KR102494777B1 - A Primer set for Diagnosis of Dengue Virus Infectious Disease based on Multiple Isothermal Amplification, A Composition for Diagnosing Denque Virus Infectious Disease including the same, and A Visual Diagnosis method for Dengue Virus Infectious Disease using the same - Google Patents

Abstract

The present invention relates to a composition for diagnosing a dengue virus infectious disease, a primer set for diagnosis based on multiple isothermal amplification, and a kit using the same with improved speed, accuracy, and portability, and a visual diagnosis method using the diagnostic kit. Specifically, by using a composition and a kit containing a primer set capable of detecting dengue virus causing dengue virus infectious diseases such as dengue fever or dengue hemorrhagic fever, dengue virus is detected more accurately, and dengue virus infectious diseases are rapidly and inexpensively can be diagnosed.

Description

A Primer set for Diagnosis of Dengue Virus Infectious Disease based on Multiple Isothermal Amplification, A Composition for Diagnosing Denque Virus Infectious Disease including the same, and A Visual Diagnosis method for Dengue Virus Infectious Disease using the same}

It relates to a composition for diagnosing a dengue virus infectious disease, a primer set for diagnosis based on multiple isothermal amplification, and a visual diagnosis method using a kit with improved speed, accuracy, and portability using the same, and a diagnostic kit. Specifically, by using the primer set included in the composition of the present invention, it is possible to more accurately detect dengue virus, which is a causative bacterium of diseases such as dengue fever or dengue hemorrhagic fever, and diagnose dengue virus infectious diseases quickly and inexpensively.

Dengue virus (DENV) is a single positive-stranded RNA virus of the genus Flaviviridae , which is transmitted by mosquitoes and is the cause of dengue fever. Dengue fever is caused by dengue mosquitoes, which live mainly in tropical and subtropical regions, biting a person with the virus during the day and then biting another person to transmit the virus.

Symptoms of dengue fever appear after an incubation period of 3 to 14 days, such as fever, rash, headache, muscle and joint pain, and loss of appetite. Dengue fever itself rarely leads to death, but when bleeding spots on the skin, epistaxis, bleeding gums, excessive menstruation, 'dengue hemorrhagic fever' in which bleeding occurs in various parts of the body, and 'dengue shock syndrome', in which blood pressure drops along with bleeding, mortality occurs it rises Dengue virus is known to have four serotypes, and a fifth was discovered, but the specific genetic sequence has not yet been confirmed.

All four serotypes of dengue virus are known to be capable of causing dengue virus-related infections. However, it is known that there are 47 strains of dengue virus, and in the case of dengue virus-infected patients, there are many cases of concurrent infection with Zika virus and chikungunya, so test results are often confused, and a means to quickly test Since this does not exist, it is very difficult to diagnose and treat patients. In addition, there is no vaccination or clear treatment yet, so we have no choice but to rely on rapid diagnosis to block local infection.

Dengue virus has increased dramatically over the past 20 years, making it one of the worst mosquito-borne human pathogens tropical countries have to deal with. Current estimates suggest that there are 390 million infections each year, and many dengue virus infections are asymptomatic and increasing.

Dengue virus infection can be determined by detecting dengue antibody through a blood test. Since the antibody is detected in the blood several days after symptoms of dengue fever appear, early diagnosis using the antibody is impossible.

Therefore, it is important to differentiate dengue virus from other mosquito-borne human pathogens and diagnose it at an early stage in order to apply appropriate treatment methods to patients and minimize local infection.

An object of the present invention is to provide a primer set for diagnosing a dengue virus infectious disease.

Another object of the present invention is to provide a composition for diagnosing dengue virus infectious diseases comprising the primer set.

Another object of the present invention is to provide a kit for diagnosing dengue virus infectious diseases including the primer set.

Another object of the present invention is to provide a method for diagnosing a dengue virus infectious disease from a biological sample using the primer set.

The terminology used herein is intended only to refer to specific embodiments and is not intended to limit the present invention. As used herein, the singular forms also include the plural forms unless the phrases clearly indicate the opposite. As used herein, the meaning of "comprising" specifies specific characteristics, regions, integers, steps, operations, elements and/or components, and other specific characteristics, regions, integers, steps, operations, elements, components and/or groups. does not exclude the presence or addition of

Although not defined differently, all terms including technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Terms defined in commonly used dictionaries are additionally interpreted as having meanings consistent with related technical literature and currently disclosed content, and are not interpreted in ideal or very formal meanings unless defined.

