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WO2021182850A1 - Procédé de détection simultanée de multiples miarn et kit de détection de miarn l'utilisant - Google Patents

Procédé de détection simultanée de multiples miarn et kit de détection de miarn l'utilisant Download PDF

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WO2021182850A1
WO2021182850A1 PCT/KR2021/002915 KR2021002915W WO2021182850A1 WO 2021182850 A1 WO2021182850 A1 WO 2021182850A1 KR 2021002915 W KR2021002915 W KR 2021002915W WO 2021182850 A1 WO2021182850 A1 WO 2021182850A1
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primer
reverse
mirna
site
reverse transcription
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이재훈
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Heimbiotek Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a method for simultaneous multiple detection of miRNA and a kit for detecting miRNA using the same, and more particularly, to a method for detecting miRNA using the same, comprising: synthesizing a plurality of cDNAs using a reverse transcription primer that hybridizes with a portion of the 3' end of each of a plurality of target miRNAs; synthesizing a plurality of PCR templates by bidirectionally extending the plurality of reverse transcribed cDNAs with a plurality of extension primers that hybridize with a portion of the 3' end of each; It relates to a simultaneous multiplex detection method of miRNA comprising: amplifying using a forward primer and a reverse primer specific for the plurality of PCR templates, and detecting miRNA with a plurality of double-labeled probes.
  • Micro RNA is an untranslated RNA composed of about 22 ribonucleotides, and its size is very small ( ⁇ 22 nt), so it is difficult to isolate or detect it. have. Conventional methods for detecting this include Northern blot analysis, detection based on hybridization using microarray, and detection method using RT-qPCR based.
  • RT-qPCR-based miRNA detection technology includes technologies from Qiagen and Applied Biosystems, and each of the above technologies uses a different reverse transcription technology.
  • Qiagen's miRNA detection technology uses Poly-A-Tailing technology using Poly-A-Polymerase to insert Poly-A at the 3' end of all miRNAs. After synthesizing the tail, general RT-primers with Poly T at the 3' end are hybridized to the Poly-A Tail at the 3' end of miRNA, and cDNA from miRNA (ssDNA PCR Template to be precise) Synthesize the library.
  • This is a technique for amplifying the synthesized library using an oligomer having the same DNA sequence as the target miRNA as a primer in the PCR step, and quantitatively detecting the amplified PCR product using SYBR Green.
  • the miRNA detection technology of Qiagen synthesizes cDNA using the same RT-primer in the RT step, so regardless of the type of miRNA, the cDNA sequence is identical except for the miRNA nucleotide sequence, and also in the PCR step. Since amplification is performed using the sequence of the target miRNA as a PCR primer, a dual-labeled probe that can hybridize with a PCR template synthesized from the target miRNA with high specificity (Dual-Labeled Probe) or TaqMan Probe There is a problem in the design of , which makes it impossible to accurately detect multiple miRNAs simultaneously.
  • ABI Applied Biosystems's miRNA detection technology uses an RT-primer having a Stem-Loop structure, and a complementary sequence that hybridizes with 6-8 sequences of the 3' end of the target miRNA at the 3' end of the RT-primer. design, and synthesize the target miRNA into cDNA (ssDNA PCR Template to be precise).
  • the cDNA synthesized in this way is amplified by using an oligomer having the same DNA sequence as the target miRNA as a primer in the PCR step, and then the amplified PCR product is quantitatively detected using a TaqMan Probe.
  • the TaqMan Probe is complementary to several nucleotide sequences at the end of the target miRNA and some nucleotide sequences at the end of Stem-Loop to specifically detect the PCR product amplified by cDNA derived from one target miRNA.
  • the RT-primer of the Stem-Loop structure has a slow reaction rate due to its long length, and has limitations in modifying or adjusting the sequence to maintain the Stem-Loop structure, so it is not suitable for simultaneous multiplex analysis.
  • the method of the present invention uses a short linear RT-primer, unlike other technologies, and synthesizes cDNA using RT-primers having different sequences according to the target miRNA, and also synthesized from a plurality of target miRNAs in the PCR step. It is possible to design extension primers complementary to cDNA with different nucleotide sequences. In addition, since it is possible to design a double-labeled probe that complementarily binds to PCR products amplified by the synthesized cDNAs with high specificity, it is confirmed that miRNA can be simultaneously detected with high accuracy, and the present invention was completed.
