Patent Application
20180346999
The invention relates to an isothermal-based dual functional oligonucleotide containing quencher and reporter dye for an isothermal nucleic acid amplification and a method for nucleic acid amplification and measurement using the same, more specifically an use of isothermal-based dual functional oligonucleotide measuring the presence or absence of the nucleic acid amplification at each of the reaction times through a real-time fluorescence monitoring and detecting the present or absence of the nucleic acid amplification reaction of the reaction product through the measurement of amount of fluorescence at the end-point step, and nucleic acid amplification method and measurement methods by using the same.
A nucleic acid amplification technique is a technique which is mainly used in a molecular biology and biotechnology fields, which can detect and analyze a small amount of nucleic acid. PCR (Polymerase chain reaction) technology analyzing DNA/RNA using a thermostable enzyme is the most widely used method for the nucleic acid amplification, wherein a double strand DNA is denatured into a single strand DNA; then a temperature is lowered to link a primer to the single strand; the Taq polymerase (thermostable enzyme) is extended to the double strand DNA; and the said procedures are repeated.
A real-time PCR method is a method that a nucleic acid is detected according to a luminescence intensity of fluorescence during PCR procedure, wherein a reporter dye is attached at oligonucleotide 5′ end having a complementary sequence corresponding to the PCR reactant intermediate position; a single strand of probe is linked to the complementary sequence during the PCR reaction by the 5′→3′ exonuclease activity of the Taq polymerase by adding a single strand of probe wherein a quencher is attached a 3′ end; the probe is hydrolyzed from 5′ end by the extension reaction of Taq polymerase to make the reporter dye to away from the quencher and thus to emit the florescence and thus mark the amount of fluorescence by the fluorescence detector of the real time PCR equipment. This is a technology which is widely used in the diagnostic field such as a pathogen detection, mutation detection, etc. since a simultaneous multiple-detection detecting two or more of targets can be made by varying the fluorescence detector of the real-time PCR equipment, and has disadvantages that the expensive real-time PCR equipment and skilled technicians are needed.
Isothermal amplification methods detecting DNA/RNA under an isothermal condition without an expensive real-time PCR equipment and detecting nucleic acid within a faster time than PCR method are being developed. For a RNA amplification, there is Transcription Mediated Amplification (TMA), and Nucleic Acid Sequence-Based Amplification (NASBA) methods using an enzyme such as RNA polymerase, reverse transcriptase, and Rnase H, and the Strand Displacement Amplification (SDA) is the method in which the exonuclease-deficient DNA polymerase displaces one existing strand so as to synthesize the double strand of nucleic acid, and Nicking and Extension Amplification Reaction (NEAR) similar to this is the method using a nicking enzyme instead of a restriction enzyme. Helicase-Dependent Amplification (HDA) is the method using the function of Helicase sets the double strand free from 5′ end and 3′ end, and Recombinase Polymerase Amplification (RPA) similar to this is the method using the recombinase sets free the double strand.
A loop-mediated isothermal amplification (LAMP) is a nucleic acid amplifying technique (Patent No. PCT/JP2000/001919) using the DNA polymerase in which has 4˜6 the specific primers and strand displacement.
A band of the smeared pattern is confirmed by the measuring method of LAMP through the electrophoresis analysis of agarose gel and the presence or absence of the nucleic acid amplification reaction of specific gene can be analyzed, but since the reaction tube is opened at the time of the electrophoresis analysis of agarose gel, said method can be a source of the nucleic acid contamination.
Therefore, technologies are developing which can measure without opening the reaction tube to block the source of the nucleic acid contamination pollution source, and a turbidimetry method being generally used in LAMP (Mori. Y. et al., Biochemical and Biophysical Research Communications, 2001, 289:150-154) can confirm the presence of the nucleic acid amplification without opening the reaction tube since the real-time detection and end-point detection are possible by measuring the turbidity having 400 nM of wavelength by the accumulation of white precipitation due to magnesium pyrophosphate (Mg2P2O7) according to the increase of the amplification reaction of the isothermal nucleic acid. However, it has the disadvantage that the multiple-detections at the same time such as a discrimination of two or more of targets is impossible, and the accuracy for the measuring method is low due to the precipitation of the magnesium pyrophosphate.
In LAMP, the fluorescence detection method for the real-time measuring the presence of the nucleic acid amplification removed the occurrence of contamination by measuring only the fluorescence material without opening the reaction tube, and has advantage which can remove the reduction of the accuracy due to the generation of precipitating materials.
