Nucleic acid aptamer for identifying tRNA, tRNA, reagent, kit and tRNA detection method

Abstract

The invention provides a nucleic acid aptamer for specifically recognizing tRNA, tRNA, a reagent, a kit and a tRNA detection method. The tRNA is mature tRNA, and the aptamer comprises a 3'end protruding part, a 4 'end protruding part and a 5' end protruding part; a stem ring structure forming portion; wherein the 3'end protruding part is complementary with a CCA sequence, a recognition site basic group and an aminoacyl tRNA synthetase receptor structural domain at the 3 'end of the mature tRNA; the nucleic acid aptamer is a single-stranded DNA (deoxyribonucleic acid). The nucleic acid aptamer disclosed by the embodiment of the invention is simple in structure, low in synthesis difficulty and low in cost, and can specifically recognize tRNA and destroy a secondary structure of the tRNA, so that the difficulty of reversely transcribing the tRNA into cDNA is reduced; compared with an existing four-leaf clover qRT-PCR method, the nucleic acid aptamer does not need to be subjected to a connection reaction with tRNA, the reverse transcription reaction step of the tRNA is simplified, the reverse transcription reaction is completely carried out with RNA as a template, the reaction efficiency is high, the detection sensitivity is high, and accurate, rapid and low-cost detection of the tRNA can be achieved.

Description

Technical Field

[0001] This invention relates to the field of tRNA detection technology, and in particular to nucleic acid aptamers that specifically recognize tRNA, tRNA, reagents, kits, and tRNA detection methods. Background Technology

[0002] Transfer RNA (tRNA) is a type of non-coding RNA with a highly structured secondary network within cells, typically 70-90 base pairs in length. All organisms, including eukaryotes, bacteria, archaea, and some viruses, contain tRNA. tRNA is a core component in gene expression, responsible for encoding genetic information from messenger RNA (mRNA) into amino acids for proteins. For example, the eukaryotic model organism *Saccharomyces cerevisiae* has 286 tRNA genes in its nuclear and mitochondrial genomes, collectively encoding 20 amino acids. Besides its classic amino acid-coding function, tRNA can also be processed into shorter tRNA fragments to perform other important molecular biological functions. In humans, abnormal tRNA expression is associated with some cancers and neurodegenerative diseases. Therefore, qualitative and quantitative analysis of intracellular tRNA expression is crucial. The most classic method for detecting tRNA is the Northern blot assay. Its basic principle involves first performing gel electrophoresis on the RNA within the cell to be tested, separating it according to molecular weight, then transferring the RNA to a nylon or nitrocellulose membrane for fixation, and finally detecting it using probes labeled with isotopes or other markers. However, this method has several significant drawbacks: the experimental process is complex and time-consuming, involving multiple steps such as gel electrophoresis, membrane transfer, probe incubation, membrane washing, and signal detection, often requiring two days or more; the experimental process involves handling radioactive isotopes, demanding high safety standards from experimental personnel; and the sensitivity for detecting tRNA is relatively low, unable to distinguish between different tRNAs with very high sequence similarity. These drawbacks limit the application of the Northern blot method.

[0003] The current four-leaf clover qRT-PCR method is based on the principle of deacylation of the RNA sample to be tested, removing the amino acids at the 3' end of the tRNA. Then, an oligonucleotide aptamer with a stem-loop structure is designed. This oligonucleotide is a complex of DNA (from the 5' end to the penultimate nucleotide) and RNA (the two nucleotides at the 3' end). The aptamer has four bases protruding from its 3' end (5'-TGGN-3'), which can complementarily pair with the four bases protruding from the 3' end of mature tRNA (5'-NCCA-3'). Next, annealing is performed. Because the oligonucleotide aptamer contains a phosphate group at its 5' end and a hydroxyl group at its 3' end, it can be ligated to the tRNA using T4 RNA ligase (Rnl2) to form a four-leaf clover-like structure. Finally, quantitative real-time PCR is performed using primers complementary to the D and T loops of the tRNA. However, this method has several obvious drawbacks: (1) The heterozygous oligonucleotide aptamers have complex structures, being chimeras composed of DNA and RNA, making their synthesis difficult and costly; (2) Since the heterozygous oligonucleotide aptamers are mostly DNA, the reverse transcription process requires DNA as a template, and the reverse transcription reaction using DNA as a template has low efficiency, resulting in low efficiency of tRNA detection; (3) During the reaction process, the heterozygous oligonucleotide aptamers need to be ligated with tRNA using T4 RNA ligase (Rnl2), which is expensive, leading to high detection costs. These drawbacks limit the application of this cloverleaf-based qRT-PCR method in tRNA detection. Therefore, there is a need to further develop accurate, simple, rapid, and low-cost tRNA detection methods. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides nucleic acid aptamers, reagents, kits, and tRNA detection methods that specifically recognize tRNA. The nucleic acid aptamers of this invention have simple structures, are easy to synthesize, and are low in cost. They can specifically recognize tRNA and disrupt its secondary structure, reducing the difficulty of reverse transcription of tRNA into cDNA. Compared with the current four-leaf cloverleaf qRT-PCR method, the nucleic acid aptamers of this invention do not require a ligation reaction with tRNA, simplifying the tRNA reverse transcription reaction steps. The reverse transcription reaction is entirely performed using RNA as a template, resulting in high reaction efficiency and high detection sensitivity, enabling accurate, rapid, and low-cost detection of tRNA.

