KR101887742B1 - LAMP based method for detecting miRNA - Google Patents

Abstract

This disclosure discloses methods, kits, and compositions that are capable of rapidly detecting the presence of miRNAs in LAMP-based samples. The method according to the present invention comprises cloning the miRNA to be detected into plasmid and using it in a LAMP reaction with the sample, substituting one of the potential miRNAs present in the sample with one of the acceleration primers, The reaction proceeds more rapidly and the presence of a specific miRNA can be detected quickly and easily.

Description

[0001] LAMP-based miRNA detection method [0002]

The present invention relates to a technique for rapidly analyzing the presence of a specific miRNA in a sample using the LAMP method.

MicroRNAs (miRNAs) are noncoding RNAs in cells that are short single-stranded nucleotides of 20-25 nucleotides and are known to contain about 1,500 microRNA genes in humans.

The production of microRNAs is derived from pri-miRNA, a precursor that is transcribed by RNA polymerase II (II). The pri-miRNA has a cap at the 5 'end and a poly A tail at the 3' end. The pri-miRNA is cleaved by ribonuclease III (Drosha) and the binding protein DGCR8 (DiGeorge critical region 8) into hairpin-like pre-miRNA. The intermediate, pre-miRNA, is transported to the cytoplasm by exportin5 Ran-GTP, a nuclear export factor, and double-stranded microRNAs composed of 20-25 nucleotides by the second cleavage enzymes Dicer and TRBP (transactivating response RNA binding protein) . Among the double strands, one strand is disassembled and combined with Ago (Argonaute) to form a RNA-induced silencing complex (RISC). TRBP induces the binding of Ago to microRNA, allowing the microRNA to regulate the expression of the target gene. In general, microRNAs bind to the 3 'untranslated region (3' UTR), which is not translated into a protein, to lower the stability of the mRNA or lower the translation rate to inhibit the expression of the target gene. MicroRNAs play an important role in basic life phenomena such as development, differentiation and cell proliferation by controlling more than 30% of total genes.

Thus, studies on cancer have focused on genes encoding proteins, but recently it has been found that genes that do not code for proteins also play an important role in cancer formation. In general, microRNAs are located in the genomic region associated with cancer, such as the minimal amplification region, the loss of heterozygocity, the fragile site, and the truncation site within or near the tumor gene or tumor suppressor gene have. There is increasing evidence that the expression of microRNAs associated with cancer or tumor suppressor function in human tumors is abnormally increased. In tumors, microRNAs can act as tumors (eg, miR-17-92) through a variety of mechanisms, including inhibition of tumor suppressor genes, increased expression of tumor genes, cell cycle and death control (eg, miR-17-92) It may also show a tumor suppressive effect (eg let-7). The potential value of microRNAs as biological markers of cancer prognosis and prediction by microRNAs appears in recent research by Schetter et al. In this study, we compared the patterns of microRNA expression in the colon adenocarcinoma and surrounding normal tissues, and some microRNAs showed prognostic value (Schetter AJ, et al. 2008. MicroRNA expression profile associated with prognosis and therapeutic outcome in colon adenocarcinoma, JAMA 299: 425-36).

 Therefore, there is a need for techniques to accurately and rapidly detect microRNAs. In the past, analysis was performed using nothern blotting, microarray, and beadbased platform. However, due to its short length, similar nucleotide sequences exist and are not satisfactory in terms of sensitivity and selectivity due to low microRNA expression levels (Waldman SA and Terzic A. 2008. MicroRNA signatures as diagnostic and therapeutic targets. Clin Chem 54: 943-4 ).

Recently, various methods for detecting microRNAs have been proposed. Conventional methods such as nanoparticle-labeling, modified invader assay, ribozyme-based methods, conjugated polymer-based methods, bioluminescence detection and electrochemical detection have improved sensitivity to detect microRNAs, but most approaches are based on probe modification, And the like. Recently, enzyme-based quantitative RT-PCR, RCA (branched rolling circle amplification), and LCR (ligase chain reaction) methods have been reported, but there are disadvantages such as high cost and complicated steps et al. 2011. One-step ultrasensitive detection of microRNAs with loop-mediated isothermal amplification (LAMP). Chem Commun 47: 2595-2597).

Therefore, it is necessary to develop a quick and convenient detection method based on new technology.