In LAMP, the double-strand chain-specific intercalater such as SYBR Green I is added to the reaction solution and the amount of fluorescence is increased according to the increase of nucleic acid amplification reaction product and thus the real-time measurement is possible. Since the SYBR Green I which is also used in the real-time PCR amplification reaction does not confirm the nucleic acid amplification product in a sequence-specific manner, the degree of fluorescence is measured even in the occurrence of primer dimer and thus the temperature is slowly raised up to 95° C. after the real-time measurement to discriminate the non-specific reaction and the annealing temperature of the nucleic acid amplification reaction product is measured for judging the non-specific reaction so that one can confirm the presence of the detection, and the same time (simultaneous) multiple-detection such as two or more target distinctions is impossible.
It has developed technologies for detecting fluorescence to the nucleic acid PCR sequence specific manner by LAMP. The assimilation probe is a method for real-time measurement (Patent No. PCR/US11/41540) using the FRET (Fluorescence Resonance Energy Transfer) principles with two strands of probe designed in a sequence-specific manner of nucleic acid amplification. One of two strands is attached to 5′ end of reporter dye, and other strand is located at 3′ end of the quencher. The number of oligonucleotides used in LAMP nucleic acid amplification includes 6 types of a number of primer, and the assimilation probe designs one strand of probe that the reporter dye is attached to 5′ end for one of the loop primers, and designs a part of the sequence complementary to this to design other strand of probe that the quencher is attached at 3′ end and thus it should synthesize 7 types of oligonucleotides; and the assimilation probe slowly decreases the temperature of two probes from the high temperature to low temperature prior to LAMP reaction to synthesize as a pair of probes and then used in the amplification of nucleic acid. In LAMP, for the real-time of detection of the gene sequence-specific real-time detection, the inhibiting effect of LAMP reaction in the nucleic acid amplification reaction is occurred by using the assimilation probe, and thus the detection time and real time measurement time get longer.
As the fluorescence detecting method using the probe similar to the assimilation probe, there is a method using a pair of probes which is designed so that the fluorophore-labeled primer/probe and quencher-labeled probe have the complementary sequence (Patent No. PCT/JP2012/077596). By designing the annealing temperature of the fluorescence-labeled probe differently from that of the quencher-labeled probe, only the fluorescence-labeled probe is annealed on the target gene-specific sequence to react during LAMP nucleic acid amplification reaction, and after the completion of LAMP nucleic acid amplification reaction, the remaining fluorescence probe is linked with the quencher probe by lowering the temperature and then detect the amount of fluorescence. The said pair of probes cannot be confirmed in real-time detection, and one oligonucleotide should also be added and designed like the assimilation probe.
Although the technologies for solving the false-positive reaction by the contamination and sensitively measuring the amplification of target nucleic acid present in a low concentration and by confirming the nucleic acid amplification without opening the reaction tube through the real-time detection and amount of fluorescence measurement in LAMP nucleic acid amplification reaction, the methods have limitations which are not specific to the target nucleic acid sequence such as turbidimetry and intercalator fluorescence measurement cannot confirm the presence or absence of the non-specific amplification, and the simultaneous multiple-nucleic acid detection is impossible, and the methods using the probes can be designed in a target gene-sequence specific manner and use various reporter dyes and quenchers, and thus the simultaneous multiple-nucleic acid detection can be carried out. However, it requires the design of oligonucleotide called as the quencher probe to form a pair of probes in addition to 6 types of oligonucleotides used in LAMP, and a process is added that a pair of probes is synthesized prior to the preparation of reactant and should be added to the reactant. Also, the design position of the pair of probes should be designed as the fluorescence probe by replacing the loop primer. Since the loop primer is proceed with the next procedure by the annealing after that the loop in the structure of dumbbell upon LAMP reaction is formed, the time that the fluorescence is measured is measured with the delayed time of the fluorescence detection than the time that the actual nucleic acid is amplified. There are disadvantages that the assimilation probe provides the reactivity of LAMP with an inhibitory effect, and in the case of the method using the fluorescence probe and quencher probe the real-time detection cannot be made, and the detection can be made under the condition that the temperature should be lowered after the completion of the reaction temperature.
The present invention is derived so as to solve said problems and meet said needs, and the purpose of the present invention is to develop the technology that for the nucleic acid amplification reaction of LAMP, in addition to 4˜6 types of oligonucleotide the oligonucleotides additionally designed are not needed; the sequence-specific detection of the target gene is possible; and the real-time detection which is not the detection after the completion of the reaction is possible, and to measure the amplification product occurred during LAMP isothermal amplification process on a real-time basis, and to provide a method that a simultaneous multiple detection is possible.
