CN116769887A

CN116769887A - Capturing nucleotide for nucleic acid amplification and application thereof

Info

Publication number: CN116769887A
Application number: CN202210226793.3A
Authority: CN
Inventors: 徐高连; 郭云飞; 徐宏; 古宏晨
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2023-09-19

Abstract

The invention discloses a capture nucleotide for nucleic acid amplification and application thereof. The capture nucleotide sequentially comprises an irrelevant sequence, a first general sequence, a folding sequence, a sequence capable of complementarily pairing with the irrelevant sequence and a binding capture sequence from a 5 'end to a 3' end; the folding sequence comprises a sequence identical to the 5' end of the target molecule; the binding capture sequence binds complementarily to the non-5' terminal sequence of the target molecule. The invention also discloses a kit comprising the capture nucleotide, a method for amplifying nucleic acid by using the same, and application of the capture nucleotide and the kit in detection of miRNA. The invention realizes single-tube multiplex detection of different miRNAs, reduces mutual interference among primers, and satisfies equivalent amplification. The primer with the special self-folding structure effectively improves the capturing efficiency of miRNA to increase the sensitivity and greatly improves the reaction specificity.

Description

Capturing nucleotide for nucleic acid amplification and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a capture nucleotide for nucleic acid amplification and application thereof.

Background

MicroRNA (miRNA) is a non-coding single-stranded RNA small molecule with the length of about 18-25bp, has stable specific sequences to regulate gene expression, and is involved in important biological processes such as cell differentiation, cell proliferation, apoptosis and immune response. The development of a high-sensitivity, high-specificity and single-tube multiplex detection method using circulating miRNA as a biomarker is urgent.

Abnormal expression of mirnas is closely related to occurrence and development of human tumors and major diseases, but association of mirnas with human diseases is intricate, occurrence of one disease usually has synergistic effects of a plurality of mirnas, and one miRNA is involved in occurrence and development of a plurality of diseases. Therefore, research on the relationship between miRNA and diseases is of great biological significance, and the miRNA can be used as a marker to provide a judgment basis in early diagnosis of tumors and prognosis thereof. Although the detection significance of miRNA is highly valued, the current detection technology still has great restriction. The detection difficulty of miRNA is that the miRNA fragment is extremely short, the concentration in a sample is extremely low, and the detection concentration range spans several orders of magnitude; it is meaningless to detect a single miRNA, and each tumor often involves aberrant expression of multiple mirnas; the homology of each miRNA family is extremely high, and specific detection is difficult to realize. Therefore, establishing an accurate, efficient and multiplex quantitative detection method is a key for taking miRNA as a tumor marker and widely popularizing.

Existing methods for detecting miRNA include Northern blotting, microarray methods, RT-PCR, RNA sequencing and the like. Northern blotting can quantitatively detect miRNA, and the types and the concentrations of miRNA are distinguished by identifying the molecular weight and the brightness of the miRNA, but the detection method is complex in operation, long in detection time, and poor in detection sensitivity on low-abundance miRNA, and meanwhile, the radioactive probe has great hidden trouble on people and the environment. The microarray method can quantitatively detect miRNA, can detect the expression of hundreds of genes in the same sample at the same time, has high flux, but has high price and complicated steps. RNA sequencing is high in flux, but is high in price, and primer design is difficult. RT-PCR includes Stem-loop RT-PCR and poly A tailing RT-PCR, both of which have certain limitations. The Stem-loop RT-PCR is characterized in that a reverse transcription primer with a Stem-loop structure is designed, the reverse transcription primer comprises a sequence complementary and paired with a target miRNA, a Stem sequence and a loop sequence complementary and paired with a universal primer, the method is high in specificity, but the primer is difficult to design, particularly single-tube multiplex detection, the Poly A tailing RT-PCR is catalyzed by Poly A polymerase, AMP converted from ATP is added to the 3' end of the miRNA under the condition of not depending on a template, and then the synthesis of cDNA is completed by reverse transcription by taking oligo-dT as a primer, and then PCR detection is carried out. In summary, these methods have a certain application prospect, but most of the design concepts of the detection methods are to detect a single miRNA, for example, only mixing each miRNA detection system to perform multiple detection, there is often mutual interference between amplification primers, and it is difficult to meet the detection requirements of high throughput and multiple detection at the same time.

Disclosure of Invention

In view of the defects of low sensitivity, poor specificity and incapability of guaranteeing single-tube multiple detection of different miRNAs in the method for detecting miRNAs in the prior art, the invention provides a fluorescent PCR technology for capturing miRNAs and amplifying universal primers based on self-folding primers (namely capturing nucleotides), and the method has the advantages of high sensitivity, good specificity and equivalent amplification on the basis of realizing multiple miRNAs detection.