WO2015103293 (published July 9, 2015) Systems, compositions and methods for detecting and analyzing micro-RNA profiles from a biological sample.

The present invention seeks to provide a rapid and accurate miRNA detection method based on LAMP.

In one embodiment, the present invention provides a miRNA detection method based on LAMP (Loop-mediated isothermal amplification), wherein the miRNA present in the sample functions as an acceleration primer and the amplification rate is increased compared to a sample not containing miRNA, Of the miRNA-containing sample.

In one embodiment, a method according to the present invention comprises the steps of: a) providing a sample of interest that requires miRNA detection; b) cloning a polynucleotide of the DNA sequence corresponding to said miRNA into a plasmid; or providing said plasmid in which said polynucleotide has been cloned; c) providing LAMP primers of four F3, B3, FIP and BIP except for the acceleration primers LB and LF, which bind to said plasmid, wherein said four primers are upstream of said cloned polynucleotide Or F3, FIP, microRNA to be detected, BIP and B3 in the downstream sequence, and does not bind to the cloned polynucleotide; d) performing a LAMP reaction for a predetermined period of time in the presence of the target sample, the plasmid and the four kinds of primers, the LAMP reaction is carried out in the same manner for the positive and / or negative control samples, And e) analyzing the result of the LAMP reaction to determine whether the target sample contains the miRNA. When the plasmid is amplified for the predetermined time, the target sample contains a predetermined miRNA .

In one embodiment according to the present disclosure, the analysis of the LAMP reaction results can be determined through measurements using one or more of colorimetric measurements or turbidity of the reactants, but is not limited thereto.

In the method according to the present invention, the LAMP reaction is performed with a time to distinguish the difference in the rate of amplification of the sample containing the miRNA and the sample not containing it, and in the embodiment, the predetermined time is performed for 30 minutes to 35 minutes .

In another embodiment, the invention also provides a plasmid in which the polynucleotide of the DNA sequence corresponding to the miRNA to be detected is cloned; And a LAMP-based miRNA detection kit comprising F3, B3, FIP, and BIP bound to the plasmid but not to the polynucleotide.

In another embodiment, the invention also provides a plasmid in which the polynucleotide of the DNA sequence corresponding to the miRNA to be detected is cloned; And a composition for detecting LAMP-based miRNA comprising F3, B3, FIP, and BIP that bind to the plasmid but do not bind to the polynucleotide.

This disclosure discloses methods, kits, and compositions that are capable of rapidly detecting the presence of miRNAs in LAMP-based samples. The method according to the present invention uses a clone of the miRNA to be detected in a plasmid, which is used in a LAMP reaction together with a sample, and replaces potential miRNAs present in the sample with one of the acceleration primers, The reaction proceeds rapidly and the presence of specific miRNAs can be detected quickly and easily.

Figure 1 (a) is a schematic representation of the location of each strand region of a target nucleic acid for which a LAMP primer is designed.
FIG. 1B schematically shows the position of each primer and the position of the cloned microRNA sequence in the plasmid into which the DNA corresponding to the microRNA has been inserted to replace the LB primer.
FIG. 2A is a table showing the combination of primers used in the LAMP reaction experiment in order to analyze the difference in the LAMP reaction rate depending on the presence or absence of the acceleration primers LB and LF used in the LAMP reaction.
FIG. 2B is a result of performing the LAMP reaction using the primer set of FIG. 2A using different concentrations of template, and FIG. 2B is a color analysis result and FIG. 2B is a table summarizing the color analysis results.
FIG. 3A shows the combination of the primers used in the LAMP reaction experiment in order to analyze the difference in the LAMP reaction speed due to the addition of the acceleration primer of the LB primer depending on whether the F3 and B3 primers were added or not based on the two kinds of primer sets FIP and BIP Respectively.
FIG. 3B is a result of performing LAMP reaction using the primer set of FIG. 3A at different reaction times. The color analysis results are shown in the upper part of FIG.
FIG. 4A is a schematic diagram illustrating a process of synthesizing a DNA corresponding to a target miRNA to be detected and cloning it into a plasmid according to an embodiment of the present invention.
Figure 4b shows the location and sequence of the primers designed around the target miRNA in the plasmid according to Figure 4a, indicating that LB was replaced by microRNA.
FIG. 5 shows the result of detection of microRNA in a sample by the LAMP reaction using the plasmid of FIG. 4A. After addition of 1 fg, 0.5 fg, and 0.1 fg of plasmid DNA containing the miR-135 base sequence, the microRNAs acted as acceleration primers in the miR-135 positive control after completion of the reaction at 63 ° C for 30 minutes, resulting in rapid rate amplification Purple to blue response occurred, but no negative reaction was observed at 30 minutes because the amplification rate was slow because no microRNA could act as an acceleration primer in the same negative control. These results indicate that the difference in the amplification rate of the LAMP reaction can determine the presence or absence of the microRNA.

