Thermostable reverse transcriptase and application thereof

Abstract

The invention relates to a thermostable reverse transcriptase and application thereof. The thermostable reverse transcriptase has at least one of mutation sites of L333, P65, Q68, P127, P175, E176, G178, G331, T332, F334 and G105, and the amino acid site of the thermostable reverse transcriptase is positioned by an amino acid sequence as shown in SEQ ID NO: 2. The reverse transcriptase has high thermal stability.

Description

A thermostable reverse transcriptase and its application Technical Field

[0001] The present invention relates to the field of enzyme engineering, and in particular to a thermostable reverse transcriptase and applications thereof. Background Art

[0002] Reverse transcriptase (RT) is a specialized DNA polymerase, also known as RNA-dependent DNA polymerase, that synthesizes DNA using RNA as a template. Reverse transcriptases were first discovered in RNA viruses and are currently the most extensively studied and widely used. These include Moloney murine leukemia virus reverse transcriptase (M-MLV RT), human immunodeficiency virus reverse transcriptase (HIV RT), and avian myeloblastosis virus reverse transcriptase (AMV RT).

[0003] Reverse transcription is a key step in many RNA reaction workflows. RNA is typically converted to more stable complementary DNA (cDNA) through reverse transcription, which is then used for research and analysis using techniques such as cloning, PCR, gene expression microarrays, and sequencing. RT-PCR is widely used to detect gene expression levels and the presence of RNA viruses in cells.

[0004] One-step reverse transcription-quantitative real-time polymerase chain reaction (One-step RT-qPCR) is a time-saving and labor-saving nucleic acid detection technology with broad application prospects in rapid food testing, pathogen detection, and infectious disease prevention and control. However, the current One-step RT-qPCR system suffers from problems such as insufficient detection sensitivity and low specificity. As one of the core components of One-step RT-qPCR, reverse transcriptase plays a key role in controlling detection sensitivity and specificity. Improving reverse transcriptase can help improve the detection sensitivity and specificity of One-step RT-qPCR.

[0005] Summary of the Invention

[0006] The present invention aims to solve, at least to some extent, one of the technical problems in the related art. To this end, one object of the present invention is to provide a reverse transcriptase that can improve the sensitivity of RT-qPCR detection, wherein the provided reverse transcriptase has high thermal stability.

[0007] In reverse transcription, increasing the reaction temperature can unravel the secondary structure of the RNA template and reduce nonspecific binding between primers and the template. However, reverse transcriptase is a room-temperature enzyme that is easily denatured and inactivated at high temperatures. Therefore, improving the heat resistance of reverse transcriptase not only allows for efficient cDNA synthesis but also facilitates the storage, packaging, and transport of the enzyme. In the present invention, the inventors mutated a wild-type M-MLV-derived reverse transcriptase to screen for a highly thermostable reverse transcriptase. This is of great significance for related industries and research requiring reverse transcription.

[0008] To this end, according to a first aspect of the present invention, a thermostable reverse transcriptase is provided. According to an embodiment of the present invention, the thermostable reverse transcriptase has at least one of the mutation sites L333, P65, Q68, P175, E176, F334, G178, G331, T332, P127, and G105, wherein the amino acid site of the thermostable reverse transcriptase is mapped based on the amino acid sequence set forth in SEQ ID NO:2. According to some specific embodiments of the present invention, the inventors studied multiple sites of wild-type M-MLV reverse transcriptase and, through experimental screening, found that the aforementioned sites have a significant effect on improving the thermostability of the reverse transcriptase. Therefore, the reverse transcriptase obtained by mutating the aforementioned sites has high thermostability and can effectively improve yield or reaction efficiency when applied to reverse transcription and related technologies.

[0009] According to an embodiment of the present invention, the above-mentioned thermostable reverse transcriptase may further include at least one of the following additional technical features:

[0010] According to an embodiment of the present invention, the thermostable reverse transcriptase further has at least one of the mutation sites Q63, Y64, M66, D200, E562 and D583.

[0011] According to an embodiment of the present invention, the thermostable reverse transcriptase has the following mutation sites or site combinations: 1) Q63; 2) Y64; 3) P65; 4) M665) Q68; 6) P127; 7) P175; 8) E176; 9) G178; 10) G331; 11) T332; 12) L333; 13) F334; 14) E176, E562 and D583; 15) L333, E562 and D583; 16) M66, Q68, G105, E562 and D583; 17) M66, Q68, E176, D200, E562 and D583; or 18) P65, M66, Q68, E69, E176, D200, E562 and D583. In some specific embodiments of the present invention, the reverse transcriptase having the mutation site or site combination has higher thermal stability than the wild-type reverse transcriptase, and can be used in reverse transcription reaction-related technologies, such as reverse transcription reaction, PCR reaction and cDNA library construction.

[0012] According to an embodiment of the present invention, the thermostable reverse transcriptase has at least one of the following mutations: i) Q63A, Q63P or Q63G; ii) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; iii) M66Y or M66V; iv) Q68A, Q68E, Q68K or Q68N; v) E69K; vi) P127D, P127R or P127T; vii) P175L, P175W or P175F; viii) E176H, E176A, E176R or E176C; ix) G178N or G178T; x) G331F, G331P, G331E or G331M; xi) T332D, T332W, T332P or T332K; xii) L333A, L333K or L333N; xiii) F334S, F334T, F334N, F334H or F334D; xiv) G105R; xv) D200N; xvi) E562Q; and xvii) D583N.

[0013] According to an embodiment of the present invention, the thermostable reverse transcriptase further has the following mutations: Y64S, Y64V or Y64F.

[0014] According to an embodiment of the present invention, the reverse transcriptase has the following mutations: I) Q63A, Q63P or Q63G; II) Y64S, Y64V or Y64F; III) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; IV) M66Y; V) Q68A, Q68E, Q68K or Q68N; VI) P127D, P127R or P127T; VII) P175L, P175W or P175F; VIII) E176H, E176A or E176C; IX) G178N or G178T; X) G331F, G331P, G331E, or G331M; XI) T332D, T332W, T332P, or T332K; XII) L333A or L333N; XIII) F334S, F334T, F334N, F334H, or F334D; XIV) E176R, E562Q, and D583N; XV) L333K, E56 2Q and D583N; XVI) M66V, Q68K, G105R, E562Q and D583N; XVII) M66V, Q68K, E176R, D200N, E562Q and D583N; or XVIII) P65K, M66V, Q68K, E69K, E176R, D200N, E562Q and D583N. The inventors screened multiple mutation sites and found that the reverse transcriptases with the above mutations were all more thermally stable than the wild-type M-MLV reverse transcriptase.

[0015] According to an embodiment of the present invention, the amino acid site positioning is based on the amino acid sequence shown in SEQ ID NO: 2.

