WO2023096373A1 - Fluorescent labeling material primer set for multiple detection in loop-mediated isothermal amplification method and molecular diagnosis method using same - Google Patents

Abstract

The present invention relates to a fluorescent labeling material primer set for multiple detection in a loop-mediated isothermal amplification method and a molecular diagnosis method using same. In particular, the primer set has a configuration in which a fluorescent agent or a quencher binds to each of the forward and reverse primers of a loop primer, wherein different ones bind to the primers.

Description

Fluorescent labeling material primer set for multiple detection in loop-mediated isothermal amplification method and molecular diagnosis method using the same

The present invention relates to a fluorescent labeling material primer set for multiple detection in a loop-mediated isothermal amplification method and a molecular diagnosis method using the same.

The national research and development projects supporting the present invention are as follows.

Assignment identification number 1465032760

Assignment No. HW20C2068

Ministry Name Ministry of Health and Welfare

Project management (specialized) organization name Korea Health Industry Development Institute

Research project name Infectious disease prevention technology development (R&D)

Research project name Development of a sample preprocessing-integrated LAMP-type molecular diagnostic device capable of rapid on-site diagnosis

Contribution rate 1/1

Name of the project performing organization Wiz Bio Solution Co., Ltd.

Research Period 2020.09.01 ~ 2023.02.28

Polymerase chain reaction (PCR) is a molecular biology technique that enables the amplification of nucleotides for various analytical purposes. Quantitative PCR (qPCR) is a type of PCR that can monitor the amplification of target nucleotides. Diagnostic qPCR has been applied to detect nucleotides representing infectious diseases, cancers and genetic abnormalities. Reverse transcription PCR (RT-PCR) is a type of qPCR that allows detection of target RNA nucleotides. Because of these capabilities, RT-PCR has the advantage of being suitable for detecting viral pathogens. However, RT-PCR requires a considerable level of equipment that cannot be used in a specific point-of-care environment, and RT-PCR has limitations in that it requires considerable resources such as trained personnel and considerable sample preparation.

In contrast, Loop-mediated isothermal amplification (LAMP) is a more simplified approach to the diagnostic identification of target nucleotides. In addition to using an isothermal heating process, LAMP can use a simple visual output test indicator such as color change instead of the complex fluorescent indicator used in PCR. Reverse transcription-LAMP (RT-LAMP) can be used to identify target nucleotides from RNA, like RT-PCR, and can therefore be used as a diagnostic method to identify the presence or absence of viral pathogens. Because LAMP is simpler, it can be performed with less equipment and sample preparation, making it more accessible for use in point-of-care settings such as clinics and emergency rooms.

The loop-mediated isothermal amplification method simply incubates a mixture of a target gene, 4 or 6 different primers, Bst DNA polymerase, and a substrate to specifically amplify under isothermal conditions (60 to 65° C.). Next, by visually evaluating the turbidity or fluorescence of the reaction mixture stored in the reaction tube, it is possible to confirm whether the target DNA is present, that is, whether the desired target nucleotide is synthesized.

However, the conventional loop-mediated isothermal amplification method has limitations in labeling due to the large number of primer sets required for the synthesis of one target, making multiplex detection difficult. However, there was difficulty in detecting multiple targets at the same time.

The present invention has been devised to solve the above problems, and an object to be solved in the present invention is to minimize labeling to smoothly implement multiplex detection.

In order to solve the above problems, the present invention provides a fluorescent labeling material primer set for multiple detection, which includes an external primer, an internal primer, and a loop primer, each of which includes forward and reverse primers, wherein the loop primer A fluorescent agent or a quencher is bound to each of the forward and reverse primers, but a technical feature is that different things bind to each other.

In order to solve the above problems, the loop primer of the present invention, i) a fluorescent agent is bound to the forward primer and a quencher is bound to the reverse primer, or ii) a quencher is bound to the forward primer and fluorescence to the reverse primer What I combine is a technical feature.

In order to solve the above problems, the fluorescent agent or quencher of the present invention is characterized in that it binds to the end or middle of the primer.

In order to solve the above problems, the fluorescent label material primer set for multiple detection of the present invention is characterized by a technical feature for detecting SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2).

In order to solve the above problems, the fluorescent marker primer set for multiple detection of the present invention targets at least two or more genes selected from the group consisting of RdRP, ORF1a, ORF1b, ORF1ab, S, E, M and N It is a technical feature that

In order to solve the above problems, the loop primers of the present invention are characterized in that they include SEQ ID NOs: 7, 8, 15 and 16.