In order to solve the technical problems, one of the technical schemes provided by the invention is as follows: a capture nucleotide (self-folding primer) for nucleic acid amplification, said capture nucleotide comprising, in order from the 5' end to the 3' end, a first universal sequence, a folding sequence, and a binding capture sequence, said first universal sequence further comprising an unrelated sequence at the 5' end, said folding sequence and said binding capture sequence further comprising a sequence capable of complementary pairing with said unrelated sequence; the folding sequence comprises a sequence identical to the 5' end of the target molecule; the binding capture sequence binds complementarily to the non-5' terminal sequence of the target molecule.

In a preferred embodiment of the invention, the sequence identical to the 5' end of the target molecule is 8-18bp in length.

The capture nucleotide according to one of the embodiments of the present invention, the unrelated sequence is any sequence having a random base composition, and the Tm value of the unrelated sequence is sufficient to allow pairing with the complementary sequence thereof, such that the capture nucleotide forms a self-folding structure.

In a preferred embodiment of the invention, the unrelated sequences comprise polyA and/or polyT.

The capture nucleotide according to one of the embodiments of the present invention further comprises a second universal sequence.

In a preferred embodiment of the invention, said second universal sequence is located between said folding sequence and said sequence capable of complementary pairing with said unrelated sequence.

In a specific embodiment of the invention, the nucleotide sequence of the first universal sequence is shown as SEQ ID NO. 1, and the nucleotide sequence of the second universal sequence is shown as SEQ ID NO. 2.

The capture nucleotide according to one of the embodiments of the present invention further comprises a nucleic acid extension blocking site.

In a preferred embodiment of the invention, the nucleic acid extension blocking site is located 3' to the folding sequence.

The nucleic acid extension blocking site modifications may be Spacer, abasic phosphate backbone (AP site) and/or uracil bases, as is conventional in the art.

In a specific embodiment of the invention, the sequence of the capture nucleotide is shown as SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11.

In order to solve the technical problems, the second technical scheme provided by the invention is as follows: a kit for nucleic acid amplification, said kit comprising a capture nucleotide according to one of the embodiments of the present invention.

The kit according to the second aspect of the present invention further comprises a pair of amplification primers, wherein the nucleotide sequence of the forward primer of the amplification primer is a subset of the first universal sequence of the capture nucleotide and the full length of the target molecule, the nucleotide sequence of the reverse primer of the amplification primer is a subset of the second universal sequence of the capture nucleotide and the sequence complementary to the unrelated sequence and the full length of the binding capture sequence, and a position for binding a specific probe is left between the forward primer and the reverse primer.

In a preferred embodiment of the invention, the nucleotide sequences of the subset are 15-25bp in length.

In a specific embodiment of the invention, the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

The kit according to the second aspect of the present invention further comprises a specific probe.

In a preferred embodiment of the invention, the specific probes are labeled with a fluorescent group and/or a quenching group.

In a preferred embodiment of the invention, the fluorescent group is labeled at the 5' end of the specific probe; and/or, the quenching group is marked at the 3' end of the specific probe.

The fluorophore may be selected from any one of FAM, VIC, JOE, TET, CY, CY5, ROX, texas Red and LC Red460, as is conventional in the art; the quenching group may be selected from any one of MGB, BHQ1, BHQ2, BHQ3, dabcy1, and Tamra.

In a specific embodiment of the invention, the nucleotide sequence of the specific probe is shown as SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12.

The kit according to a second aspect of the present invention further comprises a specific sequence blocker comprising a nucleic acid capable of complementary pairing with a non-specific product comprising a nucleic acid analogue modification.

The nucleic acid analog may be Locked Nucleic Acid (LNA), peptide Nucleic Acid (PNA), transposed base, 2'-O,4' -C-methylene bridge RNA, 2'-O-Methyl RNA, or 2' -Fluoro RNA, as is conventional in the art.

In a preferred embodiment of the invention, the nucleic acid analogue is a Locked Nucleic Acid (LNA).

In a specific embodiment of the invention, the nucleotide sequence of the specific sequence blocker is shown as SEQ ID NO. 13.

The kit further comprises DNA polymerase, dNTPs, mg according to routine in the art ²⁺ Any one or more of an enzyme digestion buffer and a PCR buffer. The kit may also include reagents required for sequencing, such as polymerase, sequencing primers, etc., as are well known to those skilled in the art.