[0016]

[0017] It should be noted that the site-directed sequence (reference sequence) SEQ ID NO: 2 of the M-MLV reverse transcriptase does not limit the reverse transcriptase produced by the present invention. In addition to SEQ ID NO: 2, the reverse transcriptase can also be used to improve other M-MLV reverse transcriptases that have undergone mutations. It is only necessary to make the mutation sites and / or site combinations described above mutate at the corresponding positions relative to the M-MLV reverse transcriptase shown in SEQ ID NO: 2, for example, at least one of the Q63, Y64, P65, M66, Q68, P127, P175, E176, G178, G331, T332, L333, F334, G105, D200, E562 and D583 positions mutate; or make the specific mutations described above mutate at the corresponding positions relative to SEQ ID NO: NO:2 is the corresponding position of the M-MLV reverse transcriptase, for example: i) Q63A, Q63P or Q63G; ii) Y64S, Y64V or Y64F; iii) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; iv) M66Y; v) Q68A, Q68E, Q68K or Q68N; vi) P127D, P127R or P127T; vii) P175L, P175W or P175F; vii At least one of i) E176H, E176A, or E176C; ix) G178N or G178T; x) G331F, G331P, G331E, or G331M; xi) T332D, T332W, T332P, or T332K; xii) L333A or L333N; xiii) F334S, F334T, F334N, F334H, or F334D; xiv) G105R; xv) D200N; xvi) E562Q; and xvii) D583N. Those skilled in the art can find the corresponding sites by conventional sequence analysis methods.

[0018] According to an embodiment of the present invention, the reverse transcriptase has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 53; or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NOs: 3 to 53; or a polypeptide having one or more amino acid substitutions, deletions, and / or additions compared to SEQ ID NOs: 3 to 53.

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] According to an embodiment of the present invention, the reverse transcriptase has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 5, 9 to 17, 19 to 22, 26 to 28, 42 to 48, and 49 to 53. The reverse transcriptase according to an embodiment of the present invention has a significantly better amplification efficiency in an RT-qPCR reaction than wild-type and commercial M-MLV reverse transcriptase.

[0028] According to an embodiment of the present invention, the reverse transcriptase is a mutant of M-MLV reverse transcriptase.

[0029] According to an embodiment of the present invention, the Tm value of the reverse transcriptase is 50°C-60°C.

[0030] According to a second aspect of the present invention, an isolated nucleic acid molecule is provided. According to embodiments of the present invention, the nucleic acid molecule encodes the thermostable reverse transcriptase described in the first aspect of the present invention. The nucleic acid molecules according to some specific embodiments of the present invention can effectively produce the reverse transcriptase, which has high thermostability and can effectively improve reaction efficiency when used in reverse transcription and related technologies.

[0031] According to a third aspect of the present invention, the present application provides an expression vector carrying the isolated nucleic acid molecule described in the second aspect of the present invention. The expression vector may include optional control sequences that are operably linked to the isolated nucleic acid molecule. The control sequences are one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vectors proposed in the embodiments of the present invention can efficiently express the thermostable reverse transcriptase in a suitable host cell.

[0032] When the nucleic acid molecule of above-mentioned separation is connected to the carrier, the nucleic acid molecule can be directly or indirectly connected to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can directly come from the carrier itself, or they can be exogenous, that is, not from the carrier itself. Herein, "operably connected" refers to that the foreign gene is connected to the carrier so that the control elements in the carrier, such as transcription control sequences and translation control sequences, etc., can bring into play the function of transcribing and translating the foreign gene of its expected regulation. Of course, the nucleic acid molecule used to encode the described thermostable reverse transcriptase can be independently inserted into different carriers respectively, and it is common to be inserted into the same carrier. Commonly used carriers can for example be plasmids, bacteriophages, etc., such as Plasmid-X plasmids.

[0033] According to an embodiment of the present invention, the isolated nucleic acid molecule is operably linked to a promoter.

[0034] According to an embodiment of the present invention, the promoter comprises one selected from the group consisting of: λ-PL promoter, tac promoter, trp promoter, araBAD promoter, T7 promoter and trc promoter.

[0035] According to a fourth aspect of the present invention, a recombinant cell is provided, comprising the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect, or expressing the thermostable reverse transcriptase of the first aspect. The host cell used to express the target gene or nucleic acid molecule can be a prokaryotic cell. In at least some embodiments, the thermostable reverse transcriptase of the present invention is expressed in a prokaryotic cell, such as Escherichia coli.

[0036] The recombinant cells are obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cells can efficiently and massively express the aforementioned thermostable reverse transcriptase under appropriate conditions. The thermostable reverse transcriptase has high thermal stability, and reverse transcription reactions, PCR reactions, and sequencing library construction performed using the reverse transcriptase all have high reaction efficiency.

[0037] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably Escherichia coli.

[0038] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the thermostable reverse transcriptase described herein. Those skilled in the art will readily appreciate that conditions suitable for the expression of a thermostable reverse transcriptase include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the reverse transcriptase based on the specific laboratory environment.

[0039] According to a fifth aspect of the present invention, a method for preparing the thermostable reverse transcriptase described in the first aspect is provided. According to an embodiment of the present invention, the method comprises: introducing the expression vector described in the third aspect into a host cell; and culturing the host cell introduced with the expression vector under conditions suitable for protein expression to obtain the thermostable reverse transcriptase. The method according to an embodiment of the present invention can effectively express and obtain the thermostable reverse transcriptase in large quantities.

[0040] According to an embodiment of the present invention, the method for preparing the thermostable reverse transcriptase according to the first aspect may further include at least one of the following additional technical features:

[0041] According to an embodiment of the present invention, the host cell is Escherichia coli. When the host cell is Escherichia coli, the expression efficiency of the thermostable reverse transcriptase is higher.

[0042] According to an embodiment of the present invention, the host cell does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0043] According to a sixth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the thermostable reverse transcriptase described in the first aspect of the present invention. The use of a kit containing the thermostable reverse transcriptase can effectively improve the efficiency of reverse transcription and related reactions.

[0044] According to an embodiment of the present invention, the above-mentioned kit may further include at least one of the following additional technical features:

[0045] According to an embodiment of the present invention, the kit further comprises at least one of the following: an RNA extraction reagent, one or more DNA polymerases, one or more buffers, one or more primers, and one or more terminators.

[0046] According to an embodiment of the present invention, the terminator comprises dideoxynucleotide.

[0047] In a seventh aspect, the present invention provides a reverse transcription method. According to an embodiment of the present invention, the method comprises the following steps: reverse transcribing at least one RNA template in the presence of at least one reverse transcriptase to obtain cDNA, wherein the reverse transcriptase comprises the aforementioned thermostable reverse transcriptase. As mentioned above, the thermostable reverse transcriptase has high thermal stability. Therefore, the reverse transcription method according to an embodiment of the present invention is more efficient than a reverse transcription reaction using a wild-type or commercial reverse transcriptase.