  • the present invention provides a method for simultaneous multiplex detection of miRNA comprising the following steps.
  • the reverse transcription primer consists of a portion that hybridizes with the 3' end of each target miRNA, a probing site consisting of an arbitrary nucleic acid sequence, and a forward primer site that is an arbitrary nucleic acid sequence,
  • the forward primer site sequence of the reverse transcription primer is represented by the same random sequence irrespective of the target miRNA type, or by a different random sequence for each target miRNA type.
  • the extension primer is a nucleic acid sequence that hybridizes with the reverse transcribed cDNA portion of the miRNA, except for the portion at which the 3' end of each target miRNA and the reverse transcription primer hybridize, and an extension annealing site and any nucleic acid sequence It consists of a reverse primer site,
  • the reverse primer site sequence of the extension primer is represented by the same arbitrary sequence regardless of the target miRNA type, or by a different arbitrary sequence for each target miRNA type.
  • the forward primer has the same sequence as the forward primer site of the reverse transcription primer, and when the forward primer site is represented by the same random sequence regardless of the type of target miRNA, a single forward primer, and the forward primer site is the target miRNA type, respectively
  • a plurality of forward primers specific for each are used.
  • the reverse primer has the same sequence as the reverse primer site of the extension primer, and when the reverse primer site is represented by the same random sequence regardless of the type of target miRNA, a single reverse primer is used, and the reverse primer site is the target miRNA type, respectively. If they are indicated by random sequences different from each other, a plurality of reverse primers specific for each of a plurality of double-stranded PCR templates are used.
  • the double-labeled probe has the same nucleic acid sequence as the probing site of the reverse transcription primer.
  • the length of the reverse transcription primer may be 40 nt or more to 60 nt or less, preferably 40 nt or more to 50 nt or less, more preferably 42 to 49 nt.
  • the length of the probing site of the reverse transcription primer may be 19 to 24 nt, and the length of the forward primer site may be 22 to 25 nt.
  • the length of the portion that hybridizes with the 3' end of the target miRNA at the probing site may be 5 to 8 nt, and any nucleic acid sequence may have a length of 14 to 16 nt.
  • the length of the extension primer may be 30 nt or more to 60 nt or less, preferably 30 nt or more to 50 nt or less, more preferably 34 to 43 nt.
  • the length of the extension hybridization site of the extension primer may be 12 to 18 nt, and the length of the reverse primer site may be preferably 22 to 25 nt.
  • the length of the forward primer or the reverse primer may be 20 to 25 nt.
  • the length of the double-labeled probe may be 20 to 24 nt, and the 5 to 8 nt portion of the miRNA 3' end and the 14 to 16 nt portion extended by the reverse transcription primer are simultaneously performed.
  • the double-labeled probe may be a combination of a quencher and a fluorescent material.
  • the quencher is Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa Black (Iowa) black) FQ, Iowa Black RQ or IRDye QC-1.
  • the fluorescent material is fluorescein, fluorescein chlorotriazinyl, rhodamine green, or rhodamine red. , tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine ) series dyes or thiadicarbocyanine dyes.
  • the present invention is also carried out by the miRNA simultaneous multiplex detection method,
  • An extension annealing site which is a nucleic acid sequence in which the remainder of the miRNA hybridizes with the reverse transcribed cDNA portion, except for the portion where the 3' end of each target miRNA and the reverse transcription primer hybridize, and an optional nucleic acid sequence reverse primer site (reverse) primer site) consisting of a plurality of extension primers;
  • It provides a kit for simultaneous multiple detection of miRNA, comprising a plurality of double-labeled probes having the same nucleic acid sequence as the probing site of the reverse transcription primer.
  • the kit may further include a DNA polymerase, a dNTP (dATP, dCTP, dGTP and dTTP) mixture, a buffer solution and distilled water.
  • a DNA polymerase a DNA polymerase
  • a dNTP dATP, dCTP, dGTP and dTTP
  • a buffer solution distilled water.
  • the miRNA simultaneous multiplex detection method of the present invention uses a shorter reverse transcription primer compared to a commercial miRNA detection kit, it is highly reactive in the reverse transcription step, thereby enabling accurate cDNA synthesis, and each reverse transcription primer for detecting a plurality of miRNAs; Since it is easy to design an extension primer, a forward primer/reverse primer for PCR amplification, and a double-labeled probe for miRNA detection, a multiplex analysis capable of simultaneously detecting a plurality of miRNAs is possible.