In order to solve the technical problems, the third technical scheme provided by the invention is as follows: a nucleic acid amplification method comprising employing a capture nucleotide according to one of the aspects of the invention to bind a target molecule and initiate specific linear amplification and amplification primer-based nucleic acid amplification detection; the nucleic acid amplification method comprises the steps of:

(1) The 5' end sequence determines the target molecule and capture nucleotide binding capture sequence binding.

(2) The capture nucleotide takes the target molecule as a template to carry out an extension reaction, and the nucleotide complementary with the target molecule is added at the 3' -end of the capture nucleotide, so that the extended capture nucleotide is formed.

(3) The extended capture nucleotide is denatured, the unrelated sequence is separated from its complementary sequence, and the extended sequence is base complementary paired with the folded sequence inside the molecule to form a new semi-hairpin product.

(4) The new semi-hairpin product is subjected to extension reaction, and nucleotide complementary to the first general sequence and the unrelated sequence in the molecule is added at the 3' -end, so that the complete hairpin product is formed.

(5) And (3) taking the complete hairpin structure product as a template, and carrying out exponential amplification by using an amplification primer to obtain an amplification product.

In a preferred embodiment of the invention, step (4) further comprises the use of a 3'-5' exonuclease, such as Exo I exonuclease, capable of cleaving single strands of DNA.

In a preferred embodiment of the invention, the exponential amplification of step (5) comprises a polymerase chain reaction.

In a preferred embodiment of the present invention, the amplification primer of step (5) is an amplification primer as defined in the kit according to the second aspect of the present invention.

The method according to claim III, further comprising the step of detecting the amplification product by binding to a specific probe.

In a preferred embodiment of the present invention, the specific probe is a specific probe defined in the kit according to the second aspect of the present invention.

The nucleic acid amplification method according to the third aspect of the present invention, wherein a specific sequence blocker defined in the kit according to the second aspect of the present invention is added to an amplification system when performing exponential amplification.

In order to solve the technical problems, the fourth technical scheme provided by the invention is as follows: the capture nucleotide according to one of the technical schemes of the invention or the kit according to the second of the technical schemes of the invention is applied to detection of miRNA.

The system for detecting miRNA mainly comprises two parts, namely cDNA conversion based on capturing miRNA sequences by self-folding primers and fluorescent PCR technology based on universal primer amplification, and the principle of the system is shown in figure 1:

the design of the self-folding primer mainly comprises 6 parts: starting from the 5' end, U3a is an irrelevant sequence, U1s is an upstream universal primer region, T1a is a folding region (preferably 11-12 bp) of a self-folding primer extension product, U2s is a downstream universal primer region, U3s is a complementary pairing sequence with U3a can enable a long primer to form a self-folding structure, T2s is a miRNA capturing region (preferably 10-11 bp), a nucleic acid extension blocking modification is preferably arranged between T1a and U2s, the modification mode can be a conventional mode in the field, so long as primer extension can be blocked, for example, a Spacer C18 modification (which can block DNA polymerase extension, can be used or not, a Spacer C18 product is purer, the primer is blocked after extension), and other modifications can also be used for blocking, for example, a plurality of uracil modifications can be used, and then a user enzyme is used for cutting to block the product.

The nucleic acid extension blocking modification enables the hairpin structure product to stop extension at the nucleic acid extension blocking modification position when the PCR amplification is carried out by using the universal primer and/or the target molecule specific primer, so that a linearized amplification template is generated for subsequent exponential amplification, and the amplification efficiency is improved. In the invention, the nucleic acid extension blocking modification is arranged between T1a and U2s, so that the self-folding of the extended capture nucleotide is not influenced, and the self-extension can be carried out by taking the capture nucleotide as a template after the self-folding, thereby forming a product with a complete hairpin structure; the hairpin structure product can be used as a template for PCR amplification by a universal primer and/or a target molecule specific primer to carry out PCR exponential amplification, thereby effectively ensuring the specificity and accuracy of the amplification.

As shown in fig. 1, the principle of miRNA detection of the present invention is mainly divided into two steps:

step one: reverse transcription based on U3a/U3s self-folding primers. When target miRNA exists, T2s can be complementarily paired with the 3 'end of the miRNA sequence through bases, under the action of reverse transcriptase, T1s complementarily paired with the 5' end of the miRNA sequence extends, when the miRNA is denatured, the miRNA is separated from the extended self-folding primer, a new half hairpin structure is formed by self-folding of the extended product through recombination of the T1s and the T1a, a complete stem-loop structure extends under the action of the reverse transcriptase, and the fallen target miRNA enters the next reverse transcription cycle.