[0048] According to an embodiment of the present invention, the reverse transcription method may further include at least one of the following additional technical features:

[0049] According to an embodiment of the present invention, during the reverse transcription process, at least one reverse transcription primer is also present. Those skilled in the art will appreciate that when performing the reverse transcription reaction, it is also necessary to use a reverse transcription primer for a specific region of the nucleic acid template, and those skilled in the art can design primers based on the target region on the nucleic acid template.

[0050] According to an embodiment of the present invention, the reverse transcription is performed at a temperature of 35°C to 65°C, preferably 37°C to 55°C, more preferably 42°C to 55°C, and even more preferably 50°C to 55°C. In some preferred embodiments, the thermostable reverse transcriptase has high thermal stability and can be used at a temperature of 35°C to 65°C. It has a high reverse transcription reaction efficiency at a temperature of 42°C to 55°C, and the reverse transcription reaction efficiency is still very high when the reverse transcription temperature is 50°C to 55°C.

[0051] According to an eighth aspect of the present invention, a method for RT-PCR is provided. According to an embodiment of the present invention, the method comprises: performing a reverse transcription reaction on at least one RNA template in the presence of at least one reverse transcriptase and at least one DNA polymerase to obtain cDNA, wherein the reverse transcriptase comprises the thermostable reverse transcriptase described in the first aspect. As previously mentioned, the thermostable reverse transcriptase described in the first aspect has high thermal stability and can effectively improve the reaction efficiency of RT-PCR. Therefore, the RT-PCR method described in this application is more efficient than a PCR reaction using a wild-type reverse transcriptase or a commercial reverse transcriptase.

[0052] According to an embodiment of the present invention, the above RT-PCR method may further include at least one of the following additional technical features:

[0053] According to an embodiment of the present invention, at least one reverse transcription primer is present during the reverse transcription process.

[0054] According to an embodiment of the present invention, the reverse transcription reaction is performed at a temperature of 35°C to 65°C.

[0055] According to an embodiment of the present invention, the reverse transcription reaction is performed at a temperature of 42°C to 55°C.

[0056] According to an embodiment of the present invention, the reverse transcription reaction is performed at a temperature of 50°C to 55°C.

[0057] According to an embodiment of the present invention, the RT-PCR method further comprises: subjecting the cDNA to a first polymerase chain reaction in the presence of at least one pair of primers and at least one DNA polymerase.

[0058] According to a ninth aspect of the present invention, a method for RT-PCR is provided. According to an embodiment of the present invention, the method comprises the following steps: performing a reverse transcription reaction and a second polymerase chain reaction on at least one RNA template in the presence of at least one reverse transcriptase and at least one DNA polymerase, wherein the reverse transcriptase comprises the thermostable reverse transcriptase described in the first aspect. As previously mentioned, the thermostable reverse transcriptase described in the first aspect has high thermal stability and can effectively improve the reaction efficiency of RT-PCR. Therefore, the RT-PCR method described in this application is more efficient than a PCR reaction using a wild-type reverse transcriptase or a commercial reverse transcriptase.

[0059] According to an embodiment of the present invention, the above-mentioned RT-PCR method further includes at least one of the following additional technical features:

[0060] According to an embodiment of the present invention, at least one pair of primers is further present in the reverse transcription reaction and the second polymerase chain reaction.

[0061] According to an embodiment of the present invention, the reverse transcription reaction includes a stage performed at a temperature of 35°C to 65°C.

[0062] According to an embodiment of the present invention, the reverse transcription reaction includes a stage performed at a temperature of 42°C to 55°C.

[0063] According to an embodiment of the present invention, the reverse transcription reaction is performed at a temperature of 50°C to 55°C.

[0064] In its tenth aspect, the present invention provides a method for constructing a sequencing library. According to an embodiment of the present invention, the method comprises enriching nucleic acid molecules connected to adapters, wherein the nucleic acid molecules are obtained by reverse transcription of the RNA sample to be tested using the reverse transcription method described above. As previously mentioned, the thermostable reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of reverse transcription-related technologies. Therefore, the method according to an embodiment of the present invention can construct a high-quality sequencing library.

[0065] According to an embodiment of the present invention, the above method for constructing a sequencing library may further include at least one of the following additional technical features:

[0066] According to an embodiment of the present invention, the enrichment treatment comprises subjecting the nucleic acid molecules connected with adapters to a third polymerase chain reaction in the presence of at least one pair of PCR primers and at least one DNA polymerase.

[0067] According to an embodiment of the present invention, the enrichment process further includes a purification process.

[0068] According to an embodiment of the present invention, the purification process is performed by magnetic bead purification.

[0069] According to embodiments of the present invention, the sources of the RNA to be tested include, but are not limited to, animals (e.g., rats, mice, cats, dogs, horses, cattle, porcine, pigs, chickens, ducks, geese, quail, pigeons, nematodes, zebrafish, etc.), plants (e.g., rice, Arabidopsis, wheat, corn, sweet potatoes, peanuts), protists, eukaryotic algae, bacteria, archaea, fungi, viroids, or viruses. For example, RNA can be obtained by processing multicellular or single cells from these biological samples.

[0070] According to an embodiment of the present invention, exemplary bacteria include but are not limited to: one or more of: Staphylococcus, Streptococcus, Listeria, Erysipelothrix, Nephrobacterium, Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia, Corynebacterium, Coccus, Bacillus anthracis, Erysipelothrix, Tetanus, Listeria, Emphysema and Anthracis, Mycobacterium tuberculosis, Escherichia coli, Proteus, Shigella dysenteriae, Klebsiella pneumoniae, Brucella, Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, and Acinetobacter.

[0071] According to an embodiment of the present invention, exemplary viruses include but are not limited to: one or more of: Adenoviridae (aden), Arenaviridae, Astroviridae, Bunyaviridae, Caliciviridae, Flaviviridae, Hepaciviridae, Unimolecular Negative-Strand RNA Virales, Nidovirales, Picornaviridae, Orthomyxoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae, Poxviridae, Reoviridae, and Retroviridae.

[0072] According to an embodiment of the present invention, the RNA sample to be tested includes but is not limited to samples from cells, tissues, soil, saliva, sweat, feces, urine, blood, serum or plasma.

[0073] In an eleventh aspect, the present invention provides a sequencing library. According to an embodiment of the present invention, the sequencing library is obtained by the method described in the tenth aspect. As previously mentioned, the reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of reverse transcription reaction-related technologies. Therefore, the method according to the embodiment of the present invention can construct a high-quality sequencing library.

