KR102071541B1 - Method for Detecting Target Nuceic Acid Based on Electrochemical Real-time PCR Using Aptamer-modified Primer - Google Patents

Abstract

The present invention relates to a method for detecting a target nucleic acid by electrochemical real-time polymerase chain reaction using a primer containing an aptamer base sequence of a signal material for generating an electrochemical signal, the aptamer-signal material according to the present invention Targeted nucleic acid detection method based on electrochemical real-time polymerase chain reaction using modified primers implements an electrochemical nucleic acid detection system that is inexpensive and easy to miniaturize analytical equipment, and is suitable for in situ diagnosis of various genetic diseases and infectious diseases. Applicable

Description

Method for Detecting Target Nuceic Acid Based on Electrochemical Real-time PCR Using Aptamer-modified Primer}

The present invention relates to a method for detecting a target nucleic acid through a real-time polymerase chain reaction using an aptamer-signal modification primer, more specifically, an aptamer base sequence of a signal material generating an electrochemical signal The present invention relates to a method for detecting target nucleic acid by electrochemical real-time polymerase chain reaction using a primer.

Polymerase chain reaction (PCR), a nucleic acid amplification technology developed by Kary Mullis in 1983, has had a profound impact on the technical and economic developments of the molecular biology of the 20th century. Because PCR technology can amplify very small amounts of nucleic acid samples in large quantities, key technologies are used throughout biotechnology, such as gene cloning, sequencing, genetic disease diagnosis, genetic fingerprint analysis, and infectious disease diagnosis. (Bartlett and Stirling, Methods Mol Biol. 3: 226, 2003).

In particular, the market has been increasing rapidly since the 1990s when real-time PCR was developed. Conventional PCR technology separates the target nucleic acid amplification and analysis process and cannot confirm the initial concentration before amplification.However, real-time PCR technology can confirm the amplification process in real time and determine the initial concentration of target nucleic acid. Also called quantitative PCR (qPCR). Real-time PCR technology is currently being evaluated as the most powerful technology in the field of genetic analysis, and its performance has been proven when it is used to confirm the pandemic of the influenza H1N1 virus.

Currently, most nucleic acid detection techniques, including real-time PCR, are based on fluorescence signal analysis. This requires expensive fluorescent materials and a large volume of fluorescence analysis equipment, which can only be performed in general hospitals or specialized diagnostic institutions equipped with specialized facilities. As a result, it took a lot of time and money to receive the final diagnosis results from the request for examination, which showed some limitations in performing rapid and efficient diagnosis in emergencies such as the 2009 influenza outbreak. In order to solve this problem, it is necessary to develop a low-cost and simple nucleic acid detection technology that can be used directly in local small hospitals and health centers, and ultimately at home. Representatively, the electrochemical nucleic acid detection method, which is inexpensive and easy to miniaturize analytical equipment, has been evaluated as a very promising alternative and related research is being actively conducted.

Accordingly, the present inventors have made efforts to develop a real-time PCR technique based on an electrochemical analysis method, using the aptamer-signal modification primer containing a signal material capable of generating an electrochemical signal, PCR In the case of performing the PCR, the signal material bound to the primer is released as the PCR proceeds, and the electrochemical signal is measured in real time, confirming that electrochemical real-time PCR can be performed and the present invention is completed. Was done.

An object of the present invention is to provide a method for detecting a target nucleic acid by an electrochemical real-time polymerase chain reaction using a primer containing an aptamer sequence of a signal material for generating an electrochemical signal.

In order to achieve the above object, the present invention is a pressure material characterized in that the signal material for generating an electrochemical signal is coupled to the aptamer modified primer comprising a nucleotide sequence and aptamer base sequence that can specifically bind to the target nucleic acid A tammer-signal modification primer is provided.

