35 results about "Reverse Transcription Process" patented technology

The present invention provides a transcriptome sequencing method, wherein primers used during a reverse transcription process from RNA to cDNA comprise a random sequence and a specific sequence, the random sequence is positioned at the 3' terminal of the specific sequence, bases from 0 to 5 exist between the random sequence and the specific sequence, the random sequence comprises 4-12 random bases, the specific sequence is a homopolymer of 5-30 bases, and the base of the specific sequence is any one selected from A, G, C and T. According to the present invention, with the design on the reverse transcription primers, the RNA having the poly (A) tail can be sequenced while the RNA with no poly (A) tail can be sequenced, the rRNA removal step is not required during the cell transcriptome sequencing, and the method is especially suitable for the sequencing on the transcriptome having the low initial RNA amount and the low cell number.

The invention belongs to the field of research and development of HIV inhibitors, and discloses a preparation method for a terpyridine pyridinium (II) complex and application thereof in HIV reverse transcriptase inhibition. The structure of a cationic moiety of the terpyridine pyridinium (II) complex is as shown in a formula I. A preparation process for the terpyridine pyridinium (II) complex is optimized, the raw material cost is low, and the reaction time is short. The obtained complex is high in purity and yield and has good water solubility and excellent spectral properties. The terpyridine pyridinium (II) complex has the capability of selective binding to a TAR region on HIV RNA, and can block the reverse transcription process of viral RNA by reverse transcriptase and inhibit the replication of viral RNA. The terpyridine pyridinium (II) complex is a highly affinitive HIV RNA selective binding reagent and a highly active HIV reverse transcriptase inhibitor, and is an HIV drug having a great application potential.

The invention provides novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers. A respiratory virus conservative area is selected, and a promoter sequence capable of being recognized by T7 RNA (ribose nucleic acid) polymerase is formed at the 5' terminal of one of the synthesized primers. The invention further provides a novel high-sensitivity respiratory virus nucleic acid NASBA detection method. T7 RNA polymerase promoter sequences are added to the 5' terminals of the two primers complementary to two ends of a respiratory virus RNA sequence, so that when the primers enter a circulation phase, RNA sequences [RNA(+)] consistent with a template sequence can be synthesized, RNA sequences [RNA(-)] complementary to a template RNA sequence can be synthesized, and the detection sensitivity of viral nucleic acid is greatly improved; a throat swab sample of a patient can be directly used for amplification, a reverse transcription process is not required, and the operation time is saved.

The invention relates to a preparation method of a quality control standard substance of a novel coronavirus detection kit. According to the invention, the avian infectious bronchitis virus of the same family as the novel coronavirus (SARS-CoV-2) is used as the quality control standard substance of the novel coronavirus nucleic acid detection kit. At present, novel coronavirus detection kits are uneven in quality level, positive quality control is plasmid samples (DNA), and the virus reverse transcription process cannot be supervised and evaluated. Meanwhile, due to the diversity and complexity of clinical samples, the instability of a detection result is also caused. The quality control product provided by the invention can provide sensitivity and specificity evaluation support in the processes of novel coronavirus detection kit development, delivery inspection and preservation period monitoring; influences of various factors on the quality of the kit in the production process are effectively monitored, and meanwhile, the kit is used for evaluating the inactivation effect of a sample preservation solution on viruses, so that thorough inactivation of the viruses in a sample is ensured, effective preservation of viral nucleic acid is also ensured, the preservation time of the nucleic acid is prolonged, and the detection sensitivity is ensured.

The invention relates to a kit for detecting deoxyribonucleic acid (DNA) of a product which is obtained after DNA of H4N6 avian influenza virus is subjected to amplification by a loop-mediated isothermal nucleic acid amplification technology by using nano gold particles, and belongs to the technical field of biological detection. According to the kit, the loop-mediated isothermal nucleic acid amplification technology and a biological nano technology are combined to improve the sensitivity of biomolecule detection, so that the kit is operated easily, quickly and accurately without special instruments and equipment and can be widely applied to the highly-sensitive detection of H4N6 avian influenza virus in fields of family diagnosis, clinical diagnosis, the control of infectious diseases, environmental monitoring, inspection and quarantine, biotechnology and the like. The kit comprises the following two parts: (1) the nano gold particles on which a molecular probe which can identify a specific sequence of the H4N6 avian influenza virus is marked; and (2) a loop-mediated isothermal nucleic acid amplification system which can perform amplification on the DNA of the H4N6 avian influenza virus in a sample to be detected or perform the amplification on ribonucleic acid (RNA) of the H4N6 avian influenza virus in the sample to be detected after the reverse transcription process is performed.