[0005] This invention is based on the inventor's discoveries and understanding of the following problems:

[0006] The presence of numerous complementary sequences within the tRNA sequence allows it to form complex secondary structures, which is one of the challenges in reverse transcription of tRNA into cDNA. To address this issue, the inventors designed and developed a novel PCR-based tRNA detection method. The reaction principle and process of this method are as follows: Figure 2 As shown in the diagram, firstly, the tRNA sample to be tested is denatured at high temperature and annealed at low temperature with a nucleic acid aptamer that specifically recognizes tRNA, thereby destroying the secondary structure of the tRNA and obtaining a complex of the tRNA sample and nucleic acid aptamer with the secondary structure removed. Secondly, cDNA is obtained by amplifying the tRNA sample with the secondary structure removed through reverse transcription. Finally, quantitative real-time PCR is performed using primer pairs complementary to the upstream and downstream bases of the tRNA. Based on the PCR amplification products, the type and / or content of tRNA in the RNA sample to be tested is determined.

[0007] Furthermore, the inventors conceived and designed a nucleic acid aptamer that can specifically recognize tRNA and disrupt the secondary structure of tRNA itself. The structure of this aptamer is as follows: Figure 1 First, to facilitate synthesis, the inventors designed the aptamer as a single-stranded DNA capable of forming a stem-loop secondary structure. Second, in the tRNA secondary structure, the longest complementary pair is located in the aminoacyl-tRNA synthetase receptor domain, totaling 8 bases (see...). Figure 1 C). Therefore, the inventors designed the 3' end of the aptamer as a protrusion with at least 11 bases, which complementarily pairs with at least 11 bases at the 3' end of each specific tRNA. These at least 11 bases include: 3 CCA sequences that recognize the CCA sequence added during tRNA maturation, 1 protruding discriminator base, and 8 or more aminoacyl-tRNA synthetase receptor domain bases. It should be specifically noted that the 8 or more aminoacyl-tRNA synthetase receptor domain bases include the discriminator base.

[0008] Experimental results show that the 3' protrusion of the nucleic acid aptamer of the present invention can complementarily pair with the 3' end of tRNA. By contacting the tRNA sample to be tested with the nucleic acid aptamer of the present invention and performing high-temperature denaturation and low-temperature renaturation, the secondary structure of tRNA itself can be effectively destroyed, significantly reducing the difficulty of tRNA reverse transcription.

[0009] Furthermore, to ensure that the nucleic acid aptamer of this invention continuously and effectively removes the secondary structure of tRNA and improves reverse transcription efficiency, the inventors improved the tRNA reverse transcription reaction process. Specifically, by controlling the temperature pulse changes in the reverse transcription reaction, the complex formed by the tRNA sample and the nucleic acid aptamer continuously and repeatedly undergoes the aforementioned high-temperature denaturation and low-temperature renaturation processes during the reverse transcription reaction. This keeps the tRNA in the tRNA sample in a state of secondary structure elimination, thereby further reducing the difficulty of tRNA reverse transcription. Experimental results show that this method has high reaction efficiency, is simple to operate, and has high detection sensitivity, enabling accurate, rapid, and low-cost detection of tRNA.

[0010] Therefore, in a first aspect, the present invention proposes a nucleic acid aptamer that specifically recognizes tRNA. According to an embodiment of the present invention, the tRNA is mature tRNA, and the aptamer includes: a 3' end overhang; a stem-loop structure forming portion; wherein the 3' end overhang is complementary to the CCA sequence, recognition base, and aminoacyl-tRNA synthetase receptor domain at the 3' end of the mature tRNA; the nucleic acid aptamer is single-stranded DNA. The nucleic acid aptamer of the embodiments of the present invention has a simple structure, is easy to synthesize, and is low in cost. It can specifically recognize tRNA and disrupt its secondary structure, reducing the difficulty of reverse transcription of tRNA into cDNA. Compared with the current four-leaf qRT-PCR method, the nucleic acid aptamer of the present invention does not require a ligation reaction with tRNA, simplifying the tRNA reverse transcription reaction steps. The reverse transcription reaction is entirely performed using RNA as a template, resulting in high reaction efficiency and high detection sensitivity, enabling accurate, rapid, and low-cost detection of tRNA.

[0011] In a second aspect, the present invention provides a tRNA. According to embodiments of the present invention, the CCA sequence, recognition base, and aminoacyl-tRNA synthetase receptor domain at the 3' end of the tRNA are complementary to the protruding portion of the aforementioned nucleic acid aptamer.

[0012] Those skilled in the art will understand that the features and advantages described above for nucleic acid aptamers that specifically recognize tRNA also apply to this tRNA, and will not be repeated here.

[0013] In a third aspect, the present invention provides a reagent. According to embodiments of the invention, the reagent comprises the aforementioned nucleic acid aptamer, and is used to specifically recognize tRNA.

[0014] Those skilled in the art will understand that the features and advantages described above for nucleic acid aptamers that specifically recognize tRNA also apply to this reagent, and will not be repeated here.

[0015] In a fourth aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises: the aforementioned nucleic acid aptamer or the aforementioned reagent, the kit being used for detecting tRNA.

[0016] Those skilled in the art will understand that the features and advantages described above for the nucleic acid aptamers and reagents that specifically recognize tRNA also apply to this kit, and will not be repeated here.