In order to solve the above problems, the present invention provides a molecular diagnosis method comprising the steps of (1) separating DNA from a biological sample; (2) amplifying the target sequence included in the separated DNA by performing loop-mediated isothermal amplification using the primer set according to any one of claims 1 to 6; and (3) detecting the amplified product.

In order to solve the above problems, the present invention provides a molecular diagnosis method comprising the steps of (1) extracting RNA from a virus and then performing reverse transcription to obtain DNA; (2) amplifying the target sequence included in the obtained DNA by performing loop-mediated isothermal amplification using the primer set according to any one of claims 1 to 6; and (3) detecting the amplified product.

In the present invention, by performing a loop-mediated isothermal amplification reaction using a fluorescent labeling primer set for multiplex detection, multiplex amplification for amplifying at least two or more target genes can be implemented through LAMP. In addition, by maximizing the LAMP method that can be implemented with simple equipment, the effect of molecular diagnosis can be maximized in the field, and more sensitive and accurate diagnosis results can be derived through multiple detection.

1 to 4 are schematic diagrams showing a loop-mediated isothermal amplification process for multiplex detection according to the present invention.

Figure 5 is a graph showing the results of double-detection LAMP

6 is a graph showing triple-detection LAMP results

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 and the experimental methods described below are those well known and commonly used in the art.

As used herein, the term "Loop-mediated isothermal amplification (LAMP)" refers to a method of synthesizing DNA while maintaining a constant temperature. Specifically, 4 types of primers (F3, B3, FIP, BIP) are basically required for the LAMP　 reaction, and 2 types of primers (LF, LB) are added to improve the reaction rate to obtain the final 6 types of different bases. An oligonucleotide primer consisting of the sequence is required for the reaction. The four basic primers are composed of two outer primers and two inner primers, and the outer primers include a forward outer (F3) primer and two backward outer (B3) primers. It is composed of and serves to unwind DNA double strands during the non-cyclic step of the reaction. The inner primer consists of two types, a forward inner primer (FIP) and a reverse inner primer (BIP), and is composed of nucleotides corresponding to the forward and reverse nucleotide sequences to form an essential loop for LAMP. It consists of The additional two primers consist of a forward loop (LF) primer and two backward loop (LB) primers, and are attached to a base sequence to which the inner primer does not bind to perform a loop-mediated isothermal amplification reaction. accelerate Amplification of the amplified DNA product is confirmed by using a reagent for detecting fluorescence or by precipitation reaction.

In one embodiment, a LAMP may use a set of four primers. These four primers can recognize 6 distinct sequences. Primers for the LAMP reaction can be internal primers (eg FIP and BIP) and external primers (eg F3 and B3). A LAMP reaction can be initiated by hybridization of each internal primer (eg FIP or BIP) to its respective priming site (eg F2c or B2c) on the target DNA. An outer primer (eg, F3 or B3) secondarily hybridizes to its priming site (eg, F3c or B3c) on the target DNA and creates a novel complementary sequence that displaces DNA sequences already extended from the inner primer. Initiate synthesis. The result is a DNA sequence capable of forming a stem-loop structure at both ends. This auto-primed "dumb-bell" structure is the starting material for LAMP auto-cycling amplification.

The LAMP reaction can also be promoted using additional primers, referred to as loop primers. A loop primer can hybridize to a section of the loop transcribed from a target DNA template. This additional priming promotes the LAMP reaction and, as it requires transcription of the correct starting material, can improve LAMP selectivity. In one embodiment, the LAMP reaction may be carried out under isothermal conditions and at a temperature value selected within a desired range, for example, from about 60°C to about 70°C, more preferably from about 60°C to about 65°C. . The amplification product can be stem-loop DNA, which usually has several inverted repeats of the target. These inverted repeats usually represent cauliflower-like structures with multiple loops.

Various mechanisms have been developed to detect positive amplification of DNA by LAMP. In one aspect, positive amplification of DNA can be monitored in real time using intercalating dyes such as SYBR Green or EvaGreen ^® .

As used herein, the term “multiplex LAMP” refers to LAMP that simultaneously detects various genes. For example, in the case of SARS-CoV-2, the presence or absence of new coronavirus infection is confirmed by confirming the amplification products of two or more genes selected from the group consisting of RdRP, ORF1a, ORF1b, ORF1ab, S, E, M, and N genes in the sample. can determine. More preferably, two or more genes among E, N, and RdRp genes may be targeted.

As used herein, the term “inner primer” refers to a single-stranded oligonucleotide capable of binding to template DNA and acting as a starting point for synthesizing a new DNA chain.

As used herein, the term “outer primer” refers to a single-stranded oligonucleotide that binds to the template DNA outside the site where the internal primer binds to the template DNA. After the chain is elongated, strand displacement occurs due to the combination of the external primer and the template DNA, so that the previously formed chain is separated.