Step two: PCR amplification based on universal primers. And (3) completing PCR index amplification and detection of the complete stem-loop structure formed in the step one under the action of the universal primer and the specific probe.

In order to improve the PCR amplification efficiency, the miRNA detection system of the invention introduces Exo I exonuclease (with 3'-5' exonuclease activity, only cuts DNA single strand), and can cut excessive self-folding primer and nonspecific single strand product after reverse transcription is completed, wherein the excessive self-folding primer is cut off by T2s to become an ineffective defective fragment.

According to the miRNA detection system, two ends of a related specific probe are respectively marked with a fluorescence detection group and a quenching group. Preferred fluorescent detection groups are any of FAM, VIC, texas Red, CY 5. The preferred quenching group may be MGB.

The miRNA detection system provided by the invention can be used for amplifying a single universal primer and a specific primer instead of double universal primers.

As shown in fig. 1, T2s and T1a of the capture nucleotide can be changed according to the length and GC content of the miRNA, for example, if the GC content of the T2s region is high, the sequence length can be shortened but the binding of reverse transcription cannot be affected, thereby increasing the length of T1 a; the length of the universal primer may also be varied as long as the PCR amplification requirements are met.

So long as amplification principles are satisfied, portions of the capture nucleotide may be deformed, e.g., there may be a portion of the base at the 5' end of U3a that is not complementary to any sequence; u3a and U3s can have a small number of mismatched bases as long as the complementary binding is achieved; t2s and T2a may have a small number of mismatched bases as long as they are complementary.

The length or proportion of the amplification primers in the second step may be adjusted conventionally according to the composition, length, desired specificity, etc. of the sequence to be amplified. In addition to the use of U1S, the primer sequence may be a subset of the full length of U1S (first universal sequence) +T1a+T2a (target molecule), or the reverse primer U2S may be a subset of the full length of U2S (second universal sequence) +U3S (sequence capable of complementary pairing with the unrelated sequence) +T2S (binding capture sequence) (the forward and reverse primers need to be set aside with respect to probe positions, i.e., if the forward primer is T1a+T2a, the reverse primer cannot include T2S or even U3S).

The complementarity of the present invention includes complete complementarity and partial complementarity. Typically, for a nucleic acid strand to be extended, the 3' end sequence is at least 90% complementary, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or is fully complementary, on the corresponding complementary strand, so long as nucleic acid strand extension is not affected.

In the invention, by arranging the second general sequence between the folding sequence and the sequence which can be complementarily matched with the unrelated sequence, after the extension and self folding of the capture nucleotide are carried out, the obtained novel hairpin structure product can adopt a specific primer and/or a general primer as an upstream primer and a downstream primer to carry out PCR amplification detection, and the effect of equivalent amplification on the multiple target molecules can be realized.

In the present invention, the first universal sequence, the second universal sequence, and the unrelated sequence may be synthetic sequences. Thus, in performing assays for different target molecules, only the folding sequence of the capture nucleotide and the binding capture sequence need be designed according to the target molecule, while the first universal sequence, the second universal sequence and the unrelated sequences may remain unchanged. In some embodiments, to reduce non-specific amplification, the folding sequence of the capture nucleotide and the binding capture sequence are designed according to different target molecules, while the first universal sequence and the second universal sequence remain unchanged.

The first universal sequence and the second universal sequence can be selected and configured differently according to the detected targets.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

(1) The miRNA detection technology of the invention utilizes the universal primer amplification technology to realize single-tube multiplex detection of different miRNAs, reduces the mutual interference between primers and satisfies the equivalent amplification.

(2) In the invention, the primer with a special self-folding structure can effectively improve the capturing efficiency of miRNA to increase the sensitivity, has the effect of secondarily identifying miRNA, firstly identifies a captured target by means of T2s, and secondly identifies the target by means of T1s folding after extending a target sequence, so that the reaction specificity can be greatly improved.

(3) The specific sequence blocker is introduced into the invention, which can inhibit the amplification of nonspecific reverse transcription products and improve the amplification of positive signals.

Drawings

Fig. 1 is a schematic diagram of miRNA detection.

Figure 2 is a single miRNA detection result.

FIG. 3 shows the result of verifying the effect of Exo I cleavage on the amplification efficiency of a PCR system.

FIG. 4 shows the results of multiplex miRNA detection.

FIG. 5 shows the results of the let-7a detection sequencing.

FIG. 6 is a schematic diagram of specific sequence LNA blockers to inhibit amplification of non-specific reverse transcription products during PCR.