The invention belongs to the field of researches and development of acquired immune deficiency syndrome virus inhibitors and discloses a preparation method of a ruthenium complex and acquired immune deficiency syndrome virus reverse transcriptase inhibition application of the ruthenium complex. The invention designs a novel synthesis method of a polypyridine ruthenium complex; the complex has highpurity and high yield and has good water solubility and excellent light spectrum property. The polypyridine ruthenium complex provided by the invention has a capability of selectively combining a TARregion on acquired immune deficiency syndrome virus RNA (Ribonucleic Acid), and can be used for blocking a reverse transcription process of the reverse transcriptase on the virus RNA to inhibit the copying of the virus RNA. The polypyridine ruthenium complex provided by the invention is an acquired immune deficiency syndrome virus RNA selective binding reagent with high affinity and an acquired immune deficiency syndrome virus reverse transcriptase inhibitor with high activity, and is an acquired immune deficiency syndrome virus medicine with extremely good application potential.

A method for expanding double-chain RNA target sequence fast is carried out by preparing dsRNA, tapping while recovering by agarose electrophoresis, separating and purifying for dsRNA, using Primer 5.0 software to obtain double-chain RNA related primer by GenBank virus gene group related sequence, taking recovered dsRNA as template, adding into double-chain RNA related primer, and PCR expanding under action of Taq enzyme to obtain the final product. It's cheap and simple, has better dsRNA stability and less reagent and saves time and powder.

The invention relates to a kit for detecting product deoxyribonucleic acid (DNA) which is amplified from coxsackie virus A16 subjected to a loop-mediated isothermal amplification technology by using nano-gold particles, belonging to the technical field of biological detection. By combining the loop-mediated isothermal amplification technology and biological nanometer technology, the sensitivity of biological molecular detection is improved, so the invention is a method with simplicity in operation, quickness and accuracy without professional instrument and equipment, and the kit can be widely applied to high sensitivity coxsackie virus A16 detection in the fields of household diagnosis, clinical diagnosis, infectious disease control, environmental monitoring, inspection and quarantine, biological technology and the like. The kit comprises two parts: (1) nano-gold particles marked with molecular probes which can identify the specific sequence of the coxsackie virus A16; and (2) a loop-mediated isothermal amplification system which can amplify the DNA of the coxsackie virus A16 in a sample to be detected, or amplify ribonucleic acid (RNA) of the coxsackie virus A16 in the sample to be detected after a reverse transcription process.

The invention provides a purple sweet potato ultraviolet receptor UVR8 gene and a cloning method thereof. A nucleotide sequence of the purple sweet potato ultraviolet receptor UVR8 gene is shown as SEQ NO. 4; the cloning method of the purple sweet potato ultraviolet receptor UVR8 gene comprises the following steps: (1) synthesizing a cDNA (complementary Deoxyribonucleic Acid) first chain; (2) carrying out homologous cloning on a UVR8 conserved sequence; (3) amplifying a 3' end of UVR8; (4) amplifying a 5' end of the UVR8; (5) splicing the sequence. By adopting the method provided by the invention, species with a relatively close genetic relationship are not needed and the cloning of a core fragment can be finished through designing various merged basic groups only if a homologous gene exists; the method is also applicable to gene cloning of other plants. In 5' end cloning, a process that a phosphoric acid group of mRNA (messenger Ribonucleic Acid) is removed through phosphorylase and ligase is connected with a connector linker primer is not needed; howeverbut, 3 to 5 C are added at the 3' end in an RNA reverse transcription process according to properties of M-MuLV reverse transcriptase; then 3 to 5 G are added at the tail end of a designed primer and are matchedfoe matching with the designed primerC and the reverse transcription process is finished. The method is simple and convenient, the time and cost are saved and the success rate is relatively high.