In order to achieve said purpose, the present invention provides an oligonucleotide, wherein parts of sequences among sequences except for 3′ end on Forward Inner Primer or Reverse Inner Primer for a loop-mediated isothermal nucleic acid amplification reaction (LAMP) for a specific sequence of a target gene are replaced with internal dT, internal dG, internal dC, internal dA, internal dU, internal dR; the reporter dye or quencher is placed in this site; and all or a part of the sequences form the double-strand at a certain temperature or lower, having one or more of bubble structures.
In one embodiment of the present invention, the oligonucleotide is preferably located within a range of 21 to 33-mer with respect to the reporter dye and the quencher interval, but is not limited thereto
In another embodiment of the present invention, it is preferable that the oligonucleotide is used separately or together with a forward internal probe and a reverse internal probe, but is not limited thereto.
In one example of the present invention, the reporter dye of the oligonucleotide is one of FAM, TET, HEX, TAMRA, ROX, TEXAS RED, CY3, and CY5, or is preferably an emission wavelength band of 450 to 685 nm; and the quencher of the oligonucleotide is TAMRA, DABCYL, Black Hole Quencher 1, or 2, or is preferably in the range of 500 to 705 nm absorption wavelength band, but is not limited thereto.
In one embodiment of the present invention, the oligonucleotide preferably has one to four bubble structures; and the oligonucleotide preferably has a melting temperature of 30 to 70° C. for unwinding from a double strand to a single strand, but is not limited thereto.
In one example of the present invention, the oligonucleotide is preferably one of the oligonucleotides described in SEQ ID NOS: 5 to 8 and SEQ ID NOS: 15 to 16, but is not limited thereto.
The present invention also provides a method for performing loop-mediated isothermal nucleic acid amplification (LAMP) or reverse transcription (RT)-LAMP reactions for fluorescence detection of real time nucleic acid amplification at isothermal temperature using the oligonucleotide of the present invention.
Also, the present invention provides a method for performing loop-mediated isothermal nucleic acid amplification (LAMP) or reverse transcription (RT)-LAMP reactions for fluorescence detection of end-point nucleic acid amplification under the condition of isothermal temperature or two or more of temperatures using the oligonucleotide of the present invention.
In the method of the present invention, it is preferable that the oligonucleotide is used separately or together with a forward internal probe and a reverse internal probe; and the isothermal temperature condition of the above method is preferably in the range of 50 to 75° C., but is not limited thereto.
In the method of the present invention, it is preferable that the method is performed on DNA and cDNA nucleic acids, and the method is preferable to perform a one step reaction on a specific gene after a reverse transcription reaction on an RNA nucleic acid, but is not limited thereto.
The present invention also provides a kit for separately or simultaneously multiplex-amplifying a specific gene for an infectious disease, hereditary disease, drug resistance, drug resistance or susceptibility specimen comprising the oligonucleotide of the present invention.
The present invention also provides a composition for amplifying an Ebola virus nucleic acid comprising the oligonucleotide of the present invention as an active ingredient.
Also, the present invention provides a composition for an isothermal nucleic acid amplification reaction comprising the oligonucleotide of the present invention as an active ingredient.
In one embodiment of the present invention, the composition preferably includes oligonucleotides of SEQ ID NOS: 1 to 18, but is not limited thereto.
The present invention also provides a method for performing isothermal nucleic acid amplification reaction on DNA, RNA, or cDNA by using the oligonucleotide of the present invention in combination with an anti-sense probe.
In one embodiment of the present invention, the anti-sense probe is preferably designed and used within 55 to 65° C., but is not limited thereto.
In another embodiment of the present invention, the oligonucleotide and the anti-sense probe are preferably used separately or together in the forward and reverse positions, but are not limited thereto.
In another embodiment of the present invention, the anti-sense probe preferably has one of the sequences set forth in SEQ ID NOS: 19 to 23, but is not limited thereto.
Hereinafter, the invention is illustrated.