FIG. 7 is the effect of specific sequence LNA blockers on specific detection results.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 (general) Single miRNA detection (mir 21, mir192, mir27a, mir 122)

(1) The synthetic miRNAs were serially diluted to 100pM,10pM,1pM,100fM,10fM,1fM, respectively.

(2) Reverse transcription reaction, the reaction system is: 2.5ul of self-folding primer (initial concentration 10 nM), 1 XM-Mulv reverse transcription buffer,200U M-Mulv reverse transcriptase, 0.5mM dNTPs, different concentrations of miRNA, total 20. Mu.L volume; the reaction conditions were 35℃1min,42℃1min,50℃1s,50 cycles, after completion of the reverse transcription, the system was heated to 95℃and incubated for 10min to heat-inactivate the M-Mulv reverse transcriptase.

(3) 20000U Exo I exonuclease, 1.6. Mu.L of 10 XExo I exonuclease buffer (Mg. Mu.L) ²⁺ The concentration reaches 1 xExo I exonuclease buffer concentration), and the total volume is 25 mu L; the reaction condition is 30min at 45 ℃; after the cleavage reaction, the system was heated to 95℃and incubated for 10 minutes to heat-inactivate Exo I.

(4) Taking a proper amount of target products to carry out PCR reaction, wherein the reaction system is as follows: complete stem-loop structured cDNA, 500nM upstream universal primer U1s,200nM downstream universal primer U2s,200nM specific Taqman probe, 1.25U Taq DNA polymerase, 0.5mM dNTP and 1 XPCR buffer, final volume of 20. Mu.l; the reaction procedure is pre-denaturation at 95 ℃ for 5min;95 ℃ 30s,63 ℃ 60s,45 cycles; real-time fluorescent PCR was performed on a LightCycler 96 (Roche) and the corresponding fluorescent values were collected.

The combination of self-folding primers (U3 a/U3s is a poly (A/T) n sequence with different lengths, only 0,4,6,8,10,12 six length probes are adopted), universal primers and specific probes comprises:

the upstream primer (SEQ ID NO: 1) was used: 5'-CTCAATTCCGCTGCTCACG-3'

Universal downstream primer (SEQ ID NO: 2): 5'-CCAGCAATCCTCCACCAAC-3'

Mir21 corresponds to the self-folding primer (SEQ ID NO: 3):

5’-GCG(A) _n CTCAATTCCGCTGCTCACGTAGCTTATCAGA-SpacerC18-CCAGCAATCCTCCACCAAC(T) _n CGCTCAACATCAGT-3’

wherein U1s is indicated by bold font, T1a is indicated by oblique font, U2s is indicated by underline with straight line, and T2s is indicated by underline with wavy line.

Mir21 corresponds to the specific probe (SEQ ID NO: 4):

21-VIC：5’-VIC-TCAACATCAGTCTGATAAGCTA-MGB-3’

mir192 corresponds to the self-folding primer (SEQ ID NO: 5):

5’-GCG(A) _n CTCAATTCCGCTGCTCACGCTGACCTATGA-SpacerC18-CCAGCAATCCTCCACCAAC(T) _n CGCGGCTGTCAATT-3’

Mir192 corresponds to the specific probe (SEQ ID NO: 6):

192-CY5:5’-CY5-CTGTCAATTCATAGGTCAG-MGB-3’

mir27a corresponds to the self-folding primer (SEQ ID NO: 7):

5’-GCG(A) _n CTCAATTCCGCTGCTCACGTTCACAGTGGC-SpacerC18-CCAGCAATCCTCCACCAAC(T) _n CGCGCGGAACTTAG-3’

Mir27a corresponds to the specific probe (SEQ ID NO: 8):

27aFAM：5’-FAM-CGGAACTTAGCCACTGTGAA-MGB-3’

mir122 corresponds to the self-folding primer (SEQ ID NO: 9):

5’-GCG(A) _n CTCAATTCCGCTGCTCACGTGGAGTGTGACA-SpacerC18-CCAGCAATCCTCCACCAAC(T) _n CGCCAAACACCATT-3’

Mir122 corresponds to the specific probe (SEQ ID NO: 10):

122-Texas：5’-Texas red-CAAACACCATTGTCACACTCCA-MGB-3’

as shown in FIG. 2, the detection sensitivities of mir21, mir192 and mir122 can all reach 1fM, and the detection sensitivity of mir27a can reach 10fM, which indicates that the invention has good sensitivity for detecting different miRNAs.

Example 2 verification of the Effect of Exo I cleavage on the amplification efficiency of PCR System

In order to improve the reaction efficiency of the invention, the system is verified whether Exo I enzyme digestion is added. The reverse transcription reaction system and the PCR reaction system were the same as in example 1 except that step (3) in example 1 was performed with or without adding Exo I after the reverse transcription system.