The invention discloses a method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA. The methylation level of nucleic acid is reflected by detecting 6-methylaminopurine in DNA and RNA, wherein substrate activity in DNA replication and RNA reverse transcription will be reduced due to the existence of 6-methylaminopurine, and in the DNA replication and RNA reverse transcription process, chain extension will not occur when methylation loca occur; therefore, deoxyribonucleoside triphosphate can be doped in for chain extension so as to differentiate the methylation sequence and the non-methylation sequence, and meanwhile the methylation level of a specific locus of DNA and RNA can be quantitatively evaluated. The method can be well used for analyzing 6-methylaminopurine in nucleotide separation of DNA and RNA and can be used for detecting the methylation level of adenine in DNA and RNA easily and conveniently.

The invention discloses an RNA (Ribonucleic Acid) 4-mercaptouridine specific labeling method and application thereof, and belongs to the technical field of nucleic acid chemistry. According to the method, a structure similar to cytosine (C) is generated under a mild condition by utilizing a reaction mechanism that hydrazine hydrate is used for carrying out addition elimination on RNA 4-mercaptouridine; in the reverse transcription process of RNA, the original A: T pairing at the position of 4sU is changed into C: G pairing, so that the position of 4sU is subjected to single base recognition. The method is combined with a sequencing technology and can be used for detecting the position of naturally existing 4sU and researching the dynamic state of a transcriptome.

The invention discloses a primer group for detecting non-coding small RNA through multiple fluorescent quantitative PCR based on a stem-loop method. The invention establishes a stem-loop method-based multiple fluorescent quantitative PCR method for detecting non-coding small RNA, and realizes PCR amplification of reverse transcription products of a plurality of targets by a pair of upstream and downstream universal primers at the same time. A reverse transcription extension primer is added in the reverse transcription process, a reverse transcription product can be prolonged, and the design of an upstream primer and a probe can be greatly facilitated due to the fact that the length of an amplification product is increased; according to the stem-loop reverse transcription primer, multiple target miRNAs can be subjected to reverse transcription in a single-tube reaction, and then multiple fluorescent quantitative PCR detection is performed, so that multiple detection of miRNAs based on the stem-loop reverse transcription primer is realized, the operation is simpler and more convenient, and clinical diagnosis application requirements of combined expression analysis of multiple target miRNAs are better met.

The invention relates to a kit for detecting swine fever viruses, which contains RNA (Ribonucleic Acid) enzyme inhibitor, an AMV (Avian Myeloblastosis Virus) reverse transcriptase, Taq enzyme (Thermal-resistant desoxyribonucleic acid polymerase), RNA-free enzyme water, One Step RNA PCR (one-step RNA Polymerase Chain Reaction) mixed solution, a positive and negative reference substance, and mixtures of primers with the sequences shown as SEQ ID NO.1 to SEQ ID NO.6. In the detection process, the reverse transcription process and the PCR amplification process are accomplished in one-step reaction, so that the detection time is shorten to 2-3 hours from the original time of 4-5 hours, thereby benefiting rapid detection. Simultaneously, as the kit uses three pairs of primers, compared with thecommon PCR method, the method has higher sensitivity, thereby contributing to prevention and control of CSFV (Classical Swine Fever Virus) and rejection of recessive poisonous animals, and avoiding resulting in large-range infection. The method is suitable for any laboratories, each-level prevention and control units in the basic government organization, veterinary stations, large, medium and small-size livestock farms and the like.

The invention provides a co-library-building method capable of distinguishing DNA and RNA sources. The co-library-building method is characterized by comprising the following steps of: in a library building process, firstly fragmenting DNA and RNA, adding polyA at the tail end of RNA, in a reverse transcription process, adding Poly (dC) at the other tail end of transcribed cDNA, respectively adding p5 and p7 connectors at two ends of cDNA, then adding p5 and p7 connectors at two ends of DNA, and carrying out library amplification and sequencing on DNA and cDNA. The co-library-building method provided by the invention has the advantages that two ends of a cDNA fragment of an RNA source can carry a section of fixed nucleotide sequence for distinguishing the source of sequencing data, so that independent library building and sequencing do not need to be carried out on DNA and RNA in a sample, and cost of NGS detection can be effectively reduced.