The present invention is based on the discovery that miRNAs can be used to detect specific miRNAs quickly and simply using LAMP (Loop Mediated Isothermal Amplification) as a kind of acceleration primer.

LAMP was amplified by strand amplification using a nucleic acid amplification method (Notomi, T. et al., 2000. Loop-Mediated Isothermal Amplification of DNA, Nucleic Acids Res 28, E63) capable of amplifying a target nucleic acid with high sensitivity and specificity under isothermal conditions Displacement activity and a set of at least 4 to 6 primers specifically designed at various sites of the target nucleic acid are used (Korean Patent No. 612551; Nagamine, K. et al., 2002. Accelerated reaction by Loop mediated isothermal amplification using loop primers, see Mol Cell Probes 16, 223-9). Four types of primers are used in the standard LAMP reaction: FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer) and B3 (backward outer primer). A total of six primers can be used for accelerated LAMP, including LF (Loop F) and LB (Loop B) as acceleration primers.

As shown in Figure 1A, the set of primers used in the LAMP as described above is designed for six distinct regions (F3, F2, F1, B1c, B2c and B3) on the target nucleic acid, and F3 and B3 are designed for two The FIP and BIP are hybrid primers consisting of two inner primers, the FIP consists of the F1c sequence and the F2 sequence, and the BIP consists of the B1c and B2 sequences.

The LAMP reaction largely involves an initiation structure production step and a cycling amplification step, and does not include a step of denaturing with a single strand unlike PCR. Initiation structure In the production phase, F2 of FIP binds to the F2c region and initiates elongation. The BIP also goes on. The F3 primer binds to the F3c region and initiates DNA synthesis by the strand displacement method, releasing the DNA strands elongated in FIP as they are exchanged. The single strand thus released forms a loop at its 5 'end through F1 / F1c binding, and the synthesis reaction proceeds also in the same manner as the BIP and B3 primers, and a single-stranded nucleic acid molecule . DNA synthesis starts from the 3 'end of the F1 region, and the cycling step proceeds.

The LB or LF primer, which is an acceleration primer, binds to the single-stranded site of the dumbbell structure described above and binds the loop between B1 and B2 or the loop between F1 and F2. LB or LF primers are used and the origin of DNA synthesis is increased, which results in faster reaction rate. In the LAMP reaction, six rings are usually formed in the process, and only two rings are used as the starting materials for synthesis. When LB and LF primers are used, all six rings are used as the starting materials for synthesis, The amount of DNA is increased.

The principles and principles of the present invention are described in the above-cited notations of the features and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) al., < / RTI > 2002.

As shown in FIGS. 1 and 2, a microRNA detection system according to the present invention comprises a vector or plasmid containing a polynucleotide of a DNA sequence corresponding to a microRNA to be detected, together with a sample to be tested, (LB or LF). When the microRNA is present in the sample to be examined, it acts as a kind of acceleration primer (LB or LF), and the synthesis reaction proceeds more rapidly than the case where it is not. It is the principle that the target microRNA can be detected.

Thus, in one aspect, the invention provides a method of detecting LAMP-based miRNAs, comprising: a) providing a sample of interest that requires miRNA detection; b) cloning a polynucleotide of the DNA sequence corresponding to said miRNA into a plasmid; or providing said plasmid in which said polynucleotide has been cloned; c) providing LAMP primers of four F3, B3, FIP and BIP, except for the acceleration primers LB and LF, which bind to said plasmid, wherein said four primers are selected from the group consisting of F3, FIP, detection target microRNA, BIP and B3 in sequence upstream or downstream, but not in the cloned polynucleotide; And d) performing a LAMP reaction for a predetermined period of time in the presence of the target sample, the plasmid, and the four kinds of primers, and the LAMP reaction is performed in the same manner for the positive and / or negative control samples e) analyzing the result of the LAMP reaction to determine whether the target sample contains the miRNA, wherein when the plasmid is amplified for the predetermined time, the target sample is judged to contain a predetermined miRNA do.