The present invention also relates to (a) (i) a target nucleic acid; (ii) the aptamer-signal modification primer of claim 1; (iii) nucleic acid polymerase; And (iv) a polymerase chain reaction using a composition for real-time polymerase chain reaction comprising deoxynucleotide triphosphate (dNTP) to extend DNA to the ends of primers as the reaction cycle proceeds. The signal is released from the aptamer-signal modification primer; And (b) detecting and / or quantifying a target nucleic acid by measuring a change in an electrochemical signal generated by the emitted signal, and electrochemical real-time polymerase using the aptamer-signal modification primer. It provides a chain reaction-based target nucleic acid detection method.

The present invention also provides for (i) target nucleic acid; (ii) the aptamer-signal modification primer of claim 1; (iii) nucleic acid polymerase; And (iv) provides a composition for real-time polymerase chain reaction containing deoxynucleotide triphosphate (dNTP).

The target nucleic acid detection method based on the electrochemical real-time polymerase chain reaction using the aptamer-signal modification primer according to the present invention implements an electrochemical nucleic acid detection system which is inexpensive and can be easily miniaturized. Applicable to in situ diagnosis of hereditary diseases and infectious diseases.

Figure 1 schematically shows a method for detecting target nucleic acids based on electrochemical real-time polymerase chain reaction using aptamer-signal modification primers.
2 shows an electrode patterned chip and an incubation chamber assembled thereon used in the present invention.
3 is a graph measuring the electrochemical current signal and the peak current analysis during the electrochemical real-time polymerase chain reaction according to the presence or absence of the target nucleic acid.
Figure 4a shows the amplification curve of the electrochemical real-time polymerase chain reaction according to the initial copy number of the hepatitis B virus (HBV) gene, Figure 4b shows the C _t value according to the initial copy number of the HBV gene will be.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, an electrochemical signal increasing through a polymerase chain reaction was measured by an electrochemical analysis device, and a method of identifying and quantifying the presence or absence of a target nucleic acid present in a sample was developed.

That is, the present invention performs an electrochemical real-time polymerase chain reaction using a primer containing an aptamer sequence capable of binding to a signal material that generates an electrochemical signal, which is bound to the aptamer at the beginning of the reaction. It is a method of quantitatively analyzing target nucleic acid using the principle that the electrochemical signal of the sample increases due to the release of the signal material as the polymerization reaction of the target nucleic acid proceeds.

Therefore, the present invention in one aspect, aptamer characterized in that the signal material for generating an electrochemical signal is coupled to the aptamer modified primer comprising a base sequence and aptamer base sequence that can specifically bind to the target nucleic acid It relates to a signal substance modification primer.

In the present invention, the signal material generating the electrochemical signal may be selected from the group consisting of mercury ions, silver ions, lead ions, electrochemical signal material having an aptamer sequence.

In the present invention, the aptamer is an aptamer including a thymine-thymine base pair, the signal material may be characterized in that the mercury ion.

The primer modified by the aptamer of the present invention includes an aptamer base sequence capable of binding to a signal material for generating an electrochemical signal. When a target nucleic acid is present, the terminal branch DNA of the primer is extended by a polymerization reaction. The structure of the aptamer is modified to emit the signal material. The signal material generates an electrochemical signal, and by measuring the electrochemical signal, it is possible to quantify the target nucleic acid as well as the presence or absence of the target nucleic acid (Fig. 1).

Thus, in another aspect, the present invention provides a kit comprising: (a) (i) a target nucleic acid; (ii) the aptamer-signal modification primer of claim 1; (iii) nucleic acid polymerase; And (iv) performing PCR using a composition for real-time polymerase chain reaction including deoxynucleotide triphosphate (dNTP) to release a signal from the aptamer-signal modification primer; And (b) detecting and / or quantifying target nucleic acids by measuring changes in electrochemical signals generated by the emitted signal material.

In the present invention, the polymerase chain reaction may be performed in a chamber on a diagnostic chip on which an electrode is patterned to detect an electrochemical signal.

In the present invention, the signal material generating the electrochemical signal may be selected from the group consisting of mercury ions, silver ions, lead ions, electrochemical signal material having an aptamer sequence.