[0074] In a twelfth aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the method comprises sequencing the sequencing library described in the eleventh aspect and performing data analysis. Those skilled in the art will appreciate that sequencing the sequencing library and performing data analysis can detect and verify the sequencing library to determine whether the sequencing library is a target library.

[0075] In a thirteenth aspect of the present invention, the present invention provides the use of the thermostable reverse transcriptase described in the first aspect in constructing a sequencing library. According to an embodiment of the present invention, the thermostable reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of reverse transcription reaction-related technologies. Therefore, all technologies applied to reverse transcription can use the thermostable reverse transcriptase. It should be noted that the sequencing library is not particularly limited. For example, RNA-seq, exon sequencing, small RNA-seq, single-cell DNA sequencing, single-cell mRNA sequencing, methylation sequencing, Moleculo long sequencing, Ribozero, and directional RNA sequencing libraries can all be constructed using the above-mentioned thermostable reverse transcriptase.

[0076] In a fourteenth aspect, the present invention provides the use of the thermostable reverse transcriptase described in the first aspect in sequencing. According to embodiments of the present invention, the thermostable reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of reverse transcription reaction-related technologies. Therefore, any technology applied to reverse transcription can use the thermostable reverse transcriptase. It should be noted that the sequencing method and sequencing platform are not particularly limited, and any sequencing that can use the thermostable reverse transcriptase is within the scope of protection of this application.

[0077] In its fifteenth aspect, the present invention provides a method for detecting gene content in a test sample. According to an embodiment of the present invention, the method comprises: performing a reverse transcription reaction on a test RNA sample using the reverse transcriptase described in the first aspect to obtain cDNA; using the cDNA as a template, performing a fourth polymerase chain reaction on the cDNA in the presence of predetermined primers, wherein the predetermined primers are designed to amplify a target gene fragment; and determining the expression level of the test gene in the test sample based on the content of the fourth polymerase chain reaction product. The method according to an embodiment of the present invention can effectively detect gene content in a test sample. The method can be used for diagnostic and / or non-diagnostic purposes, such as scientific research. Specifically, for example, it can be used to screen biological samples containing specific genes or genes at specific levels for subsequent research. It can also be used to detect gene content in a test sample from an individual, and by comparing the obtained gene content with a standard level for the gene in the relevant disease, determine whether the individual suffers from the gene-related disease. The standard level can be determined by comparing and verifying the differences in the gene content in a large number of test samples from individuals with the gene-related disease and a large number of healthy individuals.

[0078] According to an embodiment of the present invention, the above method for detecting gene content in a sample to be tested may further include at least one of the following additional technical features:

[0079] According to an embodiment of the present invention, the RNA sample to be tested includes a sample from an animal, a plant, a protist, a eukaryotic algae, a bacterium, an archaea, a fungus, a viroid or a virus.

[0080] According to an embodiment of the present invention, the RNA sample includes a sample from cells, tissues, soil, saliva, sweat, feces, urine, blood, serum or plasma.

[0081] The beneficial effects achieved by the present application are as follows: the reverse transcriptase provided by the present application has high thermal stability and can be applied to related technologies involving reverse transcription reactions to achieve amplification of complex templates and full-length cDNA, and the reaction tolerance temperature is increased, thereby improving the amplification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0083] FIG1 is a graph showing the detection results of one-step RT-qPCR of wild-type M-MLV reverse transcriptase and its mutants and commercial M-MLV reverse transcriptase according to one embodiment of the present invention. DETAILED DESCRIPTION

[0084] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0085] Furthermore, 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 the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0086] The endpoints of the ranges and any values ​​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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0087] For easier understanding, the terms in this document are explained and illustrated below. Those skilled in the art should understand that these explanations and illustrations should not be construed as limiting the scope of protection of the present invention.

[0088] As used herein, the term "reverse transcriptase" refers to a protein, polypeptide, or polypeptide fragment that exhibits reverse transcriptase activity.

[0089] The term "reverse transcriptase activity" or "reverse transcription activity" refers to the ability to synthesize a DNA chain using RNA as a template.

[0090] The term "mutant" or "mutant" refers to a sequence that has one or more mutations compared to a reference sequence (e.g., the wild-type DNA sequence set forth in SEQ ID NO: 1 or the wild-type amino acid sequence set forth in SEQ ID NO: 2). Such mutations can occur at either the nucleic acid level or the amino acid level.

[0091] As used herein, the term "identity" is used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is 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 program (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 of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0092] In this article, "Polymerase Chain Reaction (PCR)" refers to a molecular biology technique used to amplify specific DNA fragments. It is a DNA replication process in vitro. Its basic principle is that a template DNA-primer complex, under the action of DNA polymerase, uses dNTPs as raw materials and a target sequence as a template. According to the principle of complementary base pairing and the principle of semiconservative replication, the primer synthesizes a new DNA chain along the 5'→3' direction.

[0093]

[0014] Abbreviations for amino acid residues herein are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0094] As used herein, when an amino acid site is indicated, the expression "amino acid abbreviation + site" is used in accordance with common notation in the art, for example, "Q63," where "Q" represents the amino acid at that site and "63" represents the corresponding amino acid site. When a mutation site is indicated, the expression "amino acid abbreviation before mutation + site + amino acid abbreviation after mutation" is used in accordance with common notation in the art, for example, "Q63A," where "Q" represents the amino acid before mutation, "63" represents the corresponding mutation site, and "A" represents the amino acid after mutation. "Q" and "A" are both single-letter abbreviations commonly used in the art to represent amino acids.

[0095] The present invention provides reverse transcriptases, kits, and compositions comprising the reverse transcriptases.

[0096] The reverse transcriptase provided by the present invention has at least one of the mutation sites L333, P65, Q68, P175, E176, F334, G178, G331, T332, P127 and G105, wherein the amino acid site positioning of the reverse transcriptase is based on the amino acid sequence shown in SEQ ID NO: 2. In addition, the reverse transcriptase may further have at least one of the mutation sites Q63, Y64, M66, D200, E562 and D583.

[0097] In some specific embodiments of the present invention, the reverse transcriptase has the following mutation sites or site combinations: 1) Q63; 2) Y64; 3) P65; 4) M665) Q68; 6) P127; 7) P175; 8) E176; 9) G178; 10) G331; 11) T332; 12) L333; 13) F334; 14) E176, E562 and D583; 15) L333, E562 and D583; 16) M66, Q68, G105, E562 and D583; 17) M66, Q68, E176, D200, E562 and D583; or 18) P65, M66, Q68, E69, E176, D200, E562 and D583.