In the method according to the present invention, the "miRNA" or "miR" or "microRNA" or "microRNA" to be detected is a noncoding RNA present in a cell, which is a single strand of 20-25 nucleotides in length nucleotide) and about 1500 microRNA genes are known to be present in humans. In general, microRNAs bind to the 3 'untranslated region (3' UTR), which is not translated into a protein, to lower the stability of the mRNA or lower the translation rate to inhibit the expression of the target gene. MicroRNAs play an important role in basic life phenomena such as development, differentiation and cell proliferation by controlling more than 30% of total genes. Thus, miRNA detection can be useful in a variety of applications.

The miRNA in which the method according to the present invention is used is not particularly limited and is not limited as long as it functions as an acceleration primer at a length of about 20 to 25 nt. Thus, various miRNAs that require detection for a particular purpose can be detected according to the methods herein.

As used herein, the term "primer" refers to a single-stranded oligonucleotide used in a LAMP reaction in which a nucleotide is extended by addition of a covalent bond at its 3 'end in a nucleic acid amplification or synthesis reaction using a polymerase .

The LAMP primers used in the method according to the present invention are basically four kinds of F3, FIP, B3 and BIP primers, and according to the above-mentioned principle, the polynucleotides corresponding to the target miRNAs are cloned according to the present invention 0.0 > plasmid < / RTI > employed in the method.

Specifically, the primer according to the present invention is characterized in that the sequence of F3, F2, LF, F1c, B1c, B2, B3 in the upstream or downstream region thereof as shown in Fig. 1b is determined on the basis of the site where the polynucleotide corresponding to the miRNA to be detected is cloned, And F3, FIP, BIP, and B3 in this order, and the polynucleotide corresponding to the miRNA to be detected acts as an LB binding site, in which miRNAs in the target sample bind to LB And functions as a primer.

In the method according to the present invention, it is preferable that the acceleration primer exclude both LB and LF. When only one sample is excluded, the difference in the reaction rate between the sample to which the microRNA is added and the sample to which the microRNA has not been added is shorter than about 5 minutes, so that the distinction may be difficult. In Fig. 2a, lane 1 and lane 2 are detected in the case of LF only, since the +/- difference of LB can be regarded as a difference depending on whether a microRNA sample is present. Therefore, it is more accurate to observe the results with the difference in reaction rate depending on the presence or absence of microRNA after excluding both LF and LB.

However, the use of one of the accelerating primers is not excluded, and it is possible to control the condition, for example, by adjusting the concentration of the used plasmid to a low concentration, for example, 95% or less, for example, 90 The presence or absence of miRNA can be discriminated at the amplification rate by using the plasmid at a concentration of not more than 80%, for example, not more than 80%, for example, not more than 70%, for example, not more than 60%, for example, not more than 50% It is also possible to use only one of the two acceleration primers.

Therefore, a primer design as long as the above-mentioned conditions are satisfied can be designed in consideration of the matters to be adopted in the general LAMP method. Generally, the primer design can be designed in consideration of Tm value, double structure, stability of each primer end, and GC content. The Tm values are in the range of 60 占 to 68 占 폚 or 64 占 폚 to 66 占 폚 for the F1c and B1c regions and 55 占 폚 to 63 占 폚 or 59 占 폚 to 61 占 폚 for the F2, B2, F3 and B3 regions, Typical miRNA sizes range from 20-24 nt. Isothermal amplification features are suitable in the range of 40-60% for primer design and may range from 64-66 ° C, unless the proportion of abnormally high GC is exceeded.

The interval between the primers used in the method according to the present invention is such that the size of the amplification product is about 200 to 300 nucleotides, the polynucleotide having the DNA sequence corresponding to the miRNA to be detected is sandwiched, and the two outer primers F3 and B3 And if the length is increased, it can be increased considering other primer designs. The distance from the 5 'end of the F2 region to the 5' end of the B2 region is designed to be about 120 to 180 nucleotides, and the distance from the 5 'end of the F2 region that forms the loop to the 5' To 60 nucleotides, and the opposite B2 and B1 intervals are designed to be the same. The spacing between F3 and F2, F2 and F1 is designed so that the B3 and B2 spacings opposite 0 to 60 nucleotides are the same.