Abbreviations used in the present invention are as follows:
Internal dT: Internal Amino-C6 (or C2)-deoxythymine, Internal dA: Internal Amino-C6-deoxyadenocine, Internal dC: Internal Amino-C6-deoxycytidine, Internal dG: Internal Amino-C6-deoxyguanosine, Internal dU: Internal Amino-C6-deoxyuridine, Internal dR: Internal Amino-C6-deoxyribose
The present invention provides with an use of isothermal-based bifunctional oligonucleotides, a nucleic acid amplification and determination methods using such oligonucleotides, wherein Forward inner Primer (FIP) and Reverse Inner Primer (RIP) of Loop-mediated isothermal amplification (LAMP) method are designed as one oligonucleotide, one or more bubble structures are formed between two gene sites, and except for the 3′ end, a 5′ end or a thymine site is modified with internal dT and a reporter dye or a quencher is attached on the site, and one reporter dye and one quencher are positioned on one IB-DFO to perform the two functions of primer and probe, real-time fluorescence monitoring is performed in isothermal nucleic acid amplification reaction to determine whether the nucleic acid amplification is made in every time and the fluorescence for the reaction product is determined through monitoring the fluorescence in end-point step to detect whether the isothermal nucleic acid amplification reaction is made.
The present invention provides with an oligonucleotide wherein modifying Thymine base with internal dT, or modifying Adenine, Thymine, Guanine, Cytosine or Uridine base with internal dR on the sequence except 3′ end of FIP or RIP among primers for LAMP isothermal nucleic acid amplification reaction with regard to a certain sequence of the target gene, and by placing the reporter dye or quencher on such sites and all or a part of the sequence forms double strands at below a certain temperature and has one or more of bubble structure.
The oligonucleotide of the present invention provides an isothermal nucleic acid amplification reaction mixture containing at least one isothermal-based bifunctional oligonucleotide for isothermal nucleic acid amplification of a specific gene sequence of target DNA, cDNA, and RNA.
The present invention also relates to a mixture for LAMP nucleic acid amplification reaction comprising Tris-HCl, KCl, (NH4) 2SO4, MgCl2, MgSO4, Betaine, Dithiothreitol (DTT), Triton X-100, Teen-20, dNTPs, Bovine serum albumin Dimethyl sulfoxide (DMSO), Formamide, Single strand binding protein, Pyrophosphate, Pyrophosphatase, etc., and also comprising IB-DFO in combination with DNA polymerase having 5′→3′ exonuclease (−), 3′ exonuclease (−), strand displacement activity. And, it relates to an use of the mixture for RT-LAMP nucleic acid amplification reaction comprising RNase inhibitor and Reverse transcriptase such ad Avian Myeloblastosis Virus (AMV) Reverse transcriptase, Molony Murine Leukemia Virus (MMLV) Reverse transcriptase, etc., and IB-DFO in combination with DNA polymerase having thermostable Reverse transcriptase and Reverse transcriptase functions.
The oligonucleotides of the present invention provide oligonucleotides in which the reporter dye and a quencher are located within 21 to 33 mer interval, but are not limited thereto.
The present invention provides the oligonucleotide which is characterized in that FIP-IB-DFO, a forward probe of the oligonucleotide, and RIP-IB-DFO, a reverse probe, are used separately or together.
The present invention provides with an oligonucleotides comprising one of PAM, TEL HEX, TAMRA, ROX, TEXAS RED, CY3 and CY5 as a reporter dye of the oligonucleotides, and having the emission wavelength band of 450 to 685 nm.
The present invention provides with an oligonucleotide comprising TAMRA, DABCYL, and Black Hole Quencher 1, 2 as the oligonucleotide quencher and comprising an absorption wavelength band of 500 to 705 nm, but is not limited thereto.
The present invention provides LAMP and RT-LAMP reaction methods for detecting real-time nucleic acid amplification fluorescence detection using an oligonucleotide of the present invention at the isothermal temperature.
The present invention provides LAMP and RT-LAMP reaction method for detecting end-point nucleic acid amplification fluorescence at isothermal or two or more temperatures using oligonucleotides of the present invention.
The method provides a mixture comprising an IB-DFO concentration of 0.2-1.6 uM of the oligonucleotide either separately or together with PIP or RIP, but is not limited thereto.
The present invention provides a method wherein the isothermal temperature condition of the LAMP reaction method using the oligonucleotide of the present invention has a range of 50 to 75° C.° C., but is not limited thereto.
The present invention provides a method for performing LAMP reaction method on DNA and cDNA nucleic acid using oligonucleotide of the present invention.
The present invention provides a method for performing a reverse transcription reaction on RNA nucleic acid using the oligonucleotide of the present invention and then performing a one-step reaction on a specific gene.
The oligonucleotides of the present invention provide oligonucleotides having one to four bubble structures.
The present invention provides with an oligonucleotide characterized in that an annealing temperature in which a double strand is unwound into a single strand is 30 to 70° C., but is not limited thereto.