As shown in FIG. 3, taking mir21 detection system as an example, reverse transcription followed by Exo I cleavage has a higher sensitivity than that without addition, and can reach 100aM. Compared with the absence of the addition of the Exo I enzyme digestion under the same detection concentration, the Ct value can be increased by at least 3 cycles, the reaction efficiency is greatly improved, and the sensitivity is equivalent.

Example 3 multiplex miRNA detection (mir 21, mir192, mir27a, mir 122)

Multiplex miRNA detection was essentially the same as the reaction system in example 1, with the self-folding primers for the four mirnas remaining consistent with the final concentration of the singleplex system. In order to ensure amplification efficiency, this example increased the total amount of M-Mulv reverse transcriptase to 300U, the total amount of Exo I exonuclease to 40000U, the upstream universal primer U1s to 1.5. Mu.M, and the downstream universal primer U2s to 700nM.

As shown in fig. 4, a to d in fig. 4 are amplification curves of four mirnas under respective detection channels in a single tube multiplex reaction, respectively, and e in fig. 4 is a standard curve of four mirnas in a multiplex reaction.

In the multiple sensitivity detection experiment, the detection sensitivity of four miRNAs is about 10fM, and the detection channels (FAM, VIC, texad Red and Cy 5) of the four miRNAs are not interfered. From the standard curves established for the four mirnas mir21: y= -3.18x+38.22; mir122: y= -3.49x+40.44; mir192: y= -3.73x+42.96; mir27a: y= -3.38x+40.56; definition of amplification efficiency= (10) ^{(- (1/slope))} -1) 100%, mir21 with an amplification efficiency of 106.09%, mir122 with an amplification efficiency of 93.55%, mir192 with an amplification efficiency of 85.46%, mir27a with an amplification efficiency of 97.58%, substantially guaranteeing an equivalent amplification (95.67±8.58%).

The invention is illustrated to ensure the single tube multiple equivalent amplification requirement of miRNA.

Example 4 specific detection of let-7 family

The reverse transcription conditions, cleavage conditions and universal primers used were the same as in example 1.

(1) The synthetic let-7 family was serially diluted to 100pM and 10pM.

(2) Reverse transcription reaction, the reaction system is: 2.5ul of self-folding primer, 1 XM-Mulv reverse transcription buffer,200U M-Mulv reverse transcriptase, 0.5mM dNTPs, different concentrations of different miRNAs of let-7 family, total volume of 20. Mu.L; the reaction conditions were 35℃1min,42℃1min,50℃1s,50 cycles, after completion of the reverse transcription, the system was heated to 95℃and incubated for 10min to heat-inactivate the M-Mulv reverse transcriptase.

(4) Taking a proper amount of target products to carry out PCR reaction, wherein the reaction system is as follows: the final volume of the cleavage product, 500nM upstream universal primer U1s,200nM downstream universal primer U2s,200nM specific Taqman probe, 500nM specific sequence LNA blocker (with or without addition), 1.25U Taq DNA polymerase, 0.5mM dNTP and 1 XPCR buffer was 20. Mu.l; the reaction procedure is pre-denaturation at 95 ℃ for 5min;95 ℃ 30s,75 ℃ 30s,63 ℃ 60s,45 cycles; real-time fluorescent PCR was performed on a LightCycler 96 (Roche) and the corresponding fluorescent values were collected.

Let-7a corresponds to the self-folding primer (where n is the optimal length for Let-7a detection, optimized for n=4) (SEQ ID NO: 11):

5’-GCG(A) _n CTCAATTCCGCTGCTCACGTGAGGTAGTAGGT-SpacerC18-CCAGCAATCCTCCACCAAC(T) _n CGCAACTATACAA-3’

Let-7a corresponds to the specific probe (SEQ ID NO: 12): 5'-Texas red-AACTATAC AACCTACTACCTCA-MGB-3'

From the sequencing results of FIG. 5, it was found that let-7a detected a nonspecific product, and thus a specific sequence LNA blocker SEQ ID NO:13:5' -TCACG TGAGGTTGTATAGT-3' (underlined as LNA modification) to inhibit amplification of non-specific reverse transcription products during PCR, the principle is shown in FIG. 6: since both the T2s and the T1a of the capture nucleotide are derived from the detection target miRNA, the sequence of the let-7a sequence itself has a complementary pairing sequence, so that the T2s end of the capture nucleotide has a base and T1a complementary pairing, the target miRNA can be self-extended without existence under the action of reverse transcriptase, and an extension product comprises the region of the universal primers U1s and U2s, which can lead to the consumption of the primers to generate a nonspecific product. Amplification of the non-specific product can be effectively inhibited by adding a corresponding nucleic acid amplification blocking oligonucleotide comprising an LNA modification to the reaction. The blocker consists of a "part T2a" + sequenced T1a' + "part U1s" sequence and modifies LNA at certain bases toThe binding Tm is increased, and once the LNA blocker binds to a nonspecific product, the universal primer U1s cannot bind (particularly to the 3' end), and thus cannot be amplified, and the primer is not consumed.