Hereinafter, the present invention will be described.

One embodiment for solving the above problems provides a primer set for diagnosing a Dengue virus infectious disease including 6 primers. A primer set composed of the six primers includes a primer set represented by the nucleotide sequences of SEQ ID NOs: 1 to 6; Primer sets represented by the nucleotide sequences of SEQ ID NOs: 7 to 12; Primer sets represented by the nucleotide sequences of SEQ ID NOs: 13 to 18; and a primer set represented by the nucleotide sequences of SEQ ID NOs: 19 to 24.

The dengue virus infectious disease may be dengue fever or dengue hemorrhagic fever.

The primer set may be for detecting dengue virus more specifically. The dengue virus can be divided into serotypes classified into DENV-1, 2, 3, and 4, and the primer sets may be for detecting different serotypes, respectively. That is, SEQ ID NOs: 1 to 6 may detect serotype 1, SEQ ID NOs 7 to 12 may detect serotype 2, SEQ ID NOs 13 to 18 may detect serotype 3, and SEQ ID NOs 19 to 24 may detect serotype 4.

Since each primer set is designed to have specificity only for the target serotype, four primer sets can be used simultaneously, and serotypes 1 to 4 can be individually detected at the same time by attaching different markers to each primer set.

Dengue virus serotype information was obtained from GenBank (National Center for Biotechnology Information), and GenBank IDs were dengue virus serotype 1 (FJ687475), serotype 2 (KP406804), serotype 3 (KP406805) and serotype 4 (KP406806) am.

The term "primer" refers to a short polynucleotide having a free 3' hydroxyl group, capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. can Primers can initiate DNA synthesis in the presence of reagents (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) for polymerization at an appropriate buffer solution and temperature.

Each primer in the primer set is specific to the target gene, and the term "gene specific" or "complementary" refers to 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more of the target gene. It can mean that % base pairing can be achieved.

In the primer set, each primer may consist of or include one of the nucleotide sequences of SEQ ID NOs: 1 to 24, and as long as it maintains specific binding to the genetic material of dengue virus, SEQ ID NOs: 1 to 24 It may have 80% or more, 90% or more, 95% or more, 97% or more or 99% or more homology with the nucleotide sequence described in.

The primers may also be modified to include additional features within the range of not changing the basic properties to act as starting points for gene synthesis. Accordingly, the primer includes the nucleotide sequence of each defined SEQ ID NO and includes an additional nucleotide sequence of about 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, or 1 to 5 bp, at the 3' end of the primer, It may be further included at the 5' end or inside the nucleotide sequence, and if necessary, a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means may be further included at each end or a specific base. can

Examples of the label may be a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid. The chromogenic enzyme may be peroxidase, alkaline phosphatase or acid phosphatase, and the fluorescent substance may be thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6 -yl, 2', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6-yl, dihydrotetramethylrosamine-4-yl, tetramethylrhodamine-5 -yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl or 4,4- Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl, Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC) , Rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin), or rhodamine, and the radioactive isotope may be 32P, but is not limited thereto.

The primer set of the present invention may be suitable for use for isothermal amplification.

The term “isothermal amplification” refers to amplification of DNA under one temperature condition. Unlike the existing polymerase chain reaction (PCR), which requires a special PCR device as the denaturation, annealing, and extension steps require different temperatures and conditions, the isothermal amplification reaction is separate It has the advantage of not requiring special equipment. In general, the isothermal amplification method can amplify up to 10^9 target nucleic acids within 1 hour within a specific temperature range, and can be performed with a constant temperature device capable of maintaining the temperature. In addition, the isothermal amplification method has a short execution time and can be observed with the naked eye, so amplification can be easily confirmed.

In one embodiment, the isothermal amplification may be loop-mediated isothermal amplification.

The term "loop-mediated isothermal amplification (LAMP)" method refers to an amplification method performed isothermally using an external primer, an internal primer, and an additional loop primer to enhance the reaction rate.

Among the primers used in the LAMP reaction, the outer primer consists of two types of forward outer (F3) primer and reverse outer (B3) primer, and DNA double strands are formed during the non-cyclic step of the reaction. serves as a release. The inner primer consists of two types, a forward inner primer (FIP) and a backward inner primer (BIP). It is composed of the corresponding nucleotide. These internal primers are not included in the genetic material of the strain, but can be designed to further include a specific base, for example, a series of thymine bases, to increase the efficiency of the reaction. The additional two primers consist of a forward loop (LoopF) primer and two backward loop (LoopB) primers, and are attached to a nucleotide sequence (ring site) to which the inner primer does not bind, thereby intermediating the loop. Accelerates the isothermal amplification reaction.