The present invention provides a kit for amplifying a specific gene for DNA, cDNA, and RNA of an infectious disease, hereditary disease, drug resistance, drug resistance or susceptible sample including the oligonucleotide of the present invention, individually or multiple simultaneously.
The oligonucleotides of the present invention provide with oligonucleotide sequences of SEQ ID NOS: 1 to 18 for the subtypes of Ebola virus, Bundibugyo and Reston, and LAMP and RT-LAMP primer sequences.
As can be seen from the present invention, the present invention has advantages that oligonucleotides additionally designed in addition to 4 to 6 types of oligonucleotides for LAMP nucleic acid amplification reaction are not needed, the amount of fluorescence according to the nucleic acid amplification of the target gene specific sequence for DNA and RNA is detected and it can be detected even after finishing the reaction, and simultaneous multiple analysis can be made by determining the amount of fluorescence using different reporter dyes according to the target gene in a tube after finishing the reaction or in real-time, as a method that can detect the amount of fluorescence in real time.
Hereinafter, the present invention will be described in detail with reference to Examples. Provided that, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
To observe the fluorescence detection effect of LAMP isothermal nucleic acid amplification reaction according to the distance (mer number) between the reporter dye and the quencher of the IB-DFO of the present invention, 4 types of primers for LAMP isothermal nucleic acid amplification for the L segment (Polymerase) site sequence of Bundibugyo, one of the subtypes of Ebola virus, were designed by PrimerExplorer V4 Software (http://primerexplorer.jp/elamp4.0.0/index.html).
The designed primers are EB_B_F3 (SEQ ID NO: 1), EB_B_R3 (SEQ ID NO: 2), EB_B_FIP (SEQ ID NO: 3) and EB_B_RIP (SEQ ID NO: 4), and IB-DFO was made by replacing Thymine with internal dT on EB_B_FIP primer sequence and modifying FAM with a reporter dye at this site, and by replacing Thymine at the interval positions of 33 (EB_B_FIP_P2, SEQ ID NO: 5), 25 (EB_B_FIP_P2.5, SEQ ID NO: 6), 21 mer (EB_B_FIP_P3, SEQ ID NO: 7) with internal dT and attaching BHQ1 quencher at this sites. The Melting temperature at which three IB-DFOs were unwound from a double strand to a single strand was 54° C.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 1.4 mM dNTP each, 0.5 M Betaine, 8 U Bst DNA polymerase (MCLAB, CA, USA), and 0.2 uM of EB_B_F3 and EB_B_R3; adding 1.2 uM of EB_B_FIP and 1.6 uM of EB_B_RIP to the mixture; and additionally adding 0.4 uM of IB-DFO. Bundibugyo L segment (gb:FJ217161, 521 bp) of Ebola virus was constructed by gene synthesis and used as a concentration of 1×10̂7, 1×10̂5, 1×10̂3, 0 copies/reaction. The real-time fluorescence detection for isothermal nucleic acid amplification was performed at 65° C. for 60 minutes, and the amount of fluorescence was measured using CFX-96 (Bio-Rad, CA, USA) real-time fluorescence measurement equipment in every 1 minute.
In order to observe the fluorescence detection effect according to LAMP isothermal nucleic acid amplification reaction when the IB-DFO of the present invention was used in the forward or reverse position, four types of primers for LAMP isothermal nucleic acid amplification were designed by PrimerExplorer V4 Software on the L segment (Polymerase) site sequence of Bundibugyo, one of the subtypes of Ebola virus. The designed primers were EB_B_F3 (SEQ ID NO: 1), EB_B_R3 (SEQ ID NO: 2), EB_B_FIP (SEQ ID NO: 3) and EB_B_RIP (SEQ ID NO: 4); and IB-DFO used EB-B_FIP_P2 (SEQ ID NO: 5) obtained by replacing Thymine with internal dT on EB_B_FIP primer sequence, replacing FAM with the reporter dye on this site, replacing Thymine in 33 mer interval position with internal dT and attaching BHQ 1 on this site, and BHQ1-EB_B_RIP_P2 (SEQ ID NO: 8) designated by placing FAM reporter dye and BHQ1 quencher in 30 mer interval on EB_B_RIP primer sequences.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 1.4 mM dNTP each, 0.5 M Betaine, 8 U Bst DNA polymerase (MCLAB, CA, USA) and 0.2 uM of each EB_B_F3 and EB_B_R3, and adding 1.2 or 1.6 uM of EB_B_FIP and 1.2 or 1.6 uM of EB_B_RIP to the mixture, and additionally adding 0.4 uM of EB_B_FIP_P2 and EB_B_RIP_P2, separately. Bundibugyo L segment (gb:FJ217161, 521 bp) of Ebola virus was constructed through a gene synthesis and then used in the concentration of 1×10̂7, 1×10̂5, 1×10̂3, 0 copies/reaction concentration. The real-time fluorescence detection for isothermal nucleic acid amplification was performed at 65° C. for 60 minutes, and the amount of fluorescence was measured using CFX-96 (Bio-Rad, CA, USA) real-time fluorescence measurement equipment in every 1 minute.