As shown in FIG. 7, FIG. 7a shows the result of specific detection without adding LNA blocker of a specific sequence, and let-7 family could not be completely distinguished at detection concentrations of 100pM and 10pM. In FIG. 7 b is the result of specific detection of LNA blockers with specific sequences, which is evident for other members of the let-7 family at a detection concentration of 100 pM. This also demonstrates that LNA blockers also improve specific detection performance by improving reaction sensitivity.

SEQUENCE LISTING

<110> Shanghai university of transportation

<120> a capturing nucleotide for nucleic acid amplification and use thereof

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<400> 5

gcgactcaat tccgctgctc acgctgacct atganccagc aatcctccac caactcgcgg 60

ctgtcaatt 69

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Mir192 corresponding to the specific probes

<220>

<221> misc_binding

<222> (1)..(1)

<223> CY5

<220>

<221> misc_binding

<222> (19)..(19)

<223> MGB

<400> 6

ctgtcaattc ataggtcag 19

<210> 7

<211> 69

<212> DNA

<213> Artificial Sequence

<220>

<223> Mir27a corresponding self-folding primer

<220>

<221> repeat_region

<222> (4)..(4)

<223> Ploy A

<220>

<221> misc_feature

<222> (35)..(35)

<223> SpacerC18

<220>

<221> repeat_region

<222> (55)..(55)

<223> Ploy T

<400> 7

gcgactcaat tccgctgctc acgttcacag tggcnccagc aatcctccac caactcgcgc 60

ggaacttag 69

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Mir27a corresponding to the specific probe

<220>

<221> misc_binding

<222> (1)..(1)

<223> FAM

<220>

<221> misc_binding

<222> (20)..(20)

<223> MGB

<400> 8

cggaacttag ccactgtgaa 20

<210> 9

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> Mir122 corresponds to self-folding primer

<220>

<221> repeat_region

<222> (4)..(4)

<223> Ploy A

<220>

<221> misc_feature

<222> (36)..(36)

<223> SpacerC18

<220>

<221> repeat_region

<222> (56)..(56)

<223> Ploy T

<400> 9

gcgactcaat tccgctgctc acgtggagtg tgacanccag caatcctcca ccaactcgcc 60

aaacaccatt 70

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Mir122 corresponding specific probes

<220>

<221> misc_binding

<222> (1)..(1)

<223> Texas red

<220>

<221> misc_binding

<222> (22)..(22)

<223> MGB

<400> 10

caaacaccat tgtcacactc ca 22

<210> 11

<211> 70

<212> DNA

<213> Artificial Sequence

<220>

<223> Let-7a corresponding self-folding primer

<220>

<221> repeat_region

<222> (4)..(4)

<223> Ploy A

<220>

<221> misc_feature

<222> (37)..(37)

<223> SpacerC18

<220>

<221> repeat_region

<222> (57)..(57)

<223> Ploy T

<400> 11

gcgactcaat tccgctgctc acgtgaggta gtaggtncca gcaatcctcc accaactcgc 60

aactatacaa 70

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Let-7a corresponding specific probes

<220>

<221> misc_binding

<222> (1)..(1)

<223> Texas red

<220>

<221> misc_binding

<222> (22)..(22)

<223> MGB

<400> 12

aactatacaa cctactacct ca 22

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> let-7a specific sequence LNA blocker

<220>

<221> modified_base

<222> (2)..(2)

<223> LNA

<220>

<221> modified_base

<222> (4)..(5)

<223> LNA

<220>

<221> modified_base

<222> (7)..(7)

<223> LNA

<220>

<221> modified_base

<222> (9)..(10)

<223> LNA

<220>

<221> modified_base

<222> (13)..(15)

<223> LNA

<400> 13

tcacgtgagg ttgtatagt 19

Claims

1. A capture nucleotide for nucleic acid amplification, comprising, in order from the 5' end to the 3' end, a first universal sequence, a folding sequence, and a binding capture sequence, wherein the 5' end of the first universal sequence further comprises an unrelated sequence, and a sequence capable of complementarily pairing with the unrelated sequence is further included between the folding sequence and the binding capture sequence;

the folding sequence comprises a sequence identical to the 5' end of the target molecule; the binding capture sequence binds complementarily to the non-5' terminal sequence of the target molecule.