In the present invention, in the step (a) by performing a polymerase chain reaction using a composition for real-time polymerase chain reaction, the DNA is extended to the end of the primer as the reaction cycle proceeds, modifying the aptamer-signal substance It can be characterized in that the signal is released from the primer.

In the present invention, the aptamer is an aptamer including a thymine-thymine base pair, the signal material may be characterized in that the mercury ion.

In another aspect, the present invention provides a composition comprising (i) a target nucleic acid; (ii) the aptamer-signal modification primer of claim 1; (iii) nucleic acid polymerase; And (iv) relates to a composition for real-time polymerase chain reaction containing deoxynucleotide triphosphate (dNTP).

In one embodiment of the present invention, an electrochemical real-time polymerase chain reaction may be realized by utilizing a property of aptamers including mismatched thymine-thymine (T-T) base pairs with mercury ions. When the 5 'end of each primer is modified with a thymine-rich sequence, mercury ions are bound to the primer before PCR proceeds. When the sample is placed in the incubation chamber assembled on the chip and the electrode patterned chip as shown in FIG. 2 and the PCR is performed on the temperature control device, when the target nucleic acid is not present in the sample, the PCR cannot proceed and thus the electrochemical signal is changed. On the other hand, in the presence of the target nucleic acid, PCR proceeds, and thus DNA is extended to the terminal portion of the primer, thereby gradually releasing the bound mercury ions. Since the free diffusion of mercury ions in solution is higher than that of mercury ions bound to the aptamer, the current increases, and if measured in real time, the presence of the target nucleic acid in the sample and the initial copy number are quantitatively determined. It was confirmed that it can be determined (Fig. 3).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Detection of target nucleic acid by electrochemical real-time PCR

Hepatitis B virus (HBV) nucleic acid (SEQ ID NO: 1) was used to detect the presence and concentration of target nucleic acid in a sample using an electrochemical real-time polymerase chain reaction using an aptamer-signal modification primer. ) Was analyzed using target nucleic acid.

DW 28.5 μL, 10 × buffer 5 μL, 10 mM dNTPs 4 μL, 20 μM forward / reverse primer (SEQ ID NO: 2 and SEQ ID NO: 3) ₅ μL, 50 μM HgCl ₂ 1 μL, i-starmaxII polymerase 0.5 μL, various concentrations (10 ⁷ copy / 50 μL of PCR reaction solution containing 1 μL of target nucleic acid (μL, 10 ⁵ copy / μL, 10 ³ copy / μL) was prepared.

The PCR reaction solution was injected into an incubation chamber assembled on a diagnostic chip patterned with gold electrodes on a 1 mm thick glass wafer, and the cover glass was carefully covered to avoid bubbles. Then, the PCR reaction was performed on a thermostat. PCR reactions consisted of 45 repetitions of a cycle consisting of 5 min preliminary modification at 95 ° C., 20 sec denaturation at 95 ° C., 20 sec anneal at 56 ° C., and 30 sec elongation at 72 ° C., and a final 5 min at 72 ° C. Performed by kidney.

HPV nucleic acid sequence (SEQ ID NO: 1): 5 '- TGCACTTGTATTCCCATCCCATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGtTCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTTTGTCTTTGGGTATACATTTGAACCCTAATAAAACCAAACGTTGGGGCTACTCCCTTAACTTCATGGGATATGTAATTGGAAGTTGGGGTACTTTACCGCAGGAACATATTGTACAAAAACTCAAGCAATGTTTTCGAAAATTGCCTGTAAATAGACCTATTGATTGGAAAGTATGTCAAAGAATTGTGGGTCTTTTGGGCTTTGCTGCCCCTTTTACACAATGTGGCTATCCTGCCTTGATGCCTTTATATGCATGTATACAATCTAAGCAGGCTTTCACTTTCTCGCCAAC - 3'

Forward primer (SEQ ID NO: 2): 5 ' -CAGATTCTTTCTTCCCTTGTTTGTTTCTG TGCACTTGTATTCCCATCCC-3'