[0017] In a fifth aspect, the present invention provides a method for tRNA detection. According to embodiments of the present invention, the method includes: contacting a sample of RNA to be tested with the aforementioned nucleic acid aptamer, the aforementioned reagent, or the reagent in the aforementioned kit to obtain a reverse transcription template; when the sample to be tested contains tRNA, the secondary structure of the tRNA in the reverse transcription template is eliminated; performing a reverse transcription reaction based on the reverse transcription template to obtain a cDNA template; performing a PCR or qPCR reaction based on the cDNA template to obtain a PCR or qPCR reaction product; and determining the type or content of tRNA in the sample of RNA to be tested based on the PCR or qPCR reaction product. The detection method according to embodiments of the present invention has high reaction efficiency, high detection sensitivity, low detection cost, and can achieve accurate and rapid detection of tRNA, with a wide range of applications.

[0018] Those skilled in the art will understand that the features and advantages described above for nucleic acid aptamers, reagents, and kits that specifically recognize tRNA also apply to this detection method, and will not be repeated here.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0021] Figure 1 This is the design of the oligonucleotide aptamer in Example 1 of the present invention; wherein, (A) is the tRNA to be tested; (B) is the oligonucleotide aptamer; and (C) is the oligonucleotide aptamer that specifically recognizes the tRNA to be tested.

[0022] Figure 2 This is the core principle and process of the method for detecting tRNA in this invention;

[0023] Figure 3 This is a PCR gel electrophoresis image of tRNA in endogenous 20 cells of Saccharomyces cerevisiae using the method of the present invention.

[0024] Figure 4 alanine tRNA in Saccharomyces cerevisiae AGC Sanger sequencing peak diagram of PCR products of (S. cerevisiae_Ala-AGC-1-5);

[0025] Figure 5 Arginine tRNA from Saccharomyces cerevisiae ACG Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Arg-ACG-2-1);

[0026] Figure 6 Asparagine tRNA from Saccharomyces cerevisiae GTT Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Asn-GTT-1-4);

[0027] Figure 7 Aspartic acid tRNA in Saccharomyces cerevisiae GTC Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Asp-GTC-1-7);

[0028] Figure 8 Cysteine ​​tRNA of Saccharomyces cerevisiae GCA Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Cys-GCA-1-4);

[0029] Figure 9 Glutamine tRNA from Saccharomyces cerevisiae CTG Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Gln-CTG-1-1);

[0030] Figure 10 Glutamic acid tRNA from Saccharomyces cerevisiae CTC Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Glu-CTC-1-1);

[0031] Figure 11 Glycine tRNA from Saccharomyces cerevisiae GCC Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Gly-GCC-1-10);

[0032] Figure 12 Histidine tRNA of Saccharomyces cerevisiae GTG Sanger sequencing peak diagram of PCR product of (S. cerevisiae_His-GTG-1-6);

[0033] Figure 13 Isoleucine tRNA of Saccharomyces cerevisiaeAAT Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Ile-AAT-1-9);

[0034] Figure 14 leucine tRNA from Saccharomyces cerevisiae GAG Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Leu-GAG-1-1);

[0035] Figure 15 Lysine tRNA from Saccharomyces cerevisiae CTT Sanger sequencing peak diagram of PCR product of (S. cerevisiae_Lys-CTT-1-6);

[0036] Figure 16 methionine tRNA in Saccharomyces cerevisiae CAT Sanger sequencing peak diagram of PCR products of (S. cerevisiae-Met-CAT-1-2);

[0037] Figure 17 phenylalanine tRNA from Saccharomyces cerevisiae GAA Sanger sequencing peak diagram of PCR product of (S. cerevisiae-Phe-GAA-1-1);

[0038] Figure 18 Proline tRNA from Saccharomyces cerevisiae AGG Sanger sequencing peak diagram of PCR products of (S. cerevisiae-Pro-AGG-1-2);

[0039] Figure 19 For Saccharomyces cerevisiae serine tRNA AGA Sanger sequencing peak diagram of PCR product of (S. cerevisiae-Ser-AGA-2-1);

[0040] Figure 20 For threonine tRNA in Saccharomyces cerevisiae AGT Sanger sequencing peak diagram of PCR product of (S. cerevisiae-Thr-AGT-1-7);

[0041] Figure 21 Tryptophan tRNA in Saccharomyces cerevisiae CCA Sanger sequencing peak diagram of PCR product of (S. cerevisiae-Trp-CCA-1-1);

[0042] Figure 22 tyrosine tRNA of Saccharomyces cerevisiae GTASanger sequencing peak diagram of PCR products of (S. cerevisiae-Tyr-GTA-1-2);

[0043] Figure 23 valine tRNA from Saccharomyces cerevisiae AAC Sanger sequencing peak diagram of PCR product of (S. cerevisiae-Val-AAC-2-1);

[0044] Figure 24 Quantitative real-time PCR for endogenous cystine tRNA in Saccharomyces cerevisiae;

[0045] Figure 25 The relationship between Ct value and template amount in quantitative real-time PCR of endogenous cystine tRNA in Saccharomyces cerevisiae;

[0046] Figure 26 Quantitative real-time PCR for endogenous valine tRNA in Saccharomyces cerevisiae;

[0047] Figure 27 The relationship between Ct value and template amount in quantitative real-time PCR of endogenous valine tRNA in Saccharomyces cerevisiae;

[0048] Figure 28 This is a PCR gel electrophoresis image of the detection of exogenously expressed tRNA (Alv8) in Saccharomyces cerevisiae using the method of the present invention;

[0049] Figure 29 Sanger sequencing peak diagram of PCR product of exogenously expressed tRNA (Alv8) in Saccharomyces cerevisiae;

[0050] Figure 30 Quantitative real-time PCR of exogenously expressed tRNA (Alv8) in Saccharomyces cerevisiae;

[0051] Figure 31 The relationship between Ct value and template amount in quantitative real-time PCR of exogenous tRNA (Alv8) expressed in Saccharomyces cerevisiae. Detailed Implementation

[0052] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0053] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0054] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0055] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0056] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0057] Terms and Definitions

[0058] In this paper, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)). And the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (AltschμL et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0059] In this document, the term "at least 90% identity" means that the sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to its corresponding reference sequences, including nucleic acid sequences.