2. The capture nucleotide of claim 1, wherein the sequence identical to the 5' end of the target molecule is 8-18bp in length.

3. The capture nucleotide of claim 1, wherein the unrelated sequence is any sequence of random base composition and the Tm value of the unrelated sequence is sufficient to pair with its complement such that the capture nucleotide forms a self-folding structure;

preferably, the unrelated sequences comprise polyA and/or polyT.

4. A capture nucleotide according to any one of claims 1 to 3, further comprising a second universal sequence;

preferably, the second universal sequence is located between the folding sequence and the sequence capable of complementary pairing with the unrelated sequence.

5. The capture nucleotide of claim 4, wherein the capture nucleotide further comprises a nucleic acid extension blocking site;

the nucleic acid extension blocking site is preferably located 3' to the folding sequence;

the nucleic acid extension blocking site modification is preferably a Spacer, abasic phosphate backbone and/or uracil base.

6. A kit for nucleic acid amplification, comprising a capture nucleotide according to any one of claims 1 to 5.

7. The kit of claim 6, further comprising a pair of amplification primers, wherein the forward primer of the amplification primers has a nucleotide sequence of a first universal sequence of the capture nucleotides + a subset of the full length of the target molecule, and the reverse primer of the amplification primers has a nucleotide sequence of a second universal sequence of the capture nucleotides + the sequence capable of complementary pairing with the unrelated sequence + a subset of the full length of the binding capture sequence, leaving a location between the forward and reverse primers for binding of a specific probe;

preferably, the nucleotide sequences of the subset are 15-25bp in length.

8. The kit of claim 6 or 7, wherein the kit further comprises a specific probe;

preferably, the specific probe is labeled with a fluorescent group and/or a quenching group;

more preferably, the fluorophore is labeled at the 5' end of the specific probe; and/or, the quenching group is marked at the 3' end of the specific probe;

even more preferably, the fluorophore is selected from any one of FAM, VIC, JOE, TET, CY, CY5, ROX, texas Red and LC Red 460; and/or the quenching group is selected from any one of MGB, BHQ1, BHQ2, BHQ3, dabcy1 and Tamra.

9. The kit of any one of claims 6 to 8, further comprising a specific sequence blocker comprising a nucleic acid comprising a modification of a nucleic acid analogue capable of complementary pairing with a non-specific product;

preferably, the nucleic acid analogue is a Locked Nucleic Acid (LNA).

10. A nucleic acid amplification method comprising employing the capture nucleotide of any one of claims 1-5 to bind a target molecule and initiate specific linear amplification and amplification primer-based nucleic acid amplification detection; the nucleic acid amplification method comprises the steps of:

(1) Binding the target molecule determined by the 5' end sequence with a binding capture sequence of the capture nucleotide;

(2) The capture nucleotide takes the target molecule as a template to carry out an extension reaction, and the nucleotide complementary with the target molecule is added at the 3' -end of the capture nucleotide, so that the extended capture nucleotide is formed;

(3) The extended capture nucleotide is denatured, an unrelated sequence is separated from the complementary sequence, and the extended sequence is in base complementary pairing with a folding sequence in the molecule to form a new semi-hairpin structure product;

(4) The new semi-hairpin structure product is subjected to extension reaction, and nucleotide complementary with a first general sequence and an irrelevant sequence in the molecule is added at the 3' -end, so that a complete hairpin structure product is formed; preferably also including the use of 3'-5' exonucleases, such as Exo I exonuclease, capable of cleaving single strands of DNA;

(5) Taking the complete hairpin structure product as a template, and carrying out exponential amplification by using an amplification primer to obtain an amplification product; the exponential amplification preferably comprises a polymerase chain reaction; the amplification primer is preferably an amplification primer as defined in the kit of claim 7.

11. The nucleic acid amplification method of claim 10, further comprising the step of detecting the amplification product in combination with a specific probe;

preferably, the specific probe is a specific probe as defined in the kit of claim 8.

12. The method for amplifying nucleic acid according to claim 10, wherein the specific sequence blocker as defined in the kit according to claim 9 is added to the amplification system when the exponential amplification is performed.

13. Use of a capture nucleotide according to any one of claims 1 to 5, or a kit according to any one of claims 6 to 9, for the detection of a miRNA.