The primers employed in the method of the present invention can be selected from commercially available primer design software such as Primer Explorer 4 (Fujitsu, Japan), LAMP Assay Versatile Analysis (LAVA) and LAMP Designer PREMIER Biosoft) can be used.

The length of the primer according to the present invention can be appropriately determined in consideration of the length of the final amplification product, for example, a minimum of 10 nucleotides, a minimum of 15 nucleotides for F3 and B3, a minimum of 30 nucleotides for FIP and BIP, At least 15 nucleotides for LF and LB, but not limited thereto.

Specific binding or hybridization herein means that a particular nucleic acid molecule or primer binds only to the target nucleic acid without substantial binding to a nucleic acid other than the non-target nucleic acid. The method for preparing a primer according to the present invention can specifically bind to a nucleic acid containing a target single nucleotide polymorphism or a complementary sequence thereof under high stringency conditions, and is capable of specifically identifying a primer sequence length, a buffer, , Magnesium concentration, and reaction temperature.

As described above, the microRNA in the sample to be tested generally has a short single strand consisting of 19-25 nucleotides and plays the same role as LB or LF, which is a conventional accelerating primer. Therefore, when the sample to be examined contains microRNA, The microRNA can be detected by the principle that the amount of the nucleic acid synthesized for a predetermined time is different as compared with the case where the microRNA is not contained. It is therefore important to perform the reaction for a time sufficient to amplify the amount of amplified nucleic acid in a sample containing and not containing a microRNA acting as an acceleration primer.

In one embodiment, the reaction conditions are set so that amplification can be detected at 63 ° C for 30 minutes to 35 minutes by adjusting the concentration of the plasmid DNA containing the microRNA to be detected.

In another aspect, the present disclosure is directed to a composition or kit for detecting microRNA comprising a set of primers used in the method according to the present invention.

The methods, compositions or kits according to the present invention can be used for miRNA detection in various samples. The sample includes biological samples such as, for example, blood, urine, feces or body fluids, or isolated tissues, cells or extracts derived therefrom. Methods for treating samples are known in the art. For example, a sample can be collected, treated with Proteinase K, and subjected to LAMP reaction by hot water extraction. These biological samples may also be those obtained directly by the usual method of obtaining the samples immediately before the test or stored separately in advance.

In another embodiment, the sample according to the present invention can be used as a sample in which a nucleic acid separated from the above-mentioned sample is used. Separation is separation from the sample containing the nucleic acid, including separation into various purity.

Compositions or kits for LAMP reactions using primer sets according to the present invention may further comprise dNTPs, buffers, magnesium and DNA polymerases. It may further comprise a substance such as betaine or DMSO to enhance the reaction.

Magnesium is used in the form of a salt such as magnesium acetate, magnesium chloride or magnesium sulfate.

The buffer may be, for example, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer or Tricine buffer.

The DNA polymerase that can be used in the LAMP reaction according to the present invention is a polymerase derived from a thermophilic microorganism, in particular, a polymerase lacking 5 '->3' exonuclease function. Non-limiting examples include Bacillus stearothermophilus , Bst, DNA polymerase; Thermus, thermophilus , Tth, DNA polymerase; Thermus aquaticus , Taq, DNA polymerase; Thermococcus litoralis DNA polymerase; Pyrococcus furiosus , Pfu, DNA polymerase; And Bacillus caldotenax DNA polymerase.

Also, instead of dNTP, a nucleotide analog may be used as the reactant. Nucleotide analogues can be modified or co-polymerized with natural nucleotides in a template-induced DNA synthesis process that is not altered or found in nature. Examples of nucleotide-based analogues that can be polymerized through such Wasson Creek-based pairing include, for example, substituted purines or pyrimidines, deazapurines, methylpurines, methylpyrimidines, aminopurines, aminopyrimidines, But are not limited to, purine, thiopyrimidine, indole, pyrrole, 7-deazaguanine, 7-methylguanine, hypoxanthine, shodocytosine, shido isocytosine, isocytosine, isoguanine, , 6-thioguanine, nitropyrrol, nitroindole, and 4-methylindole. Nucleotides comprising a substituted deoxyribose analog include substituted or unsubstituted arabinose, substituted or unsubstituted xylose, and substituted or unsubstituted pyranose. Nucleotides containing phosphate ester analogs include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroamidate, phosphorocellinoate, phosphoroanilothioate, phosphoroanilide, phosphoroamidate, boron Phosphate, phosphotriester, and alkylphosphonates such as methylphosphonate.