As used herein, the term “loop primer” means that the initial stem loop structure chain formed by binding the internal and external primers to the template DNA increases the number of loop structures, resulting in It refers to a single-stranded oligonucleotide that can act as a starting point for nucleotide synthesis, allowing the overall reaction to be accelerated.

As used herein, the term "fluorescence" or "reporter" refers to a nucleotide containing a fluorescent agent or the fluorescent agent itself. Fluorescent agents include FAM (6-carboxyfluorescein), Cy5, SYBR, Texas red, fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4 ,7-dichlorofluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, fluorescein isothiocyanate (FITC), Oregon green (oregon green), alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein) ), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), selected from the group consisting of cyanine-based dyes and thiadicarbocyanine It may be at least one of More preferably, FAM, HEX, Cy5 and Rox can be used.

As used herein, the term “quencher” refers to a substance that inhibits the development of fluorescence.

As an embodiment of the present invention, a virus to be diagnosed is a corona virus. In some embodiments, the coronavirus is selected from the group consisting of alphacoronavirus, betacoronavirus, deltacoronavirus, and gammacoronavirus. Examples of alphacoronaviruses include bat coronavirus CDPHE15, bat coronavirus HKU10, human coronavirus 229E, human coronavirus NL63, long-winged bat coronavirus 1, long-winged bat coronavirus HKU8, mink coronavirus 1, swine epidemic diarrhea virus, horseshoe bat coronavirus HKU2, and yellow bat coronavirus 512. Examples of betacoronaviruses include betacoronavirus 1, hedgehog coronavirus 1, human coronavirus HKU1, coronavirus associated with Middle East respiratory syndrome, murine coronavirus, house bat coronavirus HKU5, russet bat coronavirus HKU9, associated with severe acute respiratory syndrome. corona virus, bamboo bat coronavirus HKU4, but is not limited thereto. Examples of deltacoronaviruses include japonica corona virus HKU11, coot coronavirus HKU21, coronavirus HKU15, kinbara coronavirus HKU13, egret coronavirus HKU19, thrush coronavirus HKU12, blackbird coronavirus HKU16, and red-headed duck coronavirus HKU20. It may include, but is not limited to. Examples of gammacoronaviruses may include, but are not limited to, avian coronavirus, beluga coronavirus SW1. Additional examples of coronaviruses may include MERS-CoV, SARS-CoV, and SARS-CoV-2. In some examples, the coronavirus may be SARS-CoV-2. That is, it is possible to diagnose viruses at the molecular level without limitation for viruses with known marker genes as previously known viruses.

To perform the LAMP according to the present invention, reagents such as reverse transcriptase, DNA polymerase, dNTPs and buffers may be included. As the DNA polymerase according to an embodiment of the present invention, Bst polymerase, GspSSD DNA polymerase, etc. may be used, but is not limited thereto. For other specific methods of performing LAMP, refer to the following literature; Mautner, L., Baillie, CK., Herold, HM et al. Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP). Virol J 17, 160 (2020).

In the present invention, amplification/presence of a gene to be amplified/confirmed can be confirmed by binding a fluorescent agent or a quencher to each of the forward and reverse primers of the loop primer. Specifically, the primary replication of the target gene is performed to form a loop, and a forward loop primer (fluorescent agent binding, FLPQ) and a reverse loop primer (quencher binding, FLPR) complementary thereto are present ( 1), a forward loop primer (linked with a fluorophore) is located in the loop of the copied genetic material (see FIG. 2), and then DNA synthesis occurs as a polymerase binds to a reverse loop primer (linked with a quencher, FLPQ). The fluorescent agent develops color as it is separated (see FIG. 3). That is, before amplification, the forward and reverse loop primers bind to maintain a state in which the quencher suppresses fluorescence, and as the amplification proceeds according to the LAMP reaction, the forward loop primer becomes part of the amplification product, and as it binds to the product, it is separated from the quencher and fluorescence emit a signal As this process is repeated, as the target gene having the loop is finally amplified, the degree of color development of the fluorescent agent increases, thereby confirming the amplification of the target gene (see FIG. 4).

Hereinafter, the present invention will be described through examples. The target genes of LAMP performed in the examples were ORF1ab and N, which are representative markers of SARS-CoV-2, and B-actin was used as a control to confirm that they were human genes.

Example 1. Primer Design

The nucleotide sequence of the primer for ORF1ab is shown in Table 1 below. Bases marked in dark color in the base sequence are portions to which a fluorescent or quencher is attached, and are the same below. For fluorescence of ORF1ab, FAM was used.