[0060] In this paper, the term "pulse" refers to a temperature-changing procedure used in reverse transcription, which involves maintaining a specific temperature for a certain period of time, then changing to another temperature, and so on in a cyclical manner.

[0061] In this paper, the term "oligonucleotide" refers to a short nucleotide sequence consisting of approximately 20–100 nt nucleotide monomers linked together.

[0062] This invention proposes oligonucleotide aptamers, reagents, kits, and tRNA detection methods that specifically recognize tRNA, which will be described in detail below.

[0063] Nucleic acid aptamers that specifically recognize tRNA

[0064] This invention proposes a nucleic acid aptamer that specifically recognizes tRNA. According to an embodiment of the invention, the tRNA is a mature tRNA, and the aptamer includes: a 3' end overhang; a stem-loop structure forming portion; wherein the 3' end overhang is complementary to the CCA sequence, recognition base, and aminoacyl-tRNA synthetase receptor domain at the 3' end of the mature tRNA; the aptamer is a single-stranded DNA (structural reference of the 3' end of the mature tRNA). Figure 1 The oligonucleotide aptamers of this invention have simple structures, are easy to synthesize, and are low in cost. They can specifically recognize tRNA and disrupt its secondary structure, reducing the difficulty of reverse transcription of tRNA into cDNA. Compared with the current four-leaf clover qRT-PCR method, the nucleic acid aptamers of this invention do not require ligation with tRNA, simplifying the tRNA reverse transcription reaction steps. The reverse transcription reaction is carried out entirely using RNA as a template, resulting in high reaction efficiency and high detection sensitivity, enabling accurate, rapid, and low-cost detection of tRNA.

[0065] In this document, the term "mature tRNA" refers to a tRNA molecule that has acquired a complete structure and function after transcription and a series of processing processes within the cell nucleus. In this application, the mature tRNA exists in at least one of the following forms: naked tRNA not bound to the amino acid to be transported, tRNA bound to the transport amino acid, and tRNA bound to a nucleic acid aptamer.

[0066] For example, the "mature tRNA" has a nucleotide sequence as shown in any of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:81 or a nucleotide sequence having at least 90% identity with it.

[0067] The tRNA has a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity to any of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:77, and SEQ ID NO:81.

[0068] In this paper, the term "CCA sequence" refers to a special structure at the 3' end of the tRNA molecule, consisting of two consecutive cytosine nucleotides and one adenine nucleotide, which is the site for amino acid attachment.

[0069] In this paper, the term "recognition site" refers to a base at the 3' end of the tRNA molecule that is linked to the CCA sequence.

[0070] In this paper, the term "aminoacyl-tRNA synthetase receptor domain base" refers to the domain base in the tRNA molecule that is recognized by aminoacyl-tRNA synthetase.

[0071] According to an embodiment of the present invention, the length of the 3' end protrusion is greater than or equal to 11 nt. This further improves the binding specificity of the nucleic acid aptamer to tRNA.

[0072] According to an embodiment of the present invention, the nucleic acid aptamer is an oligonucleotide aptamer.

[0073] According to an embodiment of the present invention, the length of the aptamer is 50 to 60 nt.

[0074] According to embodiments of the present invention, the tRNA includes: tRNA corresponding to natural amino acids and tRNA corresponding to non-standard amino acids.

[0075] According to an optional embodiment of the present invention, the tRNA includes, but is not limited to, at least one of the tRNAs corresponding to the non-standard amino acids pyrrolidone and selenocysteine.

[0076] According to embodiments of the present invention, the nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:81, or a nucleotide sequence having at least 90% identity with such sequences.

[0077] Exemplarily, the oligonucleotide aptamer has a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity to the nucleotide sequence shown in any of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, and SEQ ID NO:81.

[0078] tRNA

[0079] This invention proposes a tRNA. According to embodiments of the invention, the CCA sequence, recognition base, and aminoacyl-tRNA synthetase receptor domain at the 3' end of the tRNA are complementary to the protruding portion of the aforementioned nucleic acid aptamer at the 3' end.

[0080] Those skilled in the art will understand that the features and advantages described above for nucleic acid aptamers that specifically recognize tRNA also apply to this tRNA, and will not be repeated here.

[0081] reagents

[0082] This invention provides a reagent. According to embodiments of the invention, it comprises the aforementioned nucleic acid aptamer, and the reagent is used to specifically recognize tRNA.

[0083] Those skilled in the art will understand that the features and advantages described above for nucleic acid aptamers that specifically recognize tRNA also apply to this reagent, and will not be repeated here.

[0084] Reagent test kit

[0085] This invention provides a kit. According to embodiments of the invention, the kit includes the aforementioned nucleic acid aptamer or the aforementioned reagent, and the kit is used to detect tRNA.