The LAMP reaction is carried out at a temperature suitable for DNA polymerase activity. The reaction temperature can be determined without difficulty in view of the nucleotide sequence of the target enzyme and the enzyme used in the reaction. The reactants are then reacted for a predetermined time suitable for detection of miRNA as described above. Those skilled in the art will be able to determine the reaction time in consideration of the reaction conditions, and can be determined, for example, at a time of 20 minutes to 50 minutes, but the time may vary depending on the amount of the target nucleic acid contained in the sample. For example, the reaction time can be increased to increase the sensitivity.

LAMP assays according to the present invention can be analyzed in a variety of analytical formats, for example, in a liquid phase reaction or in a state where some components are adsorbed to a solid substrate.

A method or kit according to the present invention, comprising a primer set, is used for LAMP analysis using any biological sample to discriminate single nucleotide polymorphisms, the amplified product having a sequence corresponding to the molecule used as the template, Can be analyzed by a variety of methods known in the art.

The amplification reaction product after amplification can be detected in various ways, for example, by color change of reaction solution, turbidity change due to DNA synthesis, fluorescence and / or electrophoresis, and the detected product is positive and / or Is compared with the product of the negative control sample and used for quantitative or qualitative analysis.

In one embodiment, the amplification product according to the present invention can be detected by color change of the reaction solution according to DNA synthesis. In this case, the reaction solution may contain an appropriate indicator, and HNB (hydroxy naphthol blue) may be used as a dye whose color changes in response to the magnesium ion concentration in the reaction solution to produce a product of the positive and / or negative control sample , Clostridium perfringens can be quantitatively or qualitatively analyzed. Especially, detection is possible in the inside, and convenience is enhanced.

In other embodiments, the amplification product can be detected by a direct or indirect method. In a direct method, a detectably labeled primer can be used, and the nucleic acid amplification is detected through a signal emitted through a primer labeled with a specific binding to a nucleic acid, and real-time detection is possible. In indirect detection methods, labeled probes capable of binding to amplified nucleic acids can be used. Means a substance capable of emitting a detectable signal using any suitable method such as spectroscopic, optical, photochemical, biochemical, enzymatic, electrical and / or immunochemical methods. Means a labeled substance. Such materials include, for example, fluorescent moieties, chemiluminescent moieties, bioluminescent moieties, magnetic particles, enzymes, substrates, radioactivity and chromophore materials.

In one embodiment, the label for detection includes, but is not limited to, a compound capable of generating or erasing a detectable fluorescence, chemiluminescent, or bioluminescent signal such as light emission, light scattering, light absorbing material, For example, Garman A., Non-Radioactive Labeling, Academic Press 1997. Fluorescent materials include, but are not limited to, fluorescein (e.g., U.S. Patent 6,020,481), rhodamine (e.g., U.S. Patent 6,191,278), benzophenoxaphone (e.g., U.S. Patent 6,140,500), donors and receptors Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (for example, US Pat. No. 5,945,526) and cyanine (for example, WO1997-45539) , FluorX (Amersham), Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY- TRX, Cascade Blue, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, Tetramethylrhodamine, and / Lt; RTI ID = 0.0 > fluorescence < / RTI > The fluorescent dye is 6-carboxyfluorescein; 2 ', 4', 1,4, -tetrachlorofluorescein; And 2 ', 4', 5 ', 7', 1, 4-hexachlorofluorescein. In one embodiment, SYBR-Green, 6-carboxyfluorescein ("FAM"), TET, ROX, VIC or JOE are used as fluorescent labels. In one embodiment, a probe labeled with two fluorescent materials, a reporter fluorescent substance and an erasing fluorescent substance, is used. In this case, a fluorescent substance is used in which a fluorescent substance emits a spectrum having a distinguishable wavelength. Also, markers may include compounds capable of enhancing, stabilizing, or affecting the binding of nucleic acids, such as intercalators, minor groove associations and crosslinkable functional groups, including epidymium bromide and SYBR-Green, , But are not limited to, Blackburn et al., Eds. "DNA and RNA Structure" in Nucleic Acids in Chemistry and Biology (1996).