  • 1 is a schematic diagram schematically showing a method for simultaneous multiple detection of miRNA according to the present invention.
  • FIG. 2 is a schematic diagram showing the hybridization position and length of a reverse transcription primer, an extension primer, a forward primer, a reverse primer, and a double-labeled probe for simultaneous multiplex detection of miRNA.
  • FIG. 3 is a schematic diagram showing the hybridization position and length of a reverse transcription primer, an extension primer, a forward primer, a reverse primer, and a double-labeled probe for detecting hsa-miR-21-5p.
  • FIG. 4 is data showing the results of amplifying hsa-miR-21-5p using the miRNA simultaneous multiplex detection method according to the present invention.
  • the upper left is a graph showing the change in fluorescence value according to the concentration of sequentially diluted hsa-miR-21-5p and the number of amplification cycles, and the lower left is the number of copies of the template and the detection limit (Cq) as a result of performing real-time PCR. It is a graph showing the correlation between cycles.
  • the table on the right shows data showing Ct values according to the hsa-miR-21-5p concentration.
  • FIG. 5 is data showing the results of amplifying hsa-miR-16-5p using the miRNA simultaneous multiplex detection method according to the present invention.
  • the upper left is a graph showing the change in fluorescence value according to the concentration of sequentially diluted hsa-miR-16-5p and the number of amplification cycles, and the lower left is a real-time PCR result of the template copy number and detection limit (Cq). It is a graph showing the correlation between cycles.
  • the table on the right shows data showing Ct values according to the hsa-miR-16-5p concentration.
  • FIG. 6 is data showing the results of performing the miRNA simultaneous multiplex detection method according to the present invention in a miRNA sample in which hsa-miR-26a-5p, hsa-miR-193a-5p and hsa-miR-30b-5p are mixed.
  • the left is a graph showing the change in fluorescence value according to each miRNA concentration and the number of amplification cycles
  • the middle is a graph showing the correlation between the template copy number and the limit of detection (Cq) cycle
  • the right is a graph showing the change in the fluorescence value according to each miRNA concentration and the number of amplification cycles. This is the data showing the Ct value.
  • FIG. 7 is a miRNA simultaneous multiplex detection method according to the present invention in a miRNA sample in which hsa-miR-30c-5p, hsa-miR-29c-3p, hsa-miR-331-3p and hsa-miR-320a-3p are mixed. Data showing the results of the execution.
  • the left is a graph showing the change in fluorescence value according to each miRNA concentration and the number of amplification cycles
  • the middle is a graph showing the correlation between the template copy number and the limit of detection (Cq) cycle
  • the right is a graph showing the change in the fluorescence value according to each miRNA concentration and the number of amplification cycles. This is the data showing the Ct value.
  • the present invention relates to a method for simultaneous multiplex detection of miRNA comprising the following steps.
  • the reverse transcription primer consists of a portion that hybridizes with the 3' end of each target miRNA, a probing site consisting of an arbitrary nucleic acid sequence, and a forward primer site that is an arbitrary nucleic acid sequence,
  • the forward primer site sequence of the reverse transcription primer is represented by the same random sequence irrespective of the target miRNA type, or by a different random sequence for each target miRNA type.
  • the extension primer is a nucleic acid sequence that hybridizes with the reverse transcribed cDNA portion of the miRNA, except for the portion at which the 3' end of each target miRNA and the reverse transcription primer hybridize, and an extension annealing site and any nucleic acid sequence It consists of a reverse primer site,
  • the reverse primer site sequence of the extension primer is represented by the same arbitrary sequence regardless of the target miRNA type, or by a different arbitrary sequence for each target miRNA type.
  • the forward primer has the same sequence as the forward primer site of the reverse transcription primer, and when the forward primer site is represented by the same random sequence regardless of the type of target miRNA, a single forward primer, and the forward primer site is the target miRNA type, respectively
  • a plurality of forward primers specific for each are used.