Reverse primer (SEQ ID NO: 3): 5 ' -CAGATTCTTTCTTCCCTTGTTTGTTTCTG GTTGGCGAGAAAGTGAAAGC-3'

(Thick letters are aptamer sequences)

In the presence of the target nucleic acid, mercury ions bound to the aptamer are released as the PCR proceeds, thereby increasing the free state mercury on. SWV (Square Wave Voltammetry) was used to measure the current generated in the reduction reaction of mercury ions every three cycles. In the absence of target nucleic acid, the reduction current of mercury ions did not increase, and in the presence of target nucleic acid, the reduction current of mercury ions tended to increase in current signal according to the initial copy number of the gene (FIG. 4A). . C _t values for the initial copy numbers of 10 ⁷ , 10 ⁵ and 10 ³ at the threshold level were confirmed with 99% confidence (FIG. 4B).

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Advanced Institue of Science and Technology          BioNano Health Guard Research Center <120> Method for Detecting Target Nuceic Acid Based on Electrochemical          Real-time PCR Using Aptamer-modified Primer <130> P18-B017 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 514 <212> DNA <213> Artificial Sequence <220> <223> target nucleic acid <400> 1 tgcacttgta ttcccatccc atcatcctgg gctttcgcaa gattcctatg ggagtgggcc 60 tcagttcgtt tctcctggct cagtttacta gtgccatttg ttcagtggtt cgtagggctt 120 tcccccactg tttggctttc agctatatgg atgatgtggt attgggggcc aagtctgtac 180 aacatcttga gtcccttttt acctctatta ccaattttct tttgtctttg ggtatacatt 240 tgaaccctaa taaaaccaaa cgttggggct actcccttaa cttcatggga tatgtaattg 300 gaagttgggg tactttaccg caggaacata ttgtacaaaa actcaagcaa tgttttcgaa 360 aattgcctgt aaatagacct attgattgga aagtatgtca aagaattgtg ggtcttttgg 420 gctttgctgc cccttttaca caatgtggct atcctgcctt gatgccttta tatgcatgta 480 tacaatctaa gcaggctttc actttctcgc caac 514 <210> 2 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cagattcttt cttcccttgt ttgtttctgt gcacttgtat tcccatccc 49 <210> 3 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cagattcttt cttcccttgt ttgtttctgg ttggcgagaa agtgaaagc 49

Claims (8)

An aptamer comprising an nucleotide sequence complementary to a target nucleic acid at the 3 'end and a signal material for generating an electrochemical signal to an aptamer modified primer including an aptamer base sequence at the 5'-end; Signaling material modified primer pairs.
The primer pair of claim 1, wherein the signal material generating the electrochemical signal is selected from the group consisting of mercury ions, silver ions, and lead ions.
The primer pair of claim 1, wherein the aptamer is an aptamer including a thymine-thymine base pair, and the signal material is mercury ion.
A method for detecting target nucleic acid based on electrochemical real-time polymerase chain reaction using the aptamer-signal modification primer of claim 1, comprising the following steps:
(a) (i) target nucleic acids; (ii) the aptamer-signal modification primer pair of claim 1; (iii) nucleic acid polymerase; And (iv) performing a real-time polymerase chain reaction using a composition comprising deoxynucleotide triphosphate (dNTP) to release the signal material from the aptamer-signal modification primer; And
(b) detecting or quantifying target nucleic acids by measuring changes in electrochemical signals generated by the emitted signal material.
The method of claim 4, wherein the polymerase chain reaction is performed in a chamber on a diagnostic chip on which electrodes are patterned.
The method of claim 4, wherein the signal material generating the electrochemical signal is selected from the group consisting of mercury ions, silver ions, and lead ions.
The method of claim 4, wherein the aptamer is an aptamer including a thymine-thymine base pair, and the signal material is mercury ion.
(i) target nucleic acids; (ii) the aptamer-signal modification primer pair of claim 1; (iii) nucleic acid polymerase; And (iv) composition for real-time polymerase chain reaction containing deoxynucleotide triphosphate (dNTP).

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