In the loop-mediated isothermal amplification method, an internal primer is first bound to a template and then extended, and then an external primer is coupled to the outside of each DNA helix and then extended. At this time, strand displacement occurs due to extension of the external primer, and accordingly, the first synthesized strand is separated from the template. The separated synthetic strand forms a loop structure from the 5' end, and then also from the 3' end In the same process, a ring structure is formed, through which an amplification reaction takes place.

The isothermal amplification or loop-mediated isothermal amplification may be a reverse transcription reaction. Specifically, since the dengue virus is an RNA virus, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to which a reverse transcription process is added can be used to amplify it.

The isothermal amplification reaction may be performed at 50 to 75°C, 55 to 75°C, 60 to 72°C, 60 to 72°C, or 63 to 68°C. In addition, the isothermal amplification method may be performed for 10 minutes to 60 minutes, 10 minutes to 40 minutes, 20 minutes to 40 minutes, or 25 minutes to 35 minutes. When using the primer set, the Ct value may be 50 or less, 40 or less, or 30 or less.

In one embodiment, since the primer set has a very high detection sensitivity, when using the primer set, based on 25 μl reaction solution, 100 copies or less, 50 copies or less, 20 copies or less, 10 copies or less, 5 copies or less, Alternatively, it is possible to detect dengue virus in samples containing target genes such as DNA or RNA with 0.1 copies or more, 0.01 copies or more, or 0.001 copies or more, while less than 1 copy. In addition, it is possible to detect dengue virus in a short time under general isothermal amplification conditions.

For example, to detect dengue virus, six primer sets (F3, B3, FIP, BIP, LF, BF) that bind to the target genetic material, isothermal amplification buffer, dNTPs, and Bst 3.0 DNA polymerase are included. LAMP reaction composition, Phenol red for colorimetric analysis, SYBR™ Green I (Thermo Fisher Scientific, USA) for real-time fluorescence analysis, and the like, using 0.2 to 1.6 μM of each primer of the primer set, 63 to 1.6 μM Detection results can be confirmed by isothermal amplification at 68 °C for 25 to 35 minutes. In the primer set, each primer may be preferably used in an amount of 1.6 μM for each of the FIP and BIP primers, 0.2 μM for each of the F3 and B3 primers, and 0.4 μM for each of the FL and BL primers. Since the isothermal amplification reaction must produce results within a short time in an isothermal limited environment, the combination of primers and the ratio of each component are important factors to obtain significant reaction results.

The primer sets are SEQ ID NOs: 1 to 6 for DENV-1, SEQ ID NOs: 7 to 12 for DENV-2, SEQ ID NOs: 13 to 18 for DNEV-3, and SEQ ID NO: 19 for DNEV-4, depending on the diagnostic target serotype. to 24 can be used, and can be used as a method of simultaneously detecting all DNEV-1 to 4 serotypes using different markers for each primer set.

Another embodiment of the present invention includes a composition or kit for diagnosing a dengue virus infectious disease including the primer set.

The composition or kit for diagnosing a dengue virus infectious disease may further include appropriate reactants and reagents for performing an isothermal amplification reaction. These reactants and reagents may include, for example, additional primers, dNTPs, and enzymes (DNA polymerase or reverse transcriptase), and inorganic salts such as potassium chloride, magnesium sulfate, and ammonium sulfate for reaction efficiency. A surfactant may be further included.

In one embodiment, the reactants and reagents are 10 to 20 mM Tris-HCl, 1 to 10 mM (NH ₄ ) ₂ SO ₄ , 10 to 50 nM KCl, 0.1 to 2 mM MgSO ₄ , and 0.01 to 0.1% Tween- based on the final reaction solution. 20, more specifically 20mM Tris-HCl, 10mM (NH ₄ ) ₂ SO ₄ , 50nM KCl, 2mM MgSO ₄ , and 0.1% Tween-20, and may be a solution of pH 7 to 9, preferably pH 8.8. .

The reactants or reagents may further include 40 mM to 50 mM trehalose, 0.001 to 0.01% Triton X-100, and 0.1 w/w% to 0.3 w/w% glycerol. The combination of trehalose, Triton X-100, and glycerol helps to maintain reaction efficiency by increasing the storage stability of the composition and kit for diagnosing dengue virus infectious diseases.