Table 1

The nucleotide sequence of the primer for N is as shown in Table 2 below. For N fluorescence, Cy5 was used.

Table 2

Base sequences of primers for B-actin are shown in Table 3 below.

Table 3

Example 2. Multi-detection-LAMP A mixture was prepared with the composition shown in Table 4, and then primers having the composition shown in Table 5 (double-detection) or Table 6 (triple-detection) were added to the mixture and mixed. And LAMP was performed by reacting at a temperature of 65 ℃ for 40 minutes in CFX96™ (BIO-RAD).

Table 4

^* SARS-CoV-2 RNA standard (KRISS, 111-10-506)

^** 20 mM Tris-Cl (pH 8.8), 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Tween 20, 150 mM KCl

^*** Human Genomic DNA (Takara, Cat#636401)

table 5

table 6

The results of performing double-detection LAMP targeting ORF1ab and N are shown in Table 7 and FIG. 5 .

table 7

As a result, as shown in FIG. 5, each target gene was amplified through FAM fluorescence signal (ORF1ab label, see (A) in FIG. 5) and Cy5 fluorescence signal (N label, see (B) in FIG. 5). Confirmed. In addition, the fidelity of the amplification pattern and simultaneous amplification was confirmed by simultaneously confirming the two fluorescence signals (see FIG. 5(C)).

The results of triple-detection LAMP targeting ORF1ab, N and B-actin are shown in Table 8 and FIG. 6 .

Table 8

As a result, it was confirmed that the target genes (ORF1ab and N) were amplified, as shown in FIG. 6, and B-actin was detected through electrophoresis without fluorescent labeling.

In the present invention, diagnosis can be performed at the molecular level using such a primer set. Specifically, the molecular diagnosis method includes (1) isolating DNA from a biological sample; (2) amplifying the target sequence included in the separated DNA by performing loop-mediated isothermal amplification using the primer set according to any one of claims 1 to 6; and (3) detecting the amplified product.

In addition, the molecular diagnosis method, when the biological sample is a virus, (1) extracting RNA from the virus and then reverse transcribing it to obtain DNA; (2) amplifying the target sequence included in the obtained DNA by performing loop-mediated isothermal amplification using the primer set according to any one of claims 1 to 6; and (3) detecting the amplified product.

The biological sample in step (1) is preferably a body fluid, cell, etc. containing DNA or RNA collected from humans, non-human animals and plants, and the sample contains a specific virus or the nucleic acid of the virus. there is. In particular, in the case of viruses, since nucleic acids are included in RNA, a process of reverse transcription of viral RNA into DNA may be required.

Amplification in step (2) means performing LAMP according to the present invention, and preferably refers to performing LAMP using the primer set according to the present invention.

The detection of the amplified nucleic acid in step (3) may use at least one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement, but is not limited thereto.

Claims (8)

1. In a fluorescent labeling material primer set for multiple detection, including an external primer, an internal primer, and a loop primer, each of which includes forward and reverse primers,

   A fluorescent labeling material primer set for multiple detection, wherein a fluorescent agent or a quencher is bound to each of the forward and reverse primers of the loop primer, but different ones bind to each other.
2. The method of claim 1,

   The loop primer,

   i) a fluorescent agent binds to the forward primer and a quencher binds to the reverse primer, or

   ii) a fluorescent labeling material primer set for multiple detection, wherein a quencher binds to the forward primer and a fluorescent agent binds to the reverse primer.
3. The method of claim 1,

   The fluorescent agent or quencher is to bind to the end or middle of the primer, a fluorescent labeling material primer set for multiple detection.
4. The method of claim 2,

   The fluorescent marker primer set for multiple detection is for detecting SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a fluorescent marker primer set for multiple detection.
5. The method of claim 4,

   The fluorescent marker primer set for multiple detection targets at least two or more genes selected from the group consisting of RdRP, ORF1a, ORF1b, ORF1ab, S, E, M and N, a fluorescent label for multiple detection Substance Primer Set.
6. The method of claim 5,

   The loop primer comprises SEQ ID NOs: 7, 8, 15 and 16, a fluorescent labeling material primer set for multiple detection.
7. (1) isolating DNA from a biological sample;

   (2) amplifying the target sequence included in the separated DNA by performing loop-mediated isothermal amplification using the primer set according to any one of claims 1 to 6; and

   (3) a molecular diagnosis method comprising the step of detecting the amplified product.
8. (1) extracting RNA from the virus and then performing reverse transcription to obtain DNA;

   (2) amplifying the target sequence included in the obtained DNA by performing loop-mediated isothermal amplification using the primer set according to any one of claims 1 to 6; and

   (3) a molecular diagnosis method comprising the step of detecting the amplified product.

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