In order to observe the fluorescence detection effect according to LAMP isothermal nucleic acid amplification reaction, when the IB-DFO of the present invention was used in the forward or reverse position, six types of primers for LAMP isothermal nucleic acid amplification were designed by PrimerExplorer V4 Software on the L segment (Polymerase) site sequence of Reston, one of the subtypes of Ebola virus. The designed primers were EB_R_F3 (SEQ ID NO: 9), EB_R_R3 (SEQ ID NO: 10), EB_R_FIP (SEQ ID NO: 11), EB_R_RIP (SEQ ID NO: 12), Res_LP (SEQ ID NO: 13) and Res_RLP (SEQ ID NO: 14); and IB-DFO used EB-R_FIP_P2 (SEQ ID NO: 15) obtained by replacing Thymine with internal dT on EB_R_FIP primer sequence, replacing FAM with the reporter dye on this site, replacing Thymine in 31 mer interval position with internal dT and attaching BHQ 1 on this site, and EB_R_RIP_P2 (SEQ ID NO: 16) designated by placing FAM reporter dye and quencher in 33 mer interval on EB_R_RIP primer sequences.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 1.4 mM dNTP each, 0.5 M Betaine, 8 U Bst DNA polymerase (MCLAB, CA, USA) and 0.2 uM of each EB_R_F3 and EB_R_R3, and 0.8 Um of Res_LP and Res_RLP, adding 1.2 or 1.6 uM of EB_R_FIP and 1.2 or 1.6 Um of EB_R_RIP to the mixture, and adding 0.4 uM of EB_R_FIP_P2 and EB_R_RIP_P2, separately. EB_R_FIP_P2 and EB_R_RIP_P2 were added in amounts of 0.2 uM, respectively, under the condition that they used together, and EB_R_FIP and EB_R_RIP primer were in amounts of 1.4 uM, respectively. Reston L segment (gb:JX477166, 410 bp) of Ebola virus was constructed through a gene synthesis and then used in the concentration of 1×10̂4, 1×10̂3 copies/reaction concentration. The real-time fluorescence detection for isothermal nucleic acid amplification was performed at 65° C. for 30 minutes, and the amount of fluorescence was measured using CFX-96 (Bio-Rad, CA, USA) real-time fluorescence measurement equipment in every 1 minute.
In order to observe the fluorescence detection effect according to LAMP isothermal nucleic acid amplification reaction when the IB-DFO of the present invention and inner primer were designed with same sequences and used according to a ratio of them, four types of primers for LAMP isothermal nucleic acid amplification were designed by PrimerExplorer V4 Software on the L segment (Polymerase) site sequence of Bundibugyo, one of the subtypes of Ebola virus. The designed primers were EB_B_F3 (SEQ ID NO: 1), EB_B_R3 (SEQ ID NO: 2), EB_B_FIP (SEQ ID NO: 3) and EB_B_RIP (SEQ ID NO: 4); and IB-DFO used EB-B_FIP_P2 (SEQ ID NO: 5) obtained by replacing Thymine with internal dT on EB_B_FIP primer sequence, replacing FAM with the reporter dye on this site, replacing Thymine in 31 mer interval position with internal dT and attaching BHQ1 quencher on this site.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 1.4 mM dNTP each and 8 U Bst DNA polymerase (MCLAB, CA, USA) and 0.2 uM of each EB_B_F3 and EB_B_R3, and 1.6 Um of EB_B_RIP, and adding EB_B_FIP:EB_B_FIP_P2 in the ratios of 0:1.6 uM, 0.4:1.2 uM, 0.8:0.8 uM, 1.2:0.4 uM and 1.6:0 uM to the mixture, respectively. Bundibugyo L segment (gb:FJ217161, 521 bp) of Ebola virus was constructed through gene synthesis and then used in the concentration of 1×10̂7 copies/reaction concentration. Real-time fluorescence detection for isothermal nucleic acid amplification was performed at 65° C. for 60 minutes, and the amount of fluorescence was measured using CFX-96 (Bio-Rad, CA, USA) real-time fluorescence measurement equipment in every 1 minute. And, an end-point fluorescence detection was performed at 65° C. for 60 minutes, and at 30° C. in every 1 minute for 10 cycles to determine the amount of fluorescence.
