the cDNA of miR-21 synthesized in the above embodiment 1 is used as a template, and is quantitatively detected in real time by using SDA and PCR methods to verify the effectiveness of the cDNA synthesis method and the compatibility in the SDA isothermal and PCR temperature-variable detection system, which specifically includes the following operations.

2.1) A20. mu.L reaction system for isothermal amplification of RT-SDA was prepared as follows: mu.L of cDNA template for miR-21 synthesized in example 1, a forward primer (CGGTTGGAGTCCTTGTTAGCTTATCAGACTG (SEQ ID NO:12)) at a final concentration of 1.25mM, a reverse primer (SEQ ID NO:7) at a final concentration of 1.25mM, and a reaction buffer (2.0. mu.L, each at a final concentration of 50mM Tris-HCl (pH 8.3), 50mM NaCl, 50mM KCl, and 5mM MgCl)₂10mM DTT), dNTP at a final concentration of 0.5mM, DNA polymerase at a final concentration of 0.1U/. mu.L, endonuclease at a final concentration of 0.2U/. mu.L (Nt.BstNBI), 1. mu.L of 1 xEva Green dye, supplemented with water to 20. mu.L, and the cDNA obtained in example 1 was amplified according to the following amplification procedure: preserving the temperature at 55 ℃ for 40min, collecting fluorescence signals once every one minute, taking the fluorescence signals 40 times in total as a positive group, and setting a negative group (a cDNA template is not added in an amplification system). The results of isothermal SDA amplification are shown in FIG. 2, panel A.

2.2) preparing a 20 mu L reaction system for RT-PCR temperature-variable amplification according to the following system: example 1. mu.L of cDNA template for miR-21 synthesized in example 1, forward primer (SEQ ID NO:12) at a final concentration of 1.25mM, reverse primer (SEQ ID NO:7) at a final concentration of 1.25mM, and reaction buffer 2.0. mu.L (final concentrations: 50mM Tris-HCl (pH 8.3), 50mM NaCl, 50mM KCl, 5mM MgCl, respectively₂10mM DTT), dNTP at a final concentration of 0.5mM, DNA polymerization at a final concentration of 0.1U/. mu.LEnzyme, endonuclease (Nt.BstNBI) at a final concentration of 0.2U/. mu.L, 1. mu.L of 1 xEva Green dye, water supplemented to 20. mu.L, and the cDNA obtained in example 1 was amplified according to the following amplification procedure: heating at 95 deg.C for 10 s; heating at 95 ℃ for 5s, heating at 52 ℃ for 20s, and heating at 72 ℃ for 20s for a total of 40 cycles, collecting a fluorescence signal once after each cycle, wherein the fluorescence signal is collected for a total of 40 times to serve as a positive group, and meanwhile, setting a negative group (a cDNA template is not added in an amplification system). The PCR temperature-variable amplification result is shown in B in FIG. 2.

According to the results shown in the A and B panels in FIG. 2, it can be seen that the positive groups in the RT-SDA isothermal amplification and RT-PCR temperature-variable amplification tests both show effective amplification curves, and the negative groups do not show amplification signals, which indicates that the cDNA synthesis method provided by the invention can realize effective synthesis from miRNA to cDNA, and the synthesized cDNA can be simultaneously suitable for SDA isothermal amplification and PCR temperature-variable amplification, which indicates that the cDNA synthesis method established by the invention has high compatibility, and can simultaneously provide a cDNA template required for amplification for SDA isothermal amplification and PCR temperature-variable amplification. On the other hand, due to the compatible structure design of the universal amplification primer binding sequence (SEQ ID NO:1) at the 5' end of the universal reverse transcription primer used in the cDNA synthesis method provided by the invention, the primers (SEQ ID NO: 7-SEQ ID NO:10) comprising the SEQ ID NO:1 can be used as the universal amplification reverse primer in an SDA isothermal amplification and PCR temperature-variable amplification system taking the synthesized cDNA as an amplification template.

Example 3: cDNA synthesis kit and miRNA detection kit

Based on the results of the above example 1 and example 2, the present invention also provides a cDNA synthesis kit that can be used for synthesizing cDNA thereof from miRNA in the above example 1, and a miRNA detection kit for detecting miRNA using the synthesized cDNA as an amplification template in the above example 2.

The provided cDNA synthesis kit may comprise a universal reverse transcription primer represented by the general formula (I), preferably a universal reverse transcription primer as shown in SEQ ID NO. 2. The provided cDNA synthesis kit may further comprise components for simultaneously performing a poly (A) tailing reaction and a reverse transcription synthesis cDNA reaction in the cDNA synthesis method provided by the present invention: ATP, Poly (A) polymerase, reverseA transcriptase, dNTPs and a reaction buffer, wherein the reaction buffer comprises Tris-HCl pH 8.3, NaCl, KCl, MgCl₂And DTT. The concentrations of these ingredients at the time of use were: 1-20nM poly (T) primer (i.e., universal reverse transcription primer), 0.1-1mM ATP, 0.1-5U/. mu.L poly (A) polymerase, 5-50U/. mu.L reverse transcriptase, 0.1-1.0mM dNTP and reaction buffer (10-100mM Tris-HCl (pH 8.3), 20-100mM NaCl, 10-100mM KCl, 1-20mM MgCl₂5-50mM DTT), preferably 20-60mM Tris-HCl pH 8.3, 30-60mM NaCl, 20-60mM KCl, 5-15mM MgCl₂10-30mM DTT, 5-10nM poly (T) primer, 0.3-0.7mM ATP, 0.25-2.5U/. mu.L poly (A) polymerase, 5-20U/. mu.L reverse transcriptase, 0.4-0.8mM dNTP; wherein the reverse transcriptase can be one or more of AMV, M-MuLV and ProtoScript II. When the cDNA synthesis kit is used for carrying out cDNA synthesis of miRNA, the reaction conditions for synchronously carrying out poly (A) tailing reaction and reverse transcription synthesis cDNA reaction can be as follows: preserving heat for 20-60min at 37-42 ℃, and then preserving heat for 5-20min at 65-90 ℃.

The provided miRNA detection kit can comprise a universal reverse transcription primer represented by a general formula (I), preferably a universal reverse transcription primer shown as SEQ ID NO. 2; a general amplification reverse primer for amplifying cDNA represented by the general formula (II), preferably any one of SEQ ID NO 7 to SEQ ID NO 10, may also be included.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of science of fertilizer combination and substance science of Chinese academy of sciences

HEFEI ZHONGKE YIKANGDA BIOMEDICAL Co.,Ltd.

<120> universal reverse transcription primer, cDNA synthesis method of miRNA, synthesis kit and miRNA detection method

<130> CGCNL211089W

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagtcgtcgc accctcc 17

<210> 2

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cagtcgtcgc accctccttt tttttttttv n 31

<210> 3

<211> 5

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gagtc 5

<210> 4

<211> 5

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggatc 5

<210> 5

<211> 5

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtctc 5

<210> 6

<211> 7

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctcttc 7

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cggttggagt ccttgtcagt cgtcgcaccc tcc 33

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cggttgggat ccttgtcagt cgtcgcaccc tcc 33

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggttggtct cgtcagtcgt cgcaccctcc 30

<210> 10

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cggttggctc ttcgtcagtc gtcgcaccct cc 32

<210> 11

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

uagcuuauca gacugauguu ga 22

<210> 12

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cggttggagt ccttgttagc ttatcagact g 31