The invention relates to a kit for detecting product DNA by utilizing nano-gold particles after human immunodeficiency virus is amplified through a loop-mediated isothermal nucleic acid amplification technique, which belongs to the technical field of biological detection. The kit combines the loop-mediated isothermal nucleic acid amplification technique with a biological nanotechnology to improve the detection sensitivity of biological molecules, is a method with simple operation, rapidness, accuracy and no need for professional instruments and equipment, and can be widely applied to high-sensitivity HIV detection in the fields of household diagnosis, clinical diagnosis, infectious diseases control, environment monitoring, inspection and quarantine, biotechnology and the like. The kit comprises two parts, namely (1) the nano-gold particles marked with molecular robes capable of identifying specific sequences of HIV and (2) a loop-mediated isothermal nucleic acid amplification system capable of amplifying the escherichia coli DNA in to-be-detected samples or performing a reverse transcription process and then amplifying the RNA of the HIV in the to-be-detected samples.

The invention provides a probe composition, which comprises 20 probes, for hindering Globin mRNA reverse transcription, and discloses application of the probe composition. According to the invention, reverse transcription of a target RNA is inhibited with high efficiency and high specificity during synthesis of one-chain cDNA by using a reverse transcription hindering probe method, so that the target RNA is hindered from participating in a downstream library building process. The probe can specifically recognize and bind to the human globulin RNA and hinder the reverse transcription of the globulin RNA so as to realize the purpose of removing the globulin RNA in the RNA library building process, 99.5% or above of Globin mRNA sequences in the blood sample RNA can be effectively removed in the reverse transcription process, and the probe has the advantages of being easy in operation (one step) and short in time consumption (2 min), and is very suitable for large-scale industrial automation.

The invention discloses a linear probe. The linear probe at least comprises a region a, a region b and a region c, wherein the 3' end of the region b is connected with the 5' end of the region a, and the 3' end of the region c is connected with the 5' end of the region b; and the nucleotide sequence of the region a is complementarily paired with the 3' end of the target nucleic acid, the nucleotide sequence of the region b assists in looping by reducing steric hindrance, and the nucleotide sequence of the region c is the same as the 5' end of the target nucleic acid. After the linear probe and the target miRNA are subjected to two times of specific recognition in a reverse transcription process, cDNA of a stem-loop structure is formed under the assistance of the region b, and meanwhile, the target miRNA is released to participate in the next round of cDNA formation. Ring formation and cyclic reverse transcription are assisted by the region b, so that the required reverse transcription time is shorter, and meanwhile, effective reverse transcription is realized on a low-concentration target; and PCR (Polymerase Chain Reaction) amplification is carried out by taking the cDNA as a template to realize rapid, sensitive and specific detection of the target nucleic acid.

The invention provides a positive control composition for a reverse transcription polymerase chain reaction kit and a preparation method thereof, and the positive control composition is characterized in that a non-target virus is taken as positive control, a pair of primers are simultaneously designed for amplification, and the length of an amplified fragment is the same with that of a target gene of the reverse transcription polymerase chain reaction kit; and the non-target virus is inactivated by guanidine isothiocyanate. The positive control prepared by the method is safe, thereby avoiding virus expansion caused by direct use of a virus infection material in the prior art, playing a control role during an RNA (ribonucleic acid) extraction process and an RNA reverse transcription process when serving as the reverse transcription positive control of the RNA virus, proving that an RNA extraction reagent and substances used in a reaction system are normal in activity, facilitating the positive control when serving as the commercial kit and having very high practical value.

The invention relates to an RNA methylation m6A detection method and reagent kit. According to the detection method, a method of combining antibody capture and RNA reverse transcription is adopted, aprocess of synthesizing DNA by taking RNA as a template in the reverse transcription process is utilized, specific oligonucleotide primers are designed for a target gene m6A site region and coupled with biotin to detect the specified gene m6A level, and random primers are used for detecting the total RNAm6A level. Combination is realized by RNA fragments captured by an m6A antibody, streptavidin with an HRP label is combined with biotin for secondary signal amplification, absorbance is detected through TMB color development, and the level of the target gene m6A is calculated. The method for systematically detecting m6A incompletely depending on antibody immune enzyme-linked immunoassay is provided for the first time, has the advantages of high efficiency, sensitivity and the like, is low in detection cost, simple and convenient to operate and suitable for varied RNA samples, and solves the problems that a traditional detection method is limited in resolution, cannot perform m6A quantification and is high in RNA sample demanded quantity.