In one embodiment, the composition or kit for diagnosing a dengue virus infectious disease includes a reagent such as a colorimetric material, a fluorescence material, or a labeling compound for detection in order to enhance the convenience of detection and the clarity of result analysis, and visually confirm the reaction product. can include more. As such reagents, commonly known or commercially available ones may be used, for example, WarmStart® LAMP Mix, but is not limited thereto. These reagents may include Tris-HCl, (NH ₄ ) ₂ SO ₄ , KCl, MgSO ₄ , and Tween-20 as described above, and DNA polymerase, reverse transcription, etc. It may further include an enzyme for performing an amplification reaction such as an enzyme. For colorimetric analysis, Simpliamp ^TM Thermal Cycler/QuantStudio ^TM 3 may be used, and for fluorescence analysis, CFX96 ^TM may be used, but is not limited thereto.

In addition, methods for confirming the detection result of the composition or kit for diagnosing dengue virus infectious diseases include electrophoresis, radioactivity measurement using a detection label, etc., but are not limited thereto. For colorimetric analysis, a conventional PCR device (Thermocycler) can be used, and for fluorescence analysis, a real-time PCR device capable of detecting a fluorescent substance used for analysis can be used.

The composition or kit for diagnosing a dengue virus infectious disease may further include a negative control material or a positive control material in order to accurately read the diagnosis result. As a negative control material, template RNA or DNA of another strain unrelated to dengue virus can be used, and as a positive control material, a biological sample obtained from a patient infected with dengue virus or template RNA or DNA corresponding to a part of the genome of dengue virus is available. These control substances can be applied to the gene amplification reaction separately or together with the biological sample in one tube, if necessary.

In another embodiment of the present invention, mixing the primer set or a composition including the primer set and a biological sample; And it provides a method for diagnosing a dengue virus infectious disease, comprising the step of isothermally amplifying the mixture to amplify the gene. The method for diagnosing the dengue virus infectious disease may further include reverse transcription before the isothermal amplification reaction. In addition, the method for diagnosing the dengue virus infectious disease may further include the step of colorimetric analysis or fluorescence analysis.

The biological sample is obtained from the subject to be tested, obtained through swab or nasal irrigation from the nasopharynx or oropharynx, bronchial lavage or aspirate, intertracheal aspirate and bronchial biopsy, sputum, bronchoalveolar lavage , saliva, blood, urine, feces, sweat, etc.

The biological sample may be pretreated to increase the efficiency of the amplification reaction. Pretreatment may be 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold dilution with water, heat treatment after treatment with protenase K, anion exchange chromatography, affinity chromatography, size-specific exclusion chromatography, Genes may be extracted from biological samples or purified to a certain level using commonly known methods such as liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis, or using commercially available kits.

When RNA is extracted from a biological sample and used, a step of reverse transcription into cDNA may be further included before amplifying the target gene. The pretreatment is a lysis buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl, 5mM EDTA, and 1% NP-40, 0.1% to 0.5w/w% Brij-35, 10mM to 300mM Tris-HCl (pH 7.0 to 8.0), and CaCl ₂ 1 mM to 10 mM, specifically Brij-35 0.3 w/w%, Tris-HCl 100 mM (pH 7.5), and CaCl ₂ 5 mM. And, specifically, it may mean obtaining only genes from a sample by treating with the solution and then purifying by a conventional method. The combination of the pretreatment solutions can facilitate easy extraction of genes from samples and enhance the isothermal amplification reaction efficiency of the primer set of the present invention. The pre-treatment of samples obtained from virus-infected individuals may further include a conventional RNA extraction process using, for example, a QIAamp® Viral RNA Mini Kit (QIAGEN, Germany).

The dengue virus infectious disease diagnosis method includes detecting an amplified gene. Detection of the amplification product is fluorescence measurement using a fluorescent material or fluorescent marker such as SYBR Green I, color change of an indicator such as phenol red, turbidity, precipitate formation, DNA laddering, capillary electrophoresis, DNA chip, gel electrophoresis, It may be performed through radiometric measurement or phosphorescence measurement, but is not limited thereto.

The kit of the present invention may further include a container containing each reaction reagent, a tool for use in the test, and a user guide describing optimal reaction conditions.

Dengue virus infectious diseases can be specifically detected using the primer set of the present invention, and rapid and accurate on-site detection is possible using an isothermal amplification reaction.