In addition, when it is necessary to detect non-specific amplification by non-specific priming of a primer, a non-specific nucleic acid binding agent such as acidium bromide or picogreen is used in a control reaction not containing a template to generate nonspecific total amplification The amount of product can be determined.

The kit according to the present invention can provide the components used in the LAMP reaction as described above except for the template and the above-described four primer sets separately or in one tube. The kit according to the present invention further comprises a positive control, a negative control, and instructions for use. As a negative control, a sample containing no miRNA to be detected, and a positive control group may be a sample containing miRNA to be detected.

Hereinafter, the present invention will be described in detail with reference to the following examples, which are intended to be illustrative and not limit the scope of the present invention in any way.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, unless otherwise indicated. Further, a more detailed description of common techniques can be found in Molecular Biotechnology: (Bernard et al., ASM press 2014); Molecular Cloning: A Laboratory Manual, 4th Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2012); Short Protocols in Molecular Biology, 5th Ed. (Ausubel F. et al. Eds., John Wiley & Sons 2002).

Example  One: MicroRNA  Sample Preparation

MiR-135 was used as an example to verify the method according to the present invention.

Total RNA was isolated from tissues obtained from lung cancer cell lines using Trizol® (Life Technology, USA) and Qiagen RNA extraction kit according to the manufacturer's instructions.

Example  2: The desired microRNA Sequence of  Plasmid Cloning

As a plasmid, TOP blunt vector (Enzynomics, Korea) was used and miR-135 sequence information was obtained from miRBase (http://www.mirbase.org). As shown in Fig. 4, polynucleotides were synthesized by adding a miR-135 base sequence and the same restriction endonuclease site sequences as the vector at the anterior and posterior sides, respectively, and synthesizing them with the outside (Genentech Co., Ltd.). Specifically, BamH1 (New England Biolabs) and XhoI (New England Biolabs) restriction enzymes were treated in the same manner with the polynucleotide inserted with the plasmid. Then, miR-135 gene having the same restriction enzyme site was inserted into a vector obtained by digesting both ends with the restriction enzyme, and confirmed by sequence analysis.

Example  3: LAMP primer  design

A total of six sets (FIP, BIP, LF, LB, F3, B3) were designed for the vector, the sequence of which is shown in Table 1 and in Figure 4b.

Specifically, the vector sequence and miR-135 gene were inserted using the ClustalX multiple sequence alignment program and the results of the sequence analysis commissioned by Solgent were aligned. The site where the mir-135 gene was inserted was first selected as the LB primer site, and the remaining primer was designed around the LB primer.

[Table 1]

Example  4 Acceleration Primer  Analysis of reaction rate difference according to presence

In order to confirm the difference according to the presence or absence of microRNAs which play the same role as the accelerating primers (LF, LB), the present inventors used four primer sets (F3, B3, FIP, BIP) In order to confirm the amplification speed difference.

The total volume of the LAMP reaction was 25 μl. Four primers (F3, B3, FIP, BIP) and five primers (F3, B3, FIP, BIP, LF / F3, B3 and FIP , BIP, LB) or 6 primers (F3, B3, FIP, BIP, LF, LB). The composition and components used in the reaction were the same except for the number of primers. To a total of 25 μl was added 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO4, 1.4 mM dNTPs, 1 × ThermoPol reaction buffer Biolabs), 8 U Bst DNA polymerase (New England Biolabs), 120 μM HNB (Sigma) and plasmid DNA inserted with mir-135 sequence. FIGS. 2 and 3 were carried out by adding LB primer synthesized in Genotech instead of mir-135 RNA sample.

The LAMP reaction was carried out at 63 ° C for 45 minutes, and the Bst DNA polymers were inactivated at a temperature of 80 ° C or higher. Therefore, the reaction was terminated at 80 ° C for 5 minutes.

The results are shown in FIG. As shown in this figure, LF and LB additions (Lane 1), LF only (Lane 2), and LB only addition (Lane 3) were compared when four primers were used alone (Lane 4) The difference in amplification speed between 10 min and 15 min was observed.

The result of LAMP reaction of 5 primer sets (F3, B3, FIP, BIP, LF or LB) and results of 6 primer sets (F3, B3, FIP, BIP, LF and LB) Is short. However, the results of the LAMP reaction of the four primer sets (F3, B3, FIP, BIP) and the results of the five primer sets (F3, B3, FIP, BIP, LF or LB) Therefore, it was found to be suitable for microRNA detection.