[0086] Those skilled in the art will understand that the features and advantages described above for the nucleic acid aptamers and reagents that specifically recognize tRNA also apply to this kit, and will not be repeated here.

[0087] According to embodiments of the present invention, it further includes: reverse transcription reaction reagents and / or PCR reaction reagents.

[0088] A method for tRNA detection

[0089] This invention proposes a method for tRNA detection. According to embodiments of the invention, the method includes: contacting the aforementioned nucleic acid aptamer, the aforementioned reagent, or the reagent from the aforementioned kit with a sample of RNA to be tested to obtain a reverse transcription template; when the sample to be tested contains tRNA, the secondary structure of the tRNA in the reverse transcription template is eliminated; performing a reverse transcription reaction based on the reverse transcription template to obtain a cDNA template; performing a PCR or qPCR reaction based on the cDNA template to obtain a PCR or qPCR reaction product; and determining the type or content of tRNA in the sample of RNA to be tested based on the PCR or qPCR reaction product. The detection method according to embodiments of the invention has high reaction efficiency, high detection sensitivity, low detection cost, and can achieve accurate and rapid detection of tRNA, with a wide range of applications.

[0090] Those skilled in the art will understand that the features and advantages described above for nucleic acid aptamers, reagents, and kits that specifically recognize tRNA also apply to this detection method, and will not be repeated here.

[0091] According to an embodiment of the present invention, the contact treatment includes: mixing the nucleic acid aptamer or a reagent containing the nucleic acid aptamer with the RNA sample to be tested and dNTP Mix, followed by high-temperature denaturation and low-temperature annealing. This further eliminates the secondary structure of the tRNA itself.

[0092] According to a specific embodiment of the present invention, the high-temperature denaturation temperature is 50–75°C, preferably 65°C, and the high-temperature denaturation time is 4–15 min, preferably 5 min; the low-temperature annealing temperature is 0°C, and the low-temperature annealing time is not less than 2 min. Therefore, the secondary structure of tRNA in the test sample is effectively destroyed, and the reverse transcription efficiency is further improved.

[0093] According to an embodiment of the present invention, the reverse transcription reaction includes: the temperature is pulsed, the pulsed temperature range being 15–55°C, and each pulsed temperature range lasting for 50–105 seconds. This ensures that the aforementioned nucleic acid aptamer continuously and effectively removes the secondary structure of tRNA, further improving reverse transcription efficiency.

[0094] According to some specific embodiments of the present invention, each pulse change includes at least two pulse temperatures; wherein the first pulse temperature is 15-17°C, preferably 16°C; and the second pulse temperature is 45-55°C, preferably 50°C.

[0095] According to some specific embodiments of the present invention, the duration of the first pulse temperature is 25-35s, preferably 30s; the duration of the second pulse temperature is 25-35s, preferably 30s.

[0096] According to an embodiment of the present invention, each pulse change further includes a third pulse temperature, wherein the first pulse temperature is lower than the second pulse temperature, the second pulse temperature is higher than the third pulse temperature, and the third pulse temperature is higher than the first pulse temperature.

[0097] According to some specific embodiments of the present invention, the temperature of the third pulse is 40-50°C, preferably 45°C.

[0098] According to some specific embodiments of the present invention, the duration of the third pulse temperature is 25 to 35 seconds, preferably 30 seconds.

[0099] According to an embodiment of the present invention, in the reverse transcription process, the reverse transcriptase used is capable of withstanding temperatures greater than or equal to 50°C.

[0100] According to an embodiment of the present invention, the reverse transcription reaction is performed according to the following procedure:

[0101] 40 cycles: 16℃ for 30 seconds, 50℃ for 30 seconds, 45℃ for 30 seconds;

[0102] 42℃ for 1 hour;

[0103] 80℃ for 10 minutes;

[0104] Store at 4℃.

[0105] According to an embodiment of the present invention, the amplification system of the PCR reaction includes: a pair of primers specific to both ends of tRNA and the cDNA template.

[0106] According to some specific embodiments of the present invention, the amplification system of the PCR reaction is as follows:

[0107] Components Added amount 2X GreenTaq mix 10μL tRNA forward primer (50 μmol / L) 1μL tRNA reverse primer (50 μmol / L) 1μL The cDNA template 0.5μL <![CDATA[H2O]]> 6.5μL

[0108] According to some specific embodiments of the present invention, the amplification system of the qPCR reaction is as follows:

[0109] Components Added amount SYBR Green qPCR Mix(2X,High ROX,UDG) 10μL tRNA forward primer (50 μmol / L) 1μL tRNA reverse primer (50 μmol / L) 1μL The cDNA template 1μL <![CDATA[H2O]]> 7μL

[0110] According to some specific embodiments of the present invention, the tRNA forward primer has a nucleotide sequence as shown in at least one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:82; the tRNA reverse primer has a nucleotide sequence as shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:32, SEQ The nucleotide sequence shown in at least one of the following is SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, or SEQ ID NO:83.

[0111] According to some specific embodiments of the present invention, the PCR reaction is performed according to the following procedure: 94℃ for 3 min;

[0112] 40 cycles: 94℃ for 15 seconds, 60℃ for 15 seconds, 72℃ for 15 seconds;

[0113] 72℃ for 5 minutes;

[0114] Store at 4℃.

[0115] According to some specific embodiments of the present invention, the PCR reaction is performed according to the following procedure: 50℃ for 3 min;

[0116] 95℃ for 2 minutes;

[0117] 40 cycles: 95℃ for 15 seconds, 58℃ for 15 seconds, 68℃ for 15 seconds.