  • the reverse primer has the same sequence as the reverse primer site of the extension primer, and when the reverse primer site is represented by the same random sequence regardless of the type of target miRNA, a single reverse primer is used, and the reverse primer site is the target miRNA type, respectively. If they are indicated by random sequences different from each other, a plurality of reverse primers specific for each of a plurality of double-stranded PCR templates are used.
  • the double-labeled probe has the same nucleic acid sequence as the probing site of the reverse transcription primer.
  • the conventional poly-A-taling method used for miRNA detection in the prior art has the same cDNA sequence except for the miRNA nucleotide sequence, and it is difficult to design a double-labeled probe with high specificity.
  • the miRNA detection method using the RT-primer of the Stem-Loop structure has a slow reaction rate due to the long RT-primer length, and has limitations in modifying or adjusting the sequence to maintain the Stem-Loop structure. is not suitable for
  • a plurality of target miRNAs to be detected are separated by a straight line specific for each of the plurality of target miRNAs.
  • synthesizing a plurality of cDNAs by reverse transcription with a reverse transcription primer of the form (b) synthesizing a plurality of double-stranded templates for PCR reaction by bidirectionally extending the plurality of cDNAs with extension primers specific for each of the plurality of cDNAs; (c) amplifying using a forward primer and a reverse primer specific to the plurality of PCR templates, and detecting miRNA with a plurality of double-labeled probes;
  • the miRNA detection method of the present invention has a short reverse transcription primer length, a fast reaction rate, and no restrictions on sequence modification, so that it is possible to more easily detect various miRNAs simultaneously.
  • the miRNA simultaneous multiplex detection method of the present invention can be applied regardless of the number of miRNA primers, but 1 to 6, preferably 1 to 5 miRNAs can be simultaneously detected for the efficiency of the reverse transcription reaction and PCR reaction.
  • the reverse transcription primer consists of a portion that hybridizes with the 3' end of each target miRNA, a probing site consisting of an arbitrary nucleic acid sequence, and a forward primer site that is an arbitrary nucleic acid sequence, and the reverse transcription primer
  • the forward primer site sequence of may be represented by the same arbitrary sequence regardless of the target miRNA type, or may be represented by a different arbitrary sequence for each target miRNA type.
  • the length of the reverse transcription primer may be 40 nt or more to 60 nt or less, preferably 40 nt or more to 50 nt or less, and more preferably 42 to 49 nt.
  • the length of the probing site of the reverse transcription primer may be 19 to 24 nt, and the length of the forward primer site may be 22 to 25 nt.
  • the length of the portion that hybridizes with the 3' end of the target miRNA at the probing site may be 5 to 8 nt, and any nucleic acid sequence may have a length of 14 to 16 nt.
  • the extension primer includes an extension annealing site, which is a nucleic acid sequence in which the rest of the miRNA hybridizes with the reverse transcribed cDNA portion, except for the portion at which the 3' end of each target miRNA and the reverse transcription primer hybridize, and an optional nucleic acid sequence in the reverse direction. Consists of a reverse primer site, and the reverse primer site sequence of the extension primer may be represented by the same arbitrary sequence regardless of the target miRNA type, or may be represented by a different arbitrary sequence for each target miRNA type. .
  • the length of the extension primer may be 30 nt or more to 60 nt or less, preferably 30 nt or more to 50 nt or less, more preferably 34 to 43 nt.
  • the length of the extension hybridization site of the extension primer may be 12 to 18 nt, and the length of the reverse primer site may be preferably 22 to 25 nt.
  • the forward primer has the same sequence as the forward primer site of the reverse transcription primer, and when the forward primer site is represented by the same random sequence regardless of the type of target miRNA, a single universal forward primer site can be used, and When the primer sites are represented by different random sequences for each type of target miRNA, a plurality of forward primers specific for each of a plurality of double-stranded PCR templates may be used.
  • the reverse primer has the same sequence as the reverse primer site of the extension primer, and when the reverse primer site is represented by the same random sequence regardless of the type of target miRNA, a single reverse primer site can be used, and the reverse primer site is reversed. When the primer sites are represented by different random sequences for each type of target miRNA, a plurality of reverse primers specific for each of a plurality of double-stranded PCR templates may be used.
  • the length of the forward primer or the reverse primer may be 20 to 25 nt.