[0098] In some embodiments of the invention, the reverse transcriptase has at least one of the following mutations: i) Q63A, Q63P or Q63G; ii) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; iii) M66Y or M66V; iv) Q68A, Q68E, Q68K or Q68N; v) E69K; vi) P127D, P127R or P127T; vii) P175L, P175W or P175F; viii) P175L, P175W or P175F; ) E176H, E176A, E176R, or E176C; ix) G178N or G178T; x) G331F, G331P, G331E, or G331M; xi) T332D, T332W, T332P, or T332K; xii) L333A, L333K, or L333N; xiii) F334S, F334T, F334N, F334H, or F334D; xiv) G105R; xv) D200N; xvi) E562Q; and xvii) D583N. The thermostable reverse transcriptase further has the following mutations: Y64S, Y64V, or Y64F.

[0099] In some embodiments of the invention, the reverse transcriptase has the following mutations: I) Q63A, Q63P or Q63G; II) Y64S, Y64V or Y64F; III) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; IV) M66Y; V) Q68A, Q68E, Q68K or Q68N; VI) P127D, P127R or P127T; VII) P175L, P175W or P175F; VIII) E176H, E176A or E176C; IX) G178N or G178T; X) G331F, G331P, G331E or G33 1M; XI) T332D, T332W, T332P or T332K; XII) L333A or L333N; XIII) F334S, F334T, F334N, F334H or F334D; XIV) E176R, E562Q and D583N; XV) L333K, E562Q and D583N; XVI) M66V, Q68K, G105R, E562Q and D583N; XVII) M66V, Q68K, E176R, D200N, E562Q and D583N; or XVIII) P65K, M66V, Q68K, E69K, E176R, D200N, E562Q and D583N.

[0100] The present invention also provides a test kit. The test kit provided by the present invention can be used to generate or amplify nucleic acid molecules (single-stranded or double-stranded) or for sequencing. The test kit provided by the present application includes a loadable carrier, such as a box or a hard case, etc. These loadable carriers are equipped with one or more containers, such as vials, tubes, etc. These containers can be equipped with one or more reverse transcriptases provided by the present application. In addition, in addition to the reverse transcriptase, one or more DNA polymerases, one or more buffers suitable for nucleic acid synthesis, one or more nucleotides and / or one or more nucleic acid extraction reagents can also be installed in the same or different containers.

[0101] The compositions provided herein may comprise, in addition to one or more reverse transcriptases (e.g., two, three, four, eight, ten, fifteen, twenty, thirty, forty, fifty, etc.) as described herein, one or more nucleotides, one or more nucleic acid extraction reagents, one or more buffers, and one or more DNA polymerases. The compositions provided herein may further comprise one or more oligonucleoside primers. The compositions are obtained by mixing the above-described components.

[0102] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0103] Example 1 Construction of expression vectors for M-MLV reverse transcriptase and its mutants

[0104] 1.1 Construction of M-MLV reverse transcriptase expression vector

[0105] A nucleic acid sequence encoding wild-type M-MLV reverse transcriptase was obtained and then introduced into the pET-22b(+) expression plasmid (purchased from GenScript Technologies) between the NdeI and EcoRI restriction sites. This vector carries six His residues at the C-terminus of the M-MLV sequence to facilitate protein purification. This expression vector was named pET-22b-M-MLV.

[0106] The nucleotide sequence encoding the wild-type M-MLV reverse transcriptase (SEQ ID NO: 1) is shown below:

[0107]

[0108]

[0109] The amino acid sequence of wild-type M-MLV reverse transcriptase (SEQ ID NO: 2) is shown below:

[0110]

[0111] 1.2 Construction of expression vector for M-MLV reverse transcriptase mutant

[0112] The expression plasmids for the M-MLV reverse transcriptase mutants were constructed by site-directed mutagenesis. The inventors designed forward and reverse primer pairs for mutagenesis and performed site-directed PCR using pET-22b-M-MLV as a template and Pfu DNA polymerase (purchased from Promega) to obtain the corresponding M-MLV reverse transcriptase mutant expression vectors. The specific method is as follows:

[0113] (1) Site-directed mutagenesis was performed according to the reaction system shown in Table 1 and the reaction conditions shown in Table 2.

[0114] Table 1: PCR reaction system for constructing M-MLV reverse transcriptase mutant expression vector

[0115] Reaction components / final concentration 1× Pfu buffer (containing MgSO4) 0.2mM dNTPs 0.5μM forward primer 0.5μM reverse primer 0.05U / μL pfu DNA polymerase 1ng / μL template (pET-22b-M-MLV) HO

[0116] Table 2: PCR reaction conditions for constructing M-MLV reverse transcriptase mutant expression vector

[0117]

[0118] (2) After the reaction, DpnI was added to a final concentration of 0.4 U / μL and digested at 37°C for 1 h;

[0119] (3) Take 5 μL of the digested product and transform it into E. coli DH5α competent cells;

[0120] (4) The next day, single colonies were picked from the plate and cultured in LB medium containing ampicillin at 37°C with shaking at 200 rpm.

[0121] (5) Extract the plasmid and perform sequencing and comparison analysis to obtain clones with the correct mutation.

[0122] Example 2 Expression and purification of M-MLV reverse transcriptase and its mutants

[0123] 2.1 Small-scale expression and purification of wild-type M-MLV reverse transcriptase and its mutants to obtain crude enzyme

[0124] Wild-type M-MLV reverse transcriptase and its mutants are expressed from the pET22b promoter and are fused with six His tags at the C-terminus. Ni-column affinity purification can be performed using the His tags to obtain the corresponding crude enzyme solutions. The specific methods are as follows:

[0125] (1) The wild-type and mutant plasmids obtained in Example 1 were transformed into BL21 competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.);

[0126] (2) Then, a single colony was picked and placed in 15 mL of LB medium containing Amp resistance (100 μg / mL) and cultured at 37°C, 200 rpm / min, with shaking until the OD600 was 0.6-0.8;

[0127] (3) Add inducer IPTG (final concentration 0.5 mM) and induce at 16°C, 200 rpm / min for 16-18 h;

[0128] (4) Centrifuge at 8000 rpm / min for 5 min to collect the induced bacterial precipitate;

[0129] (5) Resuspend the cells in M-MLV reverse transcriptase Ni column A (50 mM Tris, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole, pH 7.5). Perform non-contact batch ultrasonic sterilization in an ice-water bath at an intensity of 70% for 30 min.