[0118] The sequence descriptions involved in this invention are detailed in Table 1.

[0119] Table 1. Nucleotide Sequence Description

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0128] Example 1: Qualitative detection of endogenous tRNA (using Saccharomyces cerevisiae as an example) in cells using the method of the present invention.

[0129] 1.1 Cell Culture

[0130] Select a single clone of wild-type Saccharomyces cerevisiae and add it to 5 ml of YPD medium. Place it on a shaker (220 rpm) and incubate overnight at 30 degrees Celsius.

[0131] 1.2 RNA Extraction (using the HiPure Yeast RNA Kit)

[0132] (1) When the OD of the brewer's yeast cells reaches about 600 to 1, take 2ml (~3 x 10) 7 Transfer the cells to a 2ml centrifuge tube.

[0133] (2) Centrifuge at 10,000g for 3 minutes.

[0134] (3) Add 500 μL of ATL buffer to the cells and mix well.

[0135] (4) Add 0.4g-0.5g glass beads and 500μL PHC buffer, and shake at high speed for 10 minutes.

[0136] (5) Place the centrifuge tubes in a metal bath or water bath at 65 degrees Celsius for 10 minutes.

[0137] (6) Add 500 μL of chloroform, mix well, and centrifuge at 12000g for 5 minutes.

[0138] (7) Take the upper aqueous phase into a new centrifuge tube, add 1.5 times the volume of GXP2 buffer, and mix well.

[0139] (8) Place the mixture into a Hipure RNA Mini column twice and centrifuge at 12000g for 30 seconds.

[0140] (9) Add 300 μL of RW1 buffer to wash the column and centrifuge at 12000g for 60 seconds.

[0141] (10) Add DNase (200 units in total) to the center of the column and let it stand at room temperature for 20-30 minutes.

[0142] (11) Add 500 μL of RW1 buffer, let stand at room temperature for 10 minutes, and centrifuge at 12000g for 30 seconds.

[0143] (12) Add 500 μL of RW2 buffer and centrifuge at 12000g for 30 seconds.

[0144] (13) Place the Hipure RNA Mini column into a new centrifuge tube, add 30-50 μL of water, let it stand at room temperature for 2 minutes, and centrifuge at 12000g for 60 seconds to obtain total RNA from Saccharomyces cerevisiae cells.

[0145] 1.3 Primer Design

[0146] This embodiment selected 20 endogenous tRNAs from 20 Saccharomyces cerevisiae species, each corresponding to one of the 20 natural amino acids, and one exogenous tRNA encoding pyrrolidone. The sequences of these tRNAs were obtained from the tRNA database GtRNAdb (http: / / gtrnadb.ucsc.edu / genomes / eukaryota / Scere3 / ). The matured sequences of these tRNAs, along with their corresponding sequence-specific aptamer sequences, forward qPCR primer sequences, and reverse qPCR primer sequences, are shown in Table 1.

[0147] 1.4 Reverse transcription

[0148] Prepare the following 20 μL reaction mixture in a PCR tube:

[0149] (1) 8 μL RNA (~16 ug);

[0150] (2)0.5μL 11nt Stem-loop primer (50uM);

[0151] (3) 2 μL dNTP Mix (10 mM concentration of each dNTP);

[0152] (4) Add water to a 20 μL system;

[0153] The reaction system was placed in a 65°C water bath for 5 minutes to remove the secondary structure of the tRNA;

[0154] Place on ice immediately for at least 2 minutes;

[0155] Add the following to the above reaction system respectively:

[0156] (1) 6 μL 5X reverse transcriptase buffer;

[0157] (2) 1.5 μL RNase inhibitor;

[0158] (3) 1 μL 0.1 M dTT;

[0159] (4) 1.5 μL BeyoRTMIII M-MLV reverse transcriptase;

[0160] The reaction was performed in a PCR instrument as follows (pulse PCR):

[0161] (1) 16℃ for 30s;

[0162] (2) 50℃ for 30 seconds;

[0163] (3) 45℃ for 30 seconds;

[0164] (4) Repeat step one 40 times;

[0165] (5) 42℃ for 1 hour;

[0166] (6) 80℃ for 10 min;

[0167] (7) Store at 4℃;

[0168] 1.5 PCR

[0169] The PCR reaction system in this embodiment is as follows:

[0170] (1) 10 μL 2X GreenTaq mix;

[0171] (2) 1μL Forward qPCR primer (50uM);

[0172] (3) 1μL Reverse qPCR primer (50uM);

[0173] (4) 0.5 μL of the above RT-PCR product;

[0174] (5) Add water to a 20 μL system;

[0175] The reaction was performed in a PCR instrument as follows:

[0176] (1) 94℃ for 3 min;

[0177] (2) 94℃ for 15s;

[0178] (3) 60℃ for 15 seconds;

[0179] (4) 72℃ for 15 seconds;

[0180] (5) In the second step, repeat the cycle 40 times;

[0181] (6) 72℃ for 5 minutes;

[0182] (7) Store at 4℃.

[0183] A 2% agarose gel was prepared for electrophoresis to verify the reaction products. The reaction products are as follows: Figure 3 As shown.

[0184] 1.6 Sequencing verification of PCR products

[0185] (1) The gel electrophoresis bands of tRNA were cut and the DNA of the PCR products was recovered;

[0186] (2) The recovered DNA was cloned into pMD19 using the Takara TA cloning kit and transformed into E. coli. Plasmids were extracted and Sanger sequencing was performed.