  • the double-labeled probe may have the same nucleic acid sequence as the probing site of the reverse transcription primer, and the length of the double-labeled probe may be 20 to 24 nt, and the 5 to 8 nt portion of the miRNA 3' terminal and extended by the reverse transcription primer. 15 to 16 nt moieties can be recognized simultaneously.
  • the double-labeled probe is a combination of a quencher and a fluorescence
  • the quencher is Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, and Black Hole Quencher. Hole Quencher), Qxl, Iowa black FQ, Iowa black RQ or IRDye QC-1, wherein the fluorescent material is fluorescein, fluorescein chlorotriazinyl, rhodamine.
  • rhodamine green rhodamine red
  • tetramethylrhodamine FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas red ( Texas Red), TET, TRITC, TAMRA, cyanine-based dye or thiadicarbocyanine dye, but is not limited thereto.
  • cDNA is synthesized using reverse transcription primers having different sequences according to the target miRNA, unlike the prior art, and in the PCR step, extension primers complementary to cDNA synthesized from a plurality of target miRNAs are used in different ways. Design by nucleotide sequence is possible. In addition, since it is possible to design a double-labeled probe that complementarily binds to PCR products amplified by each synthesized cDNA with high specificity, it is possible to simultaneously detect multiple miRNAs with high accuracy.
  • miRNA since miRNA is short in length, only 1 to 2 nucleotide sequences are different depending on the type and the remaining nucleotide sequences are the same. There is a problem in that simultaneous multiplex detection of miRNAs with similar sequences is not possible. Therefore, in the present invention, in order to prevent a non-specific amplification reaction from occurring, a reverse transcription primer and an extension primer were designed to recognize all parts of miRNA.
  • the reverse transcription primer sequence should be designed to be at least 40 nt in length since the hybridization portion of the double-labeled probe and the forward primer does not overlap during the PCR amplification process and the length that can be sufficiently amplified without a non-specific reaction must be secured.
  • the extension primer must also hybridize with the cDNA to which the rest of the miRNA is reverse transcribed, except for the part where the miRNA 3' end and the reverse transcription primer hybridize, and the length that can be sufficiently amplified without non-specific reaction by the reverse primer in the PCR amplification process must be secured. Therefore, it should be designed to be at least 30 nt in length.
  • the double-labeled probe since the double-labeled probe must be designed to simultaneously recognize the miRNA 3' end portion and any sequence portion extended by the reverse transcription primer to specifically detect the target miRNA from a plurality of miRNAs, also , the hybridization of the forward primer and the double-labeled probe should be designed to have an appropriate length so that they do not overlap each other.
  • the reverse transcription primer and extension primer used in the miRNA simultaneous multiplex detection method of the present invention are capable of recognizing all miRNA sequences specific in order to prevent non-specific amplification and detection reactions. Simultaneous multiplex detection of a plurality of target miRNAs in one tube is possible because parts that hybridize with each other do not overlap, and a primer length for performing an appropriate amplification reaction can be secured in the PCR process.
  • nucleotide used in the present invention is a unit molecule constituting a nucleic acid and is composed of a base, a pentose and phosphoric acid, and the base is adenine, guanine, cytosine in the case of DNA. , and thymine are present, and in the case of RNA, uracil is used instead of the thymine.
  • pentose 2'-deoxyribose is used for DNA and ribose is used for RNA.
  • oligonucleotide refers to a short single-stranded nucleic acid chain composed of up to 13 to 25 nucleotides as a polymer of the nucleotide unit molecule, and in some cases, 6-mer, 7 -mer, 8-mer, 9-mer, 10-mer, 11-mer and 12-mer, etc. may refer to a nucleic acid chain consisting of less than 13 nucleotides or more than 25 nucleotides.
  • PCR polymerase chain reaction
  • nucleic acid amplification reaction refers to a reaction for amplifying a specific target nucleic acid molecule using a thermostable DNA polymerase.
  • a reaction buffer containing divalent ions such as primers (forward primer, reverse primer), deoxynucleotide mixture (dNTP mixture), Mg2+, etc., which are oligonucleotides that can specifically hybridize to a target nucleic acid, are used in addition to DNA polymerase. do.
  • DNA molecules produced by the PCR reaction are referred to as "amplification products" in this document.
  • primer refers to an oligonucleotide or polynucleotide that is complementary to hybridization to a template nucleic acid molecule, which is used for initiation of a PCR reaction or a primer extension reaction.