The results of
In order to observe the fluorescence detection effect according to LAMP isothermal nucleic acid amplification reaction with plasmid DNA as a template when adding the IB-DFO of the present invention to FIP position, six types of primers for LAMP isothermal nucleic acid amplification were designed by PrimerExplorer V4 Software on the L segment (Polymerase) site sequence of Bundibugyo, one of the subtypes of Ebola virus. The designed primers were EB_B_F3 (SEQ ID NO: 1), EB_B_R3 (SEQ ID NO: 2), EB_B_FIP (SEQ ID NO: 3), EB_B_RIP (SEQ ID NO: 4), EB_BLP_F (SEQ ID NO: 17) and EB_B_LP_R(SEQ ID NO: 18); and IB-DFO used EB-B_FIP_P2 (SEQ ID NO: 5) obtained by replacing Thymine with internal dT on EB_B_FIP primer sequence, replacing FAM with the reporter dye on this site, replacing Thymine in 33 mer interval position with internal dT and attaching BHQ1 quencher on this site.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 1.4 mM dNTP each, 8 U Bst DNA polymerase (MCLAB, CA, USA), 0.2 uM of each EB_B_F3 and EB_B_R3, and 0.8 uM of EB_B_LP_F and EB_B_LP_R; and adding 1.2 uM of EB_B_FIP and 1.6 uM of EB_R_RIP to the mixture; and adding 0.4 uM of EB_B_FIP_P2, IB-DFO of the present invention to the mixture. Bundibugyo L segment (gb:FJ217161, 521 bp) of Ebola virus was constructed through a gene synthesis and then used in the concentration of 1×10̂7, 1×10̂6, 1×10̂5, 1×10̂4, 1×10̂3, 1×10̂2, 1×10̂1, 0 copies/reaction. The real-time fluorescence detection for isothermal nucleic acid amplification was performed at 65° C. for 60 minutes, and the amount of fluorescence was measured using CFX-96 (Bio-Rad, CA. USA) real-time fluorescence measurement equipment in every 1 minute. And, end-point fluorescence detection was performed at 65° C. for 60 minutes, and at 30° C. in every 1 minute for 10 cycles to determine the amount of fluorescence.
From the results of
In order to observe the fluorescence detection effect according to RT-LAMP isothermal nucleic acid amplification reaction with RNA transcript as a template when adding the IB-DFO of the present invention to FIP position, six types of primers for RT-LAMP isothermal nucleic acid amplification were designed by PrimerExplorer V4 Software on the L segment (Polymerase) site sequence of Bundibugyo, one of the subtypes of Ebola virus. The designed primers were EB_B_F3 (SEQ ID NO: 1), EB_B_R3 (SEQ ID NO: 2), EB_B_FIP (SEQ ID NO: 3), EB_B_RIP (SEQ ID NO: 4), EB_BLP_F (SEQ ID NO: 17) and EB_B_LP_R(SEQ ID NO: 18); and IB-DFO used EB-B_FIP_P2.5 (SEQ ID NO: 6) obtained by replacing Thymine with internal dT on EB_B_FIP primer sequence, replacing FAM with the reporter dye on this site, replacing Thymine in 25 mer interval position with internal dT and attaching BHQ1 quencher on this site.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 5 mM DTT, 5 U RNase inhibitor (New England Biolabs, MA, USA), 1.4 mM dNTP each, 8 U Bst DNA polymerase (MCLAB, CA, USA), 0.2 uM of EB_B_F3 and EB_B_R3, and 0.8 uM of EB_B_LP_F and EB_B_LP_R; adding 1.4 uM of EB_B_FIP and 1.6 uM of EB_R_RIP to the mixture and adding 0.2 uM of EB_B_FIP_P2.5, IB-DFO of the present invention to the mixture. Bundibugyo L segment (gb:FJ217161, 521 bp) of Ebola virus was constructed as the plasmid DNA through gene synthesis and then RNA transcript was synthesized using MEGAscript T7 Transcription Kit (Applied Biosystems, CA, USA) with the plasmid DNA through in vitro transcription. Bundibugyo RNA transcript was used in the concentration of 1×10̂5, 1×10̂4, 1×10̂3 and 0 copies/reaction. The real-time fluorescence detection for isothermal nucleic acid amplification was performed at 65° C. for 30 minutes, and the amount of fluorescence was measured using CFX-96 (Bio-Rad, CA, USA) real-time fluorescence measurement equipment in every 1 minute. And, end-point fluorescence detection was performed at 65° C. for 60 minutes, and at 30° C. in every 1 minute for 10 cycles to determine the amount of fluorescence.