[0187] (3) Sequencing results of PCR products of 20 endogenous tRNAs are as follows: Figure 4-23 As shown.

[0188] Example 2: Quantitative detection of endogenous tRNA (using Saccharomyces cerevisiae as an example) in cells using the method of the present invention.

[0189] Some procedures in this embodiment (cell culture, RNA extraction, primer design, and reverse transcription) are consistent with those described in Example 1. This embodiment quantifies endogenous cysteine ​​tRNA and valine tRNA in total RNA from *Saccharomyces cerevisiae*, as follows:

[0190] First, configure the following reaction:

[0191] (1) 10 μL BeyoFast TM SYBR Green qPCR Mix(2X,High ROX,UDG);

[0192] (2) 1μL Forward qPCR primer (50uM);

[0193] (3) 1μL Reverse qPCR primer (50uM);

[0194] (4) 1 μL RT-PCR template (RT-PCR template is serially diluted at a ratio of 1:4);

[0195] (5) Add water to a final volume of 20 μL;

[0196] Place the prepared reaction into a real-time PCR instrument and set it up as follows:

[0197] (1) 50℃ for 3 minutes;

[0198] (2) 95℃ for 2 min;

[0199] (3) 95℃ for 15 seconds;

[0200] (4) 58℃ for 15 seconds;

[0201] (5) 68℃ for 15 seconds;

[0202] Repeat steps 3, 4, and 5 for 40 cycles, and record the fluorescence value in step 5.

[0203] The sequence-specific aptamer sequences, forward qPCR primer sequences, and reverse qPCR primer sequences corresponding to endogenous cystine tRNA and valine tRNA are shown in Table 1 (SEQ ID NO: 18-20, SEQ ID NO: 78-80).

[0204] The results of quantitative real-time PCR of endogenous cystine tRNA in Saccharomyces cerevisiae are as follows: Figure 24 As shown, the results of real-time quantitative PCR of endogenous valine tRNA in Saccharomyces cerevisiae are as follows: Figure 26 As shown, the results indicate that quantitative real-time PCR can effectively quantify endogenous cysteine ​​tRNA and valine tRNA in Saccharomyces cerevisiae. Figure 25 and Figure 27 As shown, the Ct value and the logarithm of the RNA template amount exhibit a high linear correlation.

[0205] Example 3: Qualitative and quantitative detection of exogenously introduced pyrrolysine tRNA in cells (using Saccharomyces cerevisiae as an example) using the method of the present invention.

[0206] In addition to the 20 tRNAs endogenously expressed in *Saccharomyces cerevisiae*, this embodiment expresses an archaea-derived tRNA (Alv8) with the anticodon CUA in *Saccharomyces cerevisiae*. This tRNA encodes pyrrolidone via the codon UAG. The overall implementation process is as follows:

[0207] 3.1 Cell Culture:

[0208] The process is consistent with that described in Example 1.

[0209] 3.2 Construction of the expression vector for tRNA-Alv8:

[0210] (1) Promoter sequence: controls the initiation of gene expression, is the region where RNA polymerase recognizes and binds, and drives the transcription of downstream genes. The nucleic acid sequence is shown in SEQ ID NO:84.

[0211] (2) Linker sequence: A DNA fragment that provides restriction enzyme sites for inserting the target gene into the vector during the cloning process. The nucleic acid sequence is shown in SEQ ID NO:85.

[0212] (3) tRNA_Alv8 sequence: tRNA gene encoding pyrrolysine, specifically referring to a tRNA with anticodon CUA, derived from archaea, and its nucleic acid sequence is shown in SEQ ID NO:86.

[0213] (4) Termination sequence: This marks the end of gene transcription and prevents RNA polymerase from continuing to transcribe into non-target regions. The nucleic acid sequence is shown in SEQ ID NO:87.

[0214] 3.3 Primer design for tRNA-Alv8:

[0215] (1) tRNA-Alv8 specific aptamer sequence: The nucleic acid sequence is shown in SEQ ID No:81.

[0216] (2) tRNA-Alv8 upstream primer: the nucleic acid sequence is shown in SEQ ID No:82.

[0217] (3) tRNA-Alv8 downstream primer: the nucleic acid sequence is shown in SEQ ID No:83.

[0218] 3.4 Reverse transcription:

[0219] The process is consistent with that described in Example 1.

[0220] 3.5PCR:

[0221] The process is consistent with that described in Example 1. The results are as follows: Figure 28 As shown.

[0222] 3.6 Sequencing verification of PCR products:

[0223] The PCR electrophoresis band of tRNA (Alv8) was excised from the gel and recovered, then TA cloned. The plasmid was then extracted and subjected to Sanger sequencing (the procedure is as described in Example 1(1.6) PCR product sequencing verification). The sequencing results were obtained as follows: Figure 29 As shown. Figure 30 As shown, the results demonstrate that quantitative real-time PCR can effectively quantify exogenously introduced pyrrolysine tRNA in Saccharomyces cerevisiae. Figure 31 As shown, the Ct value and the logarithm of the RNA template amount exhibit a high linear correlation.

Claims

1. A nucleic acid aptamer that specifically recognizes tRNA, characterized in that, The tRNA is a mature tRNA, and the aptamer includes: The 3' end protruding part; The stem ring structure forms part; The 3' protrusion is adapted to pair complementaryly with the CCA sequence, recognition base, and aminoacyl-tRNA synthetase receptor domain at the 3' end of the mature tRNA. The nucleic acid aptamer is a single-stranded DNA.