  • a primer for the PCR reaction is a forward primer (or sense primer) selected from the same sense strand as the direction of the genetic code of the nucleic acid molecule to be amplified, and a reverse primer (or antisense primer) selected from an antisense strand complementary to the sense strand. used, and in the case of a primer extension reaction, a single extension primer is usually used.
  • the "sense strand” refers to a single-stranded nucleic acid molecule in the same direction as the direction of the gene code among double-stranded DNA molecules, and the “antisense strand” refers to another single-stranded nucleic acid molecule complementary to the sense strand. , irrespective of the direction of the gene's coding sequence, it is also okay to define the strand whose nucleic acid sequence is first identified as the "sense strand” and its complementary strand as the "antisense strand”.
  • the "primer extension reaction” refers to a non-chain reaction in which a DNA polymerase extends a primer hybridized to a template nucleic acid of a limited length and terminates at the 5'-end of the template nucleic acid.
  • hybridizing oligonucleotide refers to a nucleic acid molecule capable of hybridizing by complementary binding to an amplification target miRNA molecule or a cDNA reverse transcribed from the amplification target miRNA molecule.
  • forward primer refers to a primer in which the direction (5' -> 3') of the corresponding primer coincides with the direction of the gene.
  • reverse primer means a primer in which the direction of the corresponding primer is opposite to the direction of the gene.
  • a plurality of target miRNAs to be detected refers to various miRNA molecules present in a sample obtained from a detection target.
  • sample is, for example, a cultured cell, protein, nucleic acid, or a biological drug containing the same, animal or human brain, eye, heart, intestine (intestine) , tissue, blood, plasma, serum from kidney, liver, lung, muscle, spleen, or testis ( serum), urine, saliva, sweat, semen, or mucus, but is not limited thereto.
  • the present invention is carried out as a method for simultaneous multiple detection of miRNA
  • An extension annealing site which is a nucleic acid sequence in which the remainder of the miRNA hybridizes with the reverse transcribed cDNA portion, except for the portion where the 3' end of each target miRNA and the reverse transcription primer hybridize, and an optional nucleic acid sequence reverse primer site (reverse) primer site) consisting of a plurality of extension primers;
  • It relates to a kit for simultaneous multiple detection of miRNA, comprising a plurality of double-labeled probes having the same nucleic acid sequence as the probing site of the reverse transcription primer.
  • the kit may further include a DNA polymerase, a dNTP (dATP, dCTP, dGTP and dTTP) mixture, a buffer solution, and distilled water.
  • a DNA polymerase e.g., a DNA polymerase, a dNTP (dATP, dCTP, dGTP and dTTP) mixture, a buffer solution, and distilled water.
  • dNTP dATP, dCTP, dGTP and dTTP
  • the kit may include at least one container including a compartmentalized carrier means for holding a sample, a container including a primer set and a probe for MVM detection, a container containing a buffer for a PCR reaction and a DNA polymerase, wherein the primer set comprises: It may include the nucleotide sequence, functional combination, and fragments thereof shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the probe may include the nucleotide sequence, functional combination, and fragment thereof described in SEQ ID NO: 9.
  • the carrier means is suitable for containing one or more containers, such as bottles and tubes, each container comprising independent components for use in the method of the invention, and a person skilled in the art will be able to store the required formulation in the container. can be easily distributed.
  • kit of the present invention may further include a user's guide describing optimal conditions for performing the reaction.
  • a handbook is a printout that explains how to use the kit, eg, how to prepare buffers, and suggested reaction conditions.
  • Instructions may include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit.
  • the guide may include information published or provided through an electronic medium such as the Internet.
  • the hybridization position and sequence of the reverse transcription primer, the extension primer, the forward primer, the reverse primer and the double-labeled probe for the detection of hsa-miR-21-5p are prepared according to the present invention.
  • a kit for simultaneous multiplex detection of miRNA was prepared by designing according to the detection method of
  • hsa-miR-21-5p and hsa-miR-16-5p were detected according to the miRNA simultaneous multiplex detection method shown in FIGS. 1 and 2, and as shown in FIG. 3, hsa-miR-21
  • the hybridization position and sequence of the reverse transcription primer, extension primer, forward primer, reverse primer and double-labeled probe were designed according to the detection method of the present invention to prepare a kit for simultaneous multiplex detection of miRNA.