In addition, based on the two sets of FIP and BIP, the difference in the LAMP reaction rate of the primer of the LB primer according to whether the F3 or B3 primer was added was analyzed. The results are shown in FIG. 3. Whether or not the addition of F3 and B3 does not significantly affect the reaction rate, and when the LB primer is added (Lane 2, Lane 4) or not (Lane 1, Lane 3) shows a 15 minute LAMP reaction rate difference. Therefore, in the case of confirming the result between 30 minutes and 40 minutes of the LAMP reaction time, it is detected in the case of the sample containing the microRNA having the same role as the LB primer, and the sample which does not contain any microRNA or microRNA other than the desired microRNA It is not detected.

Example  5: through LAMP reaction miRNA  detection

The LAMP reaction was performed in a total volume of 25 μl and was performed using four primers except LF and LB. The composition and components used in the reaction are as follows. A total of 25 μl was added with 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.4 M betaine, 10 mM MgSO4, 1.4 mM dNTPs, 1 × ThermoPol reaction buffer (New England Biolabs) (New England Biolabs), 120 μM HNB (Sigma) and 1 μg, 0.5 μg, and 0.1 μg of the plasmid DNA inserted with miR-135 gene prepared in Example 2 were added. The same amount of miR-135 positive sample and miR-135 negative sample prepared in Example 1 were added in the same amount of 50 ng each. The LAMP reaction was inhibited at 63 ° C for 30 minutes, and the Bst DNA polymerase was inactivated at 80 ° C or higher. Therefore, the reaction was terminated at 80 ° C for 5 minutes.

Amplification was performed by color analysis as follows. Color analysis was performed by adding HNB (hydroxy naphthol blue) dye to the reaction solution. HNB acts as an indicator (indicator) of Mg ^{2 +} ion concentration. Mg ^{2 +} ion is not the case, looks light blue, Mg ^{2 +} ions, ttinda purple if. Therefore, it is present in purple before the reaction, and as the DNA synthesis reaction occurs, the concentration of Mg ^{2 +} ion decreases and the color changes from purple to blue. The change in color is a positive reaction indicating that the nucleic acid has been amplified and qualitative and quantitative analysis is possible by measuring the absorbance at a wavelength of about 650 nm. The method according to the present invention is advantageous in qualitative and quantitative analysis when absorbance is measured at a wavelength of 650 nm using a spectrophotometer.

The results are shown in FIG. When miR-135 plasmid DNA was added at 1, 0.5, and 0.1 fg, miR-135 positive sample was added at 58 ℃ for 30 min. Reaction occurred but the amplification rate was slow when the negative sample was added at the same concentration. This indicates that the difference in the amplification rate of the LAMP reaction can determine the presence or absence of the microRNA.

Claims (5)

A particular miRNA detection method based on Loop-mediated isothermal amplification (LAMP)
a) providing a sample of interest that requires specific miRNA detection;
b) cloning a polynucleotide of the DNA sequence corresponding to said miRNA into a plasmid; or providing said plasmid in which said polynucleotide has been cloned;
c) Providing LAMP primers of F3, B3, FIP and BIP with four kinds of primers that bind to the plasmid but do not bind to the cloned polynucleotide except LB and LF which are acceleration primers, Binding F3, FIP, specific miRNA to be detected, BIP and B3 in the upstream or downstream thereof on the basis of the polynucleotide cloned into the plasmid,
d) performing a LAMP reaction in the presence of the target sample, the plasmid and the four kinds of primers for a predetermined time, wherein when the specific miRNA is present in the sample, the specific miRNA acts as an LB primer, The LAMP reaction is optionally performed in the same manner for the positive and / or negative control samples,
e) analyzing the result of the LAMP reaction to determine whether the target sample contains the specific miRNA, wherein when the plasmid is amplified for the predetermined time, the specific miRNA contained in the sample is an LB primer Wherein the subject sample is determined to comprise the specific miRNA.
The method according to claim 1,
Wherein the analysis of the LAMP reaction results is determined by measurement using at least one of a color change measurement of the reactants or turbidity.
The method according to claim 1,
Wherein the predetermined time is from 30 minutes to 35 minutes.
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