The invention provides application of poloxamer in improvement of reverse transcriptase efficiency or improvement of reverse transcriptase inhibitor tolerance, and an additive mixture prepared by taking poloxamer as a core component and used in a reverse transcription process. The additive mixture not only can improve reverse transcription products (about 8 times) of normal high-purity RNA samples, but also has stronger tolerance and higher cDNA yield for RNA samples of complex sources, such as FFPE RNA or RNA containing reverse transcription inhibitors such as formaldehyde, ethanol, guanidine hydrochloride, guanidine isothiocyanate, heparin, tannic acid, formamide and phenol. The reverse transcriptase additive combination can significantly improve the sensitivity and accuracy of complex source pathological sample detection on RT-qPCR detection and RNA NGS library building, and is very suitable for the field of rapid diagnosis of diseases.

The present invention relates to a kit and a method for detecting an RNA molecule, particularly, a small-size RNA molecule such as miRNA. The present invention provides an RNA detecting kit and methodin which short primers for reverse transcription are used in a reverse transcription process with the aim of rapidly diffusing the primers in a reverse-transcription reaction solution and thus reducing the reverse-transcription reaction time, enabling the overall PCR reaction rate to be increased and RNA to be detected stably and rapidly. In addition, the present invention provides an RNA detecting kit and method in which, after a reverse transcription process, extension is performed on a single-stranded cDNA by use of a primer for extension, far longer than a primer for reverse transcription, and thus a template long enough to conduct PCR amplification can be obtained.

The invention discloses a method for positioning and quantitatively detecting 6-methylaminopurine in DNA and RNA. The methylation level of nucleic acid is reflected by detecting 6-methylaminopurine in DNA and RNA, wherein substrate activity in DNA replication and RNA reverse transcription will be reduced due to the existence of 6-methylaminopurine, and in the DNA replication and RNA reverse transcription process, chain extension will not occur when methylation loca occur; therefore, deoxyribonucleoside triphosphate can be doped in for chain extension so as to differentiate the methylation sequence and the non-methylation sequence, and meanwhile the methylation level of a specific locus of DNA and RNA can be quantitatively evaluated. The method can be well used for analyzing 6-methylaminopurine in nucleotide separation of DNA and RNA and can be used for detecting the methylation level of adenine in DNA and RNA easily and conveniently.

The invention relates to a kit for detecting swine fever viruses, which contains RNA (Ribonucleic Acid) enzyme inhibitor, an AMV (Avian Myeloblastosis Virus) reverse transcriptase, Taq enzyme (Thermal-resistant desoxyribonucleic acid polymerase), RNA-free enzyme water, One Step RNA PCR (one-step RNA Polymerase Chain Reaction) mixed solution, a positive and negative reference substance, and mixtures of primers with the sequences shown as SEQ ID NO.1 to SEQ ID NO.6. In the detection process, the reverse transcription process and the PCR amplification process are accomplished in one-step reaction, so that the detection time is shorten to 2-3 hours from the original time of 4-5 hours, thereby benefiting rapid detection. Simultaneously, as the kit uses three pairs of primers, compared with thecommon PCR method, the method has higher sensitivity, thereby contributing to prevention and control of CSFV (Classical Swine Fever Virus) and rejection of recessive poisonous animals, and avoiding resulting in large-range infection. The method is suitable for any laboratories, each-level prevention and control units in the basic government organization, veterinary stations, large, medium and small-size livestock farms and the like.