In addition, the isothermal amplification reaction does not require expensive equipment and specialized personnel required to meet specific temperatures and conditions for carrying out the amplification reaction, so it can be performed efficiently and at low cost, thereby diagnosing a large number of test subjects at an early stage. can do.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Preparation Example 1: Preparation of Primer

Primers were designed and produced to detect dengue virus using loop-mediated isothermal amplification (LAMP).

In order to obtain a primer set candidate group, the genetic information of dengue virus was confirmed through blast analysis, and the most efficient sequence among them was derived for each serum. Specifically, the entire nucleotide sequence of each type of dengue virus was obtained from GenBank (National Center for Biotechnology Information), and dengue virus serotype 1 (FJ687475), serotype 2 (KP406804), serotype 3 (KP406805) and serotype 4 ( KP406806) was provided by the Korea Centers for Disease Control (Cheongju) and the Korea Pathogenic Virus Bank (Seoul).

Primer sets predicted to have a high probability of success were selected in order of top priority, 3 sets for each serum (a total of 12 primer set candidates). The primer candidate group included an external primer, an internal primer, and a loop primer, and four thymines were included inside the FIP primer and the BIP primer to promote loop formation and increase the reaction rate.

Primers for specifically amplifying DENV RNA were designed using the Primer Explorer V5 program on the Eiken website (http://primerexplorer.jp/e/). Primers for specific amplification were identified using BLAST (basic local alignment search tool), and all designed primers were synthesized from Bionics.

Test Example 1: Selection of primer sets

The primer set was finally selected by conducting the following tests on the primer set candidates derived in Preparation Example 1.

(1) Isothermal amplification reaction

The reverse transcription loop-mediated isothermal amplification reaction was performed using a reaction solution in which the following solutions were mixed in a 25 μl PCR tube; 1 μl each of forward and backward outer primers (F3 and B3, 0.2 μM), 1 μl each of inner primers (FIP and BIP, 1.6 μM), loop primers (Loop primer: LF and LB, 0.4 μM) 1 μl each, 2X isothermal amplification solution 12.5 μl, SYBR Green I (25X) or Phenol red (250 μM) 1 μl, purified water (DW) 3.5 μl, and purified DENV RNA solution 2.0 μl Thus, a LAMP reaction mixture was prepared in 25 μl for each reaction.

Purified viral RNA of dengue virus was purchased from ATCC (American Type Culture Collection, USA). Viral RNA was extracted using the QIAamp® Viral RNA Mini Kit (QIAGEN, Germany), and primers for diagnosing dengue virus infection were synthesized from Bionics (Daejeon, Korea).

2X Master Mix (isothermal amplification reaction solution) was prepared and used by the company, and based on the final reaction solution, 20mM Tris-HCl, 10mM (NH ₄ ) ₂ SO ₄ , 50nM KCl, 2mM MgSO ₄ , and 0.1% Tween-20, pH 8.8 becomes Bst 3.0 DNA polymerase and dNTPs were purchased from New England Biolabs (USA). Phenol red, a colorimetric reagent, was purchased from Sigma-Aldrich (USA), and SYBR™ Green I, a fluorescent dye, was purchased from Thermo Fisher Scientific (USA). Distilled water added to the preparation of the reaction solution and the reaction was produced and used through an ultrapure water production device (New-P.NIX® Power, Daehan Science, Korea).

Purified water was used as a negative control, and the LAMP reaction was performed using a Simpliamp ^TM Thermal Cycler (colorimetric analysis)/QuantStudio ^TM 3 and CFX96 ^TM (fluorescence analysis) at a temperature of 65° C. for 30 minutes. RNA concentration was measured using a spectrophotometer (Epoch, Agilent Biotek, USA), and the instruments used for the isothermal amplification reaction were Simpliamp ^TM Thermal Cycler (Thermo Fisher Scientific, USA) for colorimetric analysis and QuantStudio ^TM 3 (for fluorescence analysis). Thermo Fisher Scientific, USA) and Bio-Rad CFX96 ^TM (BIO-RAD, USA) were used.

primer 10X concentration (STOCK) (μM) 1X concentration (FINAL) (μM) FIP 16 1.6 BIP 16 1.6 F3 2 0.2 B3 2 0.2 FL 4 0.4 BL 4 0.4

(2) Occurrence of non-specific reactions (false negative and false positive)

Using negative/positive controls and other mosquito-borne infection samples (Zika virus, Chikungunya virus, and Malaria), primer pairs that did not cause nonspecific reactions in all test items were selected. did The concentration of the sample used for the test was 10 copies/rxn based on the standard material, and the reaction time was 30 minutes.