  • Reverse transcription reagent usage sample volume (ul) final concentration 2X Reaction Buffer 10 1X Reverse Transcriptase (RTase) One 0.5 U dNTP mix (10 mM) 2 1 mM Reverse primer (100 ⁇ M) One 5 uM template One - Distilled water 5 - total volume 20 -
  • the hsa-miR-21-5p concentration was serially diluted to 1 ⁇ g to 1 pg, and reverse transcription reaction and real-time PCR reaction were performed using Bio-Rad CFX96 as in Table 3 and Table 4 conditions.
  • the reverse transcription reaction was performed at 37° C. for 60 minutes, and then reacted at 95° C. for 5 minutes to inactivate the reverse transcriptase.
  • Primer extension reaction and PCR were reacted at 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C, and then Ct values were measured.
  • kit for simultaneous multiplex detection of miRNAs of the present invention can specifically detect each miRNA in a sample in which a plurality of miRNAs are mixed.
  • the experiment was performed by dividing it into set A and set B, and the miRNA types and sequence information used in the experiment are shown in Tables 5 and 6 below.
  • the mimic miRNA mixed with the sample was used by sequentially diluting 10 7 to 10 9 ⁇ g, and the reverse transcription reaction and real-time PCR reaction were performed under the conditions of Table 3 and Table 4 in the same manner as in Example 1 using Bio-Rad CFX96. was performed.
  • the reverse transcription reaction was performed at 37° C. for 60 minutes, and then reacted at 95° C. for 5 minutes to inactivate the reverse transcriptase.
  • Primer extension reaction and PCR were reacted at 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C, and then Ct values were measured.
  • each miRNA was detected even in a sample in which a plurality of miRNAs were mixed. It was confirmed that it can be specifically detected and quantified.

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Abstract

La présente invention concerne un procédé de détection simultanée de multiples miARN et un kit de détection de miARN l'utilisant et, plus particulièrement, un procédé de détection simultanée de multiples miARN, le procédé comprenant les étapes suivantes : synthèse d'une pluralité d'ADNc avec des amorces inverses s'hybridant respectivement avec des parties 3' terminales d'une pluralité de miARN cibles ; synthèse d'une pluralité de matrices de PCR par extension dans des directions opposées à l'aide d'une pluralité d'amorces d'extension s'hybridant respectivement avec des parties 3' terminales de la pluralité des ADNc transcrites en sens inverse ; et amplification de la pluralité de matrices de PCR avec des amorces sens et antisens spécifiques de celles-ci et détection des miARN avec une pluralité de sondes à double marquage. Le procédé de détection simultanée de multiples miARN selon la présente invention présente non seulement une réactivité élevée dans l'étape de transcription inverse en raison de l'utilisation d'amorces de transcription inverse plus courtes que celles dans des kits de détection de miARN disponibles dans le commerce, ce qui permet une synthèse précise de l'ADNc, mais permet également des amorces de transcription inverse respectives pour la détection d'une pluralité de miARN, des amorces d'extension, des ensembles d'amorces pour l'amplification par PCR, et des sondes à double marquage pour la détection de miARN pouvant être facilement conçues, permettant ainsi une analyse multiplex pour détecter simultanément une pluralité de miARN.
PCT/KR2021/002915 2020-03-09 2021-03-09 Procédé de détection simultanée de multiples miarn et kit de détection de miarn l'utilisant Ceased WO2021182850A1 (fr)

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WO2023027444A1 (fr) 2021-08-27 2023-03-02 주식회사 엘지화학 Système et procédé de prédiction de propriétés physiques d'un matériau multicouche

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US20140295434A1 (en) * 2011-03-08 2014-10-02 Biovue Technology Ltd. Detection method of micro-rna with high specificity
US20140045188A1 (en) * 2011-04-20 2014-02-13 Shenzhen University Primer and method for quantitative assay of microrna and application of same
KR20160098097A (ko) * 2015-02-09 2016-08-18 (주) 하임바이오텍 마이크로알앤에이 검출용 키트 및 방법
KR20180010549A (ko) * 2016-07-21 2018-01-31 (주) 하임바이오텍 알앤에이 검출용 키트 및 방법

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Publication number Priority date Publication date Assignee Title
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