2. The nucleic acid aptamer according to claim 1, characterized in that, The length of the protruding portion at the 3' end is greater than or equal to 11nt; Optionally, the nucleic acid aptamer is an oligonucleotide aptamer; Optionally, the oligonucleotide aptamer is 50–60 nt in length; Optionally, the tRNA includes: tRNA corresponding to natural amino acids and tRNA corresponding to non-standard amino acids; Optionally, the tRNA includes at least one of tRNAs corresponding to 20 natural amino acids; Optionally, one tRNA is adapted to transport one or more non-standard amino acids; Optionally, the tRNA includes, but is not limited to, at least one of the tRNAs corresponding to the non-standard amino acids pyrrolidone, selenocysteine, Nα-Boc-L-lysine, and 4-methoxy-L-phenylalanine. Optionally, the nucleic acid aptamer has a nucleotide sequence as shown in any of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:81, or a nucleotide sequence having at least 90% identity with it.

3. A tRNA, characterized in that, The CCA sequence, recognition base, and aminoacyl-tRNA synthetase receptor domain at the 3' end of the tRNA are complementary to the protruding portion of the 3' end of the nucleic acid aptamer according to any one of claims 1 to 2.

4. A reagent, characterized in that, include: The nucleic acid aptamer according to any one of claims 1 to 2, wherein the reagent is used to specifically recognize tRNA.

5. A reagent kit, characterized in that, include: The nucleic acid aptamer according to any one of claims 1 to 2 or the reagent according to claim 4, wherein the kit is used to detect tRNA; Optionally, it also includes: reverse transcription reaction reagents and / or PCR reaction reagents.

6. A method for detecting tRNA, characterized in that, include: The nucleic acid aptamer according to any one of claims 1 to 2, the reagent according to claim 4, or the reagent in the kit according to claim 5 are brought into contact with the RNA sample to be tested to obtain a reverse transcription template; when the sample to be tested contains tRNA, the secondary structure of the tRNA is eliminated in the reverse transcription template; A reverse transcription reaction was performed based on the reverse transcription template to obtain a cDNA template. PCR or qPCR reaction is performed based on the cDNA template to obtain PCR or qPCR reaction products; Based on the PCR or qPCR reaction products, the type or content of tRNA in the RNA sample to be tested is determined.

7. The method according to claim 6, characterized in that, The contact process includes: The nucleic acid aptamer or a reagent containing the nucleic acid aptamer is mixed with the RNA sample to be tested and dNTP Mix, and then subjected to high-temperature denaturation and low-temperature renaturation. Optionally, the high-temperature denaturation temperature is 50–75°C, preferably 65°C, and the high-temperature denaturation time is 4–15 min, preferably 5 min; the low-temperature regeneration temperature is 0°C, and the low-temperature regeneration time is not less than 2 min.

8. The method according to claim 6, characterized in that, The temperature of the reverse transcription reaction is pulsed, with the pulsed temperature range being 15–55°C, and each pulse lasting for 50–105 seconds. Optionally, each pulse change includes at least two pulse temperatures; wherein the first pulse temperature is 15-17°C, preferably 16°C; and the second pulse temperature is 45-55°C, preferably 50°C. Optionally, the duration of the first pulse temperature is 25-35 seconds, preferably 30 seconds; the duration of the second pulse temperature is 25-35 seconds, preferably 30 seconds.

9. The method according to claim 8, characterized in that, Each pulse change also includes a third pulse temperature, wherein the first pulse temperature is lower than the second pulse temperature, the second pulse temperature is higher than the third pulse temperature, and the third pulse temperature is higher than the first pulse temperature; Furthermore, the temperature of the third pulse is 40–50°C, preferably 45°C; Furthermore, the duration of the third pulse temperature is 25–35 seconds, preferably 30 seconds; Optionally, in the reverse transcription process, the reverse transcriptase used is capable of withstanding temperatures of 50°C or higher.

10. The method according to claim 8, characterized in that, The reverse transcription reaction was performed according to the following procedure: 40 cycles: 16℃ for 30 seconds, 50℃ for 30 seconds, 45℃ for 30 seconds; 42℃1h； 80℃ for 10 minutes; Store at 4℃.

11. The method according to claim 6, characterized in that, The amplification system for the PCR or qPCR reaction includes: specific primer pairs at both ends of the tRNA and the cDNA template; Furthermore, the amplification system for the PCR reaction is as follows: Furthermore, the amplification system for the qPCR reaction is as follows: Further, the tRNA forward primer has a nucleotide sequence as shown in at least one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:82; the tRNA reverse primer has a nucleotide sequence as shown in at least one of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:82, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:39, SEQ ID NO:3 ... The nucleotide sequence shown in at least one of the following sequences is NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, or SEQ ID NO:83; Furthermore, the PCR reaction was performed according to the following procedure: 94℃ for 3 minutes; 40 cycles: 94℃ for 15 seconds, 60℃ for 15 seconds, 72℃ for 15 seconds; 72℃ for 5 minutes; Store at 4℃; Furthermore, the PCR reaction was performed according to the following procedure: 50℃ for 3 minutes; 95℃ for 2 minutes; 40 cycles: 95℃ for 15 seconds, 58℃ for 15 seconds, 68℃ for 15 seconds.

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