The invention relates to a kit adopting gold nanoparticle to detect DNA formed by amplifying H5N2 avian influenza virus DNA through the loop-mediated isothermal nucleic acid amplification technology, and belongs to the technical field of biological detection. The kit combines the loop-mediated isothermal nucleic acid amplification technology and the bio-nanotechnology, improves the sensitivity of biomolecule detection, is simple, rapid and accurate to operate, does not require special instrument and equipment, and can be widely applied to the detection of high-sensitivity H5N2 avian influenza virus in fields of family diagnosis, clinical diagnosis, communicable disease control, environment monitoring, inspection and quarantine, biotechnology and the like. The kit comprises two parts; the first part is gold nanoparticle, on which a molecular probe capable of identifying H5N2 avian influenza virus specific sequences is marked; and the second part is a loop-mediated isothermal nucleic acid amplification system capable of amplifying H5N2 avian influenza virus DNA in samples to be detected or capable of amplifying H5N2 avian influenza virus RNA after a reverse transcription process.

The invention relates to a kit for detecting DNA (deoxyribonucleic acid) of an amplification product obtained by carrying out loop-mediated isothermal nucleic acid amplification technology on avian H3N8 influenza virus DNA by using nanogold particles, belongs to the technical field of biological detection. By combining loop-mediated isothermal nucleic acid amplification technology and biological nano technology, the invention enhances the biological molecule detection sensitivity, and is a method which is simple, quick and accurate to operate and does not need special instruments or equipment; and the invention can be widely used in high-sensitivity avian H3N8 influenza virus detection in the fields of household diagnosis, clinical diagnosis, infectious disease control, environmental monitoring, quarantine inspection, biotechnology and the like. The kit provided by the invention comprises the following two parts: (1) nanogold particles labeled with molecular probes capable of identifying specific sequence of avian H3N8 influenza virus; and (2) a loop-mediated isothermal nucleic acid amplification system capable of amplifying avian H3N8 influenza virus DNA in a sample to be detected or capable of amplifying avian H3N8 influenza virus DNA in the sample to be detected after a reverse transcription process.

The invention provides novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers. A respiratory virus conservative area is selected, and a promoter sequence capable of being recognized by T7 RNA (ribose nucleic acid) polymerase is formed at the 5' terminal of one of the synthesized primers. The invention further provides a novel high-sensitivity respiratory virus nucleic acid NASBA detection method. T7 RNA polymerase promoter sequences are added to the 5' terminals of the two primers complementary to two ends of a respiratory virus RNA sequence, so that when the primers enter a circulation phase, RNA sequences [RNA(+)] consistent with a template sequence can be synthesized, RNA sequences [RNA(-)] complementary to a template RNA sequence can be synthesized, and the detection sensitivity of viral nucleic acid is greatly improved; a throat swab sample of a patient can be directly used for amplification, a reverse transcription process is not required, and the operation time is saved.

The invention belongs to the field of research and development of HIV inhibitors, and discloses a preparation method for a terpyridine pyridinium (II) complex and application thereof in HIV reverse transcriptase inhibition. The structure of a cationic moiety of the terpyridine pyridinium (II) complex is as shown in a formula I. A preparation process for the terpyridine pyridinium (II) complex is optimized, the raw material cost is low, and the reaction time is short. The obtained complex is high in purity and yield and has good water solubility and excellent spectral properties. The terpyridine pyridinium (II) complex has the capability of selective binding to a TAR region on HIV RNA, and can block the reverse transcription process of viral RNA by reverse transcriptase and inhibit the replication of viral RNA. The terpyridine pyridinium (II) complex is a highly affinitive HIV RNA selective binding reagent and a highly active HIV reverse transcriptase inhibitor, and is an HIV drug having a great application potential.

The invention provides a method for constructing an immune repertoire high-throughput sequencing library for removing a chimera sequence in a sample, a group of primers and a kit. Based on a multiplex PCR library preparation method, a set of primers is designed for each of a heavy chain and a light chain of BCR, and the primers can amplify all different alleles of IgG, IgM, IgA, IgD, IgE, IgK and / or IgL found at present. According to the method, 3'-terminal UMI is added in a reverse transcription process, then 5'-terminal UMI is added in a first round of PCR process, so that two ends of each amplified template molecule are marked by uniquely paired UMI sequences, and then a specific outer Barcode primer is used for carrying out second round of PCR amplification. According to the sequencing data obtained by constructing the library by using the method, the sample chimera sequence in the high-throughput sequencing data can be discriminated and removed by using the abundance distribution of the paired UMI in the analysis process, so that more accurate immune repertoire data can be obtained.