From the results of
An anti-sense probe was designed by adding IB-DFO of the present invention to FIP position, and designing the complementary anti-sense sequence from the reporter dye sequence of IB-DFO to 3′ end according to the temperature (55, 60, 65 and 70° C.) to position the quencher on 3′ end of the anti-sense sequence. Then, DB-DFO was added to RIP position and the anti-sense probe was designed at 60° C. In order to observe the real-time fluorescence detection effect of the anti-sense probe according to the design by the temperature in LAMP isothermal nucleic acid amplification reaction with the plasmid DNA as the template by using it, six types of primers for LAMP isothermal nucleic acid amplification were designed by PrimerExplorer V4 Software on the L segment (Polymerase) site sequence of Bundibugyo, one of the subtypes of Ebola virus. The designed primers were EB_B_F3 (SEQ ID NO: 1), EB_B_R3 (SEQ ID NO: 2), EB_B_FIP (SEQ ID NO: 3), EB_B_RIP (SEQ ID NO: 4), EB_BLP_F (SEQ ID NO: 17) and EB_B_LP_R(SEQ ID NO: 18); and IB-DFO used EB-B_FIP_P2 (SEQ ID NO: 5) obtained by replacing Thymine with internal dT on EB_B_FIP primer sequence, replacing FAM with the reporter dye on this site, replacing Thymine in 25 mer interval position with internal dT and attaching BHQ1 quencher on this site. The anti-sense probe used EB_B_FIP_P2_Q2_55 (SEQ ID NO: 19) designed at 55° C., EB_B_FIP_P2_Q2_60 (SEQ ID NO: 20) designed at 60° C., EB_B_FIP_P2_Q2_65 (SEQ ID NO: 21) designed at 65 t and EB_B_FIP_P2_Q2_70 (SEQ ID NO: 22) designed at 70° C. And, EB_B_RIP_P2_Q60 (SEQ ID NO: 23) obtained by designing the anti-sense probe complementary to IB-DFO of EB_B_RIP_P2 (SEQ ID NO: 8) at 60° C. was used.
The isothermal nucleic acid amplification reaction for the IB-DFO oligonucleotides was performed by using 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO4, 10 mM (NH4) 2SO4, 0.1% Tween-20, 1.4 mM dNTP each, 8 U Bst DNA polymerase (MCLAB, CA, USA), 0.2 uM of EB_B_F3 and EB_B_R3, and 0.4 uM of EB_B_LP_F and EB_B_LP_R; and adding 1.2 uM of EB_B_FIP and 1.6 uM of EB_R_RIP to the mixture; and the mixture was prepared using 0.4 uM of EB_B_FIP_P2, IB-DFO of the present invention and 1.6 uM of the anti-sense probe, together with each of EB_B_FIP_P2_Q2_55, EB_B_FIP_P2_Q2_60, EB_B_FIP_P2_Q2_65, EB_B_FIP_P2_Q2_70. Then, IB-DFO was replaced with 0.4 uM of EB_B_RIP_P2, 1.6 uM of EB_B_RIP_P2_Q_60, the anti-sense probe for it was mixed, IB-DFO was mixed with EB_B_FIP_P2, each 0.4 uM of EB_B_RIP_P2 was mixed with 1.6 uM of EB_B_FIP_P2_Q2_60, and EB_B_RIP_P2_Q_60. Bundibugyo L segment (gb:FJ217161, 521 bp) of Ebola virus was constructed through gene synthesis and plasmid DNA was used in the concentration of 1×10̂5, 1×10̂4 and 1×10̂3 copies/reaction. The real-time fluorescence detection for isothermal nucleic acid amplification reaction was performed at 65° C. for 60 minutes, and the amount of fluorescence was measured using AB7500 (Applied Biosystems, CA, USA) real-time fluorescence measurement equipment in every 1 minute.
As a result of RN vs Min (non-normalization) type, as in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2015-0169857 | Dec 2015 | KR | national |
| 10-2016-0060283 | May 2016 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2016/013944 | 11/30/2016 | WO | 00 |