[0130] (6) Centrifuge the disrupted bacterial solution at 13,000 rpm and 4°C for 10 min, and collect the supernatant;

[0131] The crude M-MLV enzyme supernatant prepared in the previous step was subjected to Ni affinity purification. The main steps were as follows: the supernatant was added to the wells of the equilibrated His MuLtiTrap HP purification plate (purchased from GE), and the solution was removed using a vacuum pressure device after standing for 15 minutes; 2 mL of M-MLV reverse transcriptase Ni column A solution was added for rinsing and the solution was removed using a vacuum pressure device; a 96-well plate was placed under the His MuLtiTrap HP purification plate and fixed, and then 100 μL of M-MLV reverse transcriptase Ni column B solution (50 mM Tris, 500 mM NaCl, 5% Glycerol, 500 mM Imidazole, pH 7.5) was added for elution, and the eluted protein was collected by centrifugation at 500 g for 2 minutes; 100 μL of glycerol was added to the sample wells of the 96-well plate, mixed thoroughly, transferred to a 1.5 mL centrifuge tube and stored at -20°C for subsequent screening experiments.

[0132] Example 3 Thermal Stability Detection of Wild-Type M-MLV Reverse Transcriptase and Its Mutants

[0133] In this example, the wild-type M-MLV reverse transcriptase and mutants obtained in Example 2 were subjected to thermal stability assay.

[0134] Using the Protein Thermal Shift assay kit TMAfter preparation using the Dye Kit (purchased from Thermo), detection was performed using the StepOne Plus Real-Time PCR System. Specific reaction conditions were set in accordance with the kit instructions. Protein Thermal Shift Software was then used to analyze the wild-type M-MLV reverse transcriptase and mutants, and the Tm value of each protein was derived. The specific detection principle is: as the temperature rises, the protein structure changes, exposing the hydrophobic domain, which binds to the fluorescent dye to produce fluorescence. The change between temperature (melt curve) and fluorescence value is detected in real time by a qPCR instrument. The Tm values ​​of the wild-type M-MLV reverse transcriptase and its mutants are compared to determine their stability. Mutants with Tm values ​​higher than the wild-type are screened. The specific experimental results after screening are shown in Table 3, and the specific mutation pattern and sequence of each mutant are shown in Table 4.

[0135] Table 3: Thermal stability screening results of wild-type M-MLV reverse transcriptase and mutant crude enzyme solutions

[0136] No. Tm(℃)No. Tm(℃)RT-155.42RT-2757.09RT-256.11RT-2856.65RT-355.32RT-2957.05RT-456.93RT-3057.58RT-558.09RT-3157.29RT-652.13RT-3257.11RT-753.36RT-3356.91RT-852.55RT-3457.13RT-954.15RT-3556 .25RT-1053.75RT-3656.11RT-1153.75RT-3756.28RT-1253.75RT-3856.86RT-1352.55RT-3957.15RT-1453. 40RT-4056.96RT-1554.20RT-4157.55RT-1656.63RT-4256.70RT-1756.42RT-4357.26RT-1856.82RT-4457.11

[0137] RT-1956.03RT-4555.80RT-2056.43RT-4656.68RT-2158.17RT-4757.38RT-2258.97RT-4858. 23RT-2350.15RT-4954.42RT-2457.50RT-5057.59RT-2556.48RT-5154.26RT-2657.21WT49.06

[0138] Table 4: M-MLV reverse transcriptase mutation information

[0139]

[0140] Example 4 Detection of polymerization activity of wild-type M-MLV reverse transcriptase and its mutants

[0141] M-MLV reverse transcriptase is a normothermic enzyme, and its polymerization activity decreases with increasing temperature. The polymerization activity of the mutant was tested by comparing the amount of polymerization product produced by the mutant with that of the wild-type M-MLV RT at the same reaction temperature.

[0142] poly(rA) 50 Using oligo(dT) as a template and oligo(dT) as a primer, chain extension occurred under the action of the crude wild-type M-MLV reverse transcriptase and its mutants obtained in Example 2, yielding hybrid chains of extended RNA and cDNA. Polymerization reactions were performed under different reaction temperature conditions. A fluorescent molecule was added to the reaction products, and reverse transcriptase activity was calculated by measuring the RNA / cDNA ratio. The specific reaction system is shown in Table 5.

[0143] Table 5: Polymerization reaction system

[0144] Components / Final Concentration 1x First Strand Buffer 10mM DTT 0.5mM dTTP 1.5mM oligo(dT) 10 2U / μL RNase Inhibitor25ng / μL Poly(rA) 50 0.2 ng / μL M-MLV reverse transcriptase or its mutant DEPC HO

[0145] The above reaction system was incubated at 37°C or 55°C for 10 minutes, followed by the addition of 25 mM EDTA to terminate the reaction. A 2 μL aliquot of the reaction product was quantified using the Qubit dsDNA HS Assay Kit. The polymerization activity of the M-MLV reverse transcriptase mutant relative to wild-type M-MLV reverse transcriptase was calculated. The specific experimental results are shown in Table 6.

[0146] Table 6: Polymerase activity of M-MLV mutants compared to their wild type

[0147]

[0148] Example 5: Verification of the heat resistance of M-MLV reverse transcriptase and its mutants

[0149] The untreated M-MLV reverse transcriptase and its mutants prepared in Example 2 and the M-MLV reverse transcriptase and its mutants treated at 60°C for 10 minutes were tested for polymerization activity according to the polymerization activity detection method described in Example 3. The heat resistance of the M-MLV reverse transcriptase and its mutants was verified by comparing the polymerization activity of the M-MLV reverse transcriptase and its mutants after treatment at 60°C for 10 minutes with that of the M-MLV reverse transcriptase and its mutants that had not been heat treated. The residual polymerization activity of the M-MLV reverse transcriptase and its mutants after treatment at 60°C for 10 minutes compared with that of the M-MLV reverse transcriptase and its mutants that had not been heat treated is shown in Table 7.

[0150] Table 7: Residual activity of M-MLV reverse transcriptase and its mutants after heat treatment at 60°C for 10 min

[0151] No. Residual activity No. Residual activity RT-119.2% RT-2434.7% RT-240.3% RT-2536.7% RT-321.9% RT-2637.0% RT-458.8% RT-2745.1% RT-538.9% RT-2853.7% RT-647.9% RT-2940.2% RT-716.2% RT-3087.3% RT-813.5% RT-3152.6% RT-92.2% RT-3247.1% RT-1022.0% RT-3330.8% RT-1110.2% RT-3434.0% RT-1226.9% RT-3537.9% RT-1314.1% RT-3648.7%

[0152] RT-1410.8% RT-3738.4% RT-1524.3% RT-3863.9% RT-1646.9% RT-3952.8% RT-1767.0% RT-4098.6% RT-1855.1% RT-4160.8% RT-1946. 7%RT-4237.2%RT-2065.6%RT-4343.4%RT-2136.5%RT-4434.6%RT-2266.4%RT-4547.8%RT-2365.9%RT-4649.0%RT-2434.7%WT30.7%

[0153] Example 6 Detection of the Effect of Templates and Primers on the Thermal Stability of M-MLV Reverse Transcriptase and Its Mutants

[0154] The inventors further tested the effects of template and primer on the thermal stability of the P65 site mutant of the M-MLV reverse transcriptase described in this application. The detection method used the method described in Example 3, and the thermal stability of the M-MLV reverse transcriptase mutant was tested in the presence of a template / primer (T / P) system. The specific experimental method is as follows: the reaction system shown in Tables 8 and 9 was prepared, and the M-MLV reverse transcriptase mutants W313F and L435G, which have been shown to improve the thermal stability of the enzyme in the reaction by increasing the binding of T / P, were used as positive controls. The detection was performed using the StepOne Plus Real-Time PCR System, and then the Protein Thermal Shift Software was used for analysis and the Tm value of each protein was derived. At the same time, the T / P-free, i.e., oligo(dT)-removed, was detected. 16 The thermal stability of M-MLV reverse transcriptase under the above-mentioned system with poly(rA) 50 was compared. The test results are shown in Table 10.

[0155] Table 8: Reaction system including template and primers

[0156] Reagent volume / μLProtein Thermal Shift Buffer58×Diluted Protein Thermal Shift Dye2.510mM dTTP110μM oligo(dT) 16 0.5500ng / μL poly(rA) 50 0.40.1mg / mL M-MLV reverse transcriptase or its mutant 2DEPC H2O8.6 Total 20

[0157] Table 9: Reaction system without template and primers

[0158] Reagent volume / μL Protein Thermal Shift Buffer 58× Diluted Protein Thermal Shift Dye 2.5 10mM dTTP 10.1mg / mL M-MLV reverse transcriptase or its mutant 2DEPC H2O 9.5 Total 20

[0159] Table 10: Effects of templates / primers on the thermal stability of M-MLV reverse transcriptase and its mutants

[0160] No. Tm 有T / P -Tm 无T / P(℃)WT-0.4W313F1.8L435G3.0RT-72.7RT-82.8RT-91.4RT-101.7RT-112.4RT-122.2RT-130.8RT-142.1RT-151.0

[0161] As shown in Table 10, the Tm value of the M-MLV reverse transcriptase P65 mutant increased in the presence of T / P compared to the absence of T / P; however, there was no significant difference in the Tm value of the wild-type M-MLV reverse transcriptase with or without T / P. This suggests that the M-MLV reverse transcriptase P65 mutant may enhance the enzyme's thermal stability during the reaction by improving its binding to the template / primer (T / P) rather than improving the enzyme's inherent heat resistance.

[0162] Example 7 Verification of the heat resistance of M-MLV reverse transcriptase and its mutants

[0163] In this example, the heat resistance of the M-MLV reverse transcriptase and its P65 site mutant in the reaction system was determined by measuring the relative activity of the M-MLV reverse transcriptase and its P65 site mutant obtained in Example 2 at 55°C compared to at 37°C. Specifically, polymerization activity was detected using the polymerization activity detection method described in Example 4, wherein the reaction temperatures were 37°C and 55°C. The relative activity of the 55°C reaction compared to the 37°C reaction is shown in Table 11. The relative activity of the P65 site mutant at 55°C / 37°C was higher than that of the WT, indicating that the P65 site mutant (in the presence of T / P) has higher thermal stability.

[0164] Table 11: Relative activities of M-MLV reverse transcriptase and its mutants (55°C / 37°C)

[0165] No. Relative activity (55℃ / 37℃) WT 14.2% RT-7 21.2% RT-8 16.9% RT-9 23.3% RT-10 22.8% RT-11 19.3% RT-12 17.3% RT-13 23.7% RT-14 17.6% RT-15 20.0%

[0166] Example 8 Comparison of the application of M-MLV reverse transcriptase and its mutants in one-step RT-qPCR

[0167] One-step RT-qPCR test was performed using the M-MLV reverse transcriptase and its mutants obtained in Example 2. The reaction system is shown in Table 12, and the reaction procedure is shown in Table 13. The primer and probe sequences used in the one-step RT-qPCR were:

[0168] GADPH-F (upstream primer): 5′-CAACGGATTTGGTCGTATTGG-3′ (SEQ ID NO: 54).

[0169] GADPH-R (downstream primer): 5'-GCAACAATATCCACTTTACCAGAGTTAA-3' (SEQ ID NO: 55).

[0170] GADPH-P (probe): 5′-HEX-CGCCTGGTCACCAGGGCTGC-BHQ1-3′ (SEQ ID NO: 56).

[0171] The results of one-step RT-qPCR for reverse transcription of M-MLV reverse transcriptase and its mutants at 50°C and 55°C are shown in Table 14. ΔCt(Mut-WT) represents the difference between the Ct values ​​of the M-MLV reverse transcriptase mutant and the wild-type M-MLV reverse transcriptase under the same conditions. A ΔCt(Mut-WT) less than 0 indicates that the M-MLV reverse transcriptase mutant reaches the threshold earlier than the wild-type M-MLV reverse transcriptase and performs better in one-step RT-qPCR than the wild-type. The results show that most mutants perform better than the wild-type M-MLV reverse transcriptase in one-step RT-qPCR.

[0172] Table 12: One-step RT-qPCR reaction system

[0173] Components / Final Concentration 1x PCR Buffer 0.27 ng / μL M-MLV Reverse Transcriptase or its mutant 0.5 U / μL HostStart Taq DNA Polymerase 0.2 U / μL RNase Inhibitor 5 mU / μL Heat-labile UDG 0.5 μM GADPH-F 0.5 μM GADPH-R 0.25 μM GADPH-P 16.7 pg / μL HEK293T Total RNA DEPC H2O

[0174] Table 13: One-step RT-qPCR reaction conditions

[0175]

[0176]

[0177] Table 14: One-step RT-qPCR data of M-MLV reverse transcriptase (WT) and its mutant (Mut)

[0178]

[0179]

[0180] Example 9 Comparison of the application of M-MLV reverse transcriptase and its mutants in one-step RT-qPCR

[0181] This example further tested the M-MLV reverse transcriptase and its mutants and commercial M-MLV RH using the method described in Example 8. - (Purchased from Takara) One-step RT-qPCR amplification effect with different RNA template concentrations, the final RNA concentrations in the system were 16.7pg / μL, 1.67pg / μL, and 0.167pg / μL, respectively. The test results are shown in Figure 1. The commercial M-MLV RH - The Ct values ​​of the M-MLV reverse transcriptase mutants RT-12, RT-40, RT-44, RT-47 to RT-51 were lower than those of the commercial M-MLV RH. - Low, its amplification effect is better than the commercial M-MLV RH - reverse transcriptase and wild-type M-MLV reverse transcriptase.

[0182] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0183] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A thermostable reverse transcriptase, characterized in that The thermostable reverse transcriptase has at least one of the mutation sites L333, P65, Q68, P175, E176, F334, G178, G331, T332, P127 and G105, wherein the amino acid site positioning of the thermostable reverse transcriptase is based on the amino acid sequence shown in SEQ ID NO:

2.

2. The thermostable reverse transcriptase according to claim 1, characterized in that The thermostable reverse transcriptase further has at least one of the mutation sites Q63, Y64, M66, D200, E562 and D583.

3. The thermostable reverse transcriptase according to any one of claims 1 to 2, characterized in that The thermostable reverse transcriptase has the following mutation sites or site combinations: 1)Q63； 2)Y64； 3)P65； 4)M66 5)Q68； 6)P127； 7)P175； 8)E176； 9)G178； 10)G331； 11)T332； 12)L333； 13)F334； 14) E176, E562, and D583; 15) L333, E562, and D583; 16) M66, Q68, G105, E562, and D583; 17) M66, Q68, E176, D200, E562, and D583; or 18)P65, M66, Q68, E69, E176, D200, E562 and D583.

4. The thermostable reverse transcriptase according to claim 1, characterized in that The thermostable reverse transcriptase has at least one of the following mutations: i) Q63A, Q63P or Q63G; ii) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; iii) M66Y or M66V; iv) Q68A, Q68E, Q68K or Q68N; v)E69K; vi) P127D, P127R or P127T; vii) P175L, P175W or P175F; viii) E176H, E176A, E176R, or E176C; ix) G178N or G178T; x) G331F, G331P, G331E or G331M; xi)T332D, T332W, T332P or T332K; xii) L333A, L333K or L333N; xiii) F334S, F334T, F334N, F334H or F334D; xiv)G105R; xv)D200N; xvi)E562Q; and xvii)D583N; Optionally, the thermostable reverse transcriptase further has the following mutations: Y64S, Y64V or Y64F.

5. The thermostable reverse transcriptase according to claim 3, characterized in that The thermostable reverse transcriptase has the following mutations: 1) Q63A, Q63P, or Q63G; II) Y64S, Y64V, or Y64F; III)P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; IV)M66Y; V) Q68A, Q68E, Q68K or Q68N; VI) P127D, P127R, or P127T; VII) P175L, P175W or P175F; VIII) E176H, E176A, or E176C; IX) G178N or G178T; X) G331F, G331P, G331E or G331M; XI)T332D, T332W, T332P or T332K; XII) L333A or L333N; XIII) F334S, F334T, F334N, F334H or F334D; XIV) E176R, E562Q, and D583N; XV) L333K, E562Q, and D583N; XVI) M66V, Q68K, G105R, E562Q, and D583N; XVII) M66V, Q68K, E176R, D200N, E562Q, and D583N; or XVIII) P65K, M66V, Q68K, E69K, E176R, D200N, E562Q and D583N.

6. The thermostable reverse transcriptase according to any one of claims 1 to 5, characterized in that The reverse transcriptase has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 53; or A polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NOs: 3-53; or Polypeptides having one or more amino acid substitutions, deletions, and / or additions compared to SEQ ID NOs: 3 to 53; Preferably, the reverse transcriptase has an amino acid sequence shown in any one of SEQ ID NOs: 3-5, 9-17, 19-22, 26-28, 42-48 and 49-53.

7. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the thermostable reverse transcriptase according to any one of claims 1 to 6.

8. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 7.

9. A recombinant cell, characterized in that The method comprises the nucleic acid molecule according to claim 7, the expression vector according to claim 8, or the thermostable reverse transcriptase according to any one of claims 1 to 6.

10. A method for preparing a thermostable reverse transcriptase, characterized in that: The steps include: introducing the expression vector according to claim 8 into a host cell; and The host cell introduced with the expression vector is cultured under conditions suitable for protein expression to obtain the thermostable reverse transcriptase.

11. The preparation method according to claim 10, characterized in that: The host cell includes Escherichia coli.

12. A kit, characterized in that The invention comprises the thermostable reverse transcriptase according to any one of claims 1 to 6.

13. The kit according to claim 12, characterized in that Also includes at least one of the following: RNA extraction reagent, one or more DNA polymerases, one or more buffers, one or more primers and one or more terminators; Optionally, the terminator comprises a dideoxynucleotide.

14. A reverse transcription method, characterized in that: The method comprises the following steps: Performing a reverse transcription reaction on at least one RNA template in the presence of at least one reverse transcriptase to obtain cDNA, wherein the reverse transcriptase includes the thermostable reverse transcriptase according to any one of claims 1 to 6.

15. The reverse transcription method according to claim 14, characterized in that During the reverse transcription reaction, at least one reverse transcription primer is also present; Optionally, the reverse transcription reaction is carried out at a temperature of 35°C to 65°C, preferably 42°C to 55°C, more preferably 50°C to 55°C.

16. A RT-PCR method, characterized in that The method comprises: Performing a reverse transcription reaction on at least one RNA template in the presence of at least one reverse transcriptase to obtain cDNA, wherein the reverse transcriptase includes the thermostable reverse transcriptase according to any one of claims 1 to 6.

17. The RT-PCR method according to claim 16, characterized in that During the reverse transcription process, at least one reverse transcription primer is also present; Optionally, the reverse transcription reaction is carried out at a temperature of 35°C to 65°C, preferably 42°C to 55°C, more preferably 50°C to 55°C; Optionally, the RT-PCR method further comprises: subjecting the cDNA to a first polymerase chain reaction in the presence of at least one pair of PCR primers and at least one DNA polymerase.

18. A RT-PCR method, characterized in that: The following steps are involved: The at least one RNA template is subjected to a reverse transcription reaction and a second polymerase chain reaction in the presence of at least one reverse transcriptase and at least one DNA polymerase, wherein the reverse transcriptase comprises the thermostable reverse transcriptase according to any one of claims 1 to 6.

19. The method according to claim 18, characterized in that At least one pair of primers is also present in the reverse transcription reaction and the second polymerase chain reaction; Optionally, the reverse transcription reaction includes a stage performed at a temperature of 35°C to 65°C, preferably 42°C to 55°C, more preferably 50°C to 55°C.

20. Use of the thermostable reverse transcriptase according to any one of claims 1 to 6 in constructing a sequencing library.

21. Use of the thermostable reverse transcriptase according to any one of claims 1 to 6 in sequencing.

22. A method for detecting gene expression in a sample to be tested, characterized in that: include: Reverse transcription is performed on the RNA sample to be tested using the thermostable reverse transcriptase according to any one of claims 1 to 6 to obtain cDNA; Using the cDNA as a template, performing a fourth polymerase chain reaction on the cDNA in the presence of predetermined primers, wherein the predetermined primers are designed with the gene fragment to be detected as the target amplification fragment; and The expression level of the gene to be tested in the sample to be tested is determined according to the content of the fourth polymerase chain reaction product.

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