As a result of repeated examination of three different samples three times, a primer pair was selected with a false diagnosis rate of less than 0.3%.

(3) Colorimetric analysis

Specifically, using Test Example 1 and 10 copies/rnx of RNA of each strain as a template, a mixture solution for an amplification reaction was prepared as in Test Example 1-(1), and 30 minutes, 35 minutes, 40 minutes, 50 minutes, and The interval was divided into 60 minutes, and an isothermal amplification reaction was performed. The method for selecting the reaction time is based on the time at which the color of the reaction solution changes from red to yellow in more than 93/100 reactions per test, and three different samples for each primer are tested 3 times each. Reproducibility was secured as a result of repeated tests.

(4) Fluorescence analysis

The reaction efficiency of the primer was confirmed by setting a sample concentration of 10 copies/rnx and 1 cycle for 1 minute. The method for selecting the reaction time was selected as having a C _t value of 30 or less based on the fluorescent substance detection value threshold of 500 in 93/100 or more reactions per test.

Reproducibility was secured as a result of repeated inspection of three different samples for each designed primer three times.

(5) Sensitivity of isothermal amplification reaction (detection limit)

Purified RNA standards of each serotype of dengue virus (DENV1-4) were purchased from ATCC (American Type Culture Collection, USA), and the concentration (Copy number) of each material was 1,000, 100, 50, 40, 30, Divided into 25, 20, and 10 copy/rxn, the detection limit of the isothermal amplification reaction was confirmed. The concentration showing 93/100 or more reactions based on one test with a reaction time of 30 minutes was set as the limit of detection (LOD).

Reproducibility was secured as a result of repeated inspection of three different samples for each designed primer three times.

(6) Selection of primers

The results are summarized in Table 2, and the base sequences of each primer of the primer sets of Examples 1, 4, 7, and 10, which are selected primers satisfying all conditions, are shown in Table 3.

serotype Base sequence (5'-3') note Example 1 DENV1-F3 TGGGGTAGCAGACTAGTGG SEQ ID NO: 1 DENV1-B3 TCTGTGCCTGGAATGATGC SEQ ID NO: 2 DENV1-FIP CCACCAGGGTACATTTTCCCGACCCCTCCCAAAACACAA SEQ ID NO: 3 DENV1-BIP AGAGGTTAGAGGAGTTTTCCCAGCAGGATCTCTGGTCTCTC SEQ ID NO: 4 DENV1-LF TGGTGTTGGGCCCCGCT SEQ ID NO: 5 DENV1-LB ACAGCATATTGACGCTG SEQ ID NO: 6 Example 4 DENV2-F3 CCCGTCCAAGGACGTTAA SEQ ID NO: 7 DENV2-B3 CTGCTGCGTTGTGTCATG SEQ ID NO: 8 DENV2-FIP ACGACGGAGCTACATTTTGAAGAAGTCAGGCCCAAA SEQ ID NO: 9 DENV2-BIP GGGACGTAAAGCCTGGTTTTCTCTAACCACTAGTCTGCTA SEQ ID NO: 10 DENV2-LF GTTTGCTCAAACCGTGGC SEQ ID NO: 11 DENV2-LB AACCGTGGAAGCTGTACG SEQ ID NO: 12 Example 7 DENV3-F3 GCTGTACGCACGGTGTAG SEQ ID NO: 13 DENV3-B3 CCTGGAATGATGCTGAGGAG SEQ ID NO: 14 DENV3-FIP GGTACAGCTTCCCTCAGTTTTCGTGGTTAGAGGAGACCCCT SEQ ID NO: 15 DENV3-BIP AGAGGTTAGAGGAGACCCTTTTGCAGGATCTCTGGTCTCTC SEQ ID NO: 16 DENV3-LF CTGCTGCGTTGTGTCAT SEQ ID NO: 17 DENV3-LB CAGCATATTGACGCTGG SEQ ID NO: 18 Example 10 DENV4-F3 GCTCCTTTCGAGAGTGAAG SEQ ID NO: 19 DENV4-B3 AGTACAGCTTCCTCCTGG SEQ ID NO: 20 DENV4-FIP CGTTATTGGCGGAGCTATTTTGAGGCTATTGAAGTCAGGC SEQ ID NO: 21 DENV4-BIP GGAGGCGTTAAATTTTCAGGGGTCTCCTCTAACCGCTAGT SEQ ID NO: 22 DENV4-LF CAGCACGGTTTGCTCAAG SEQ ID NO: 23 DENV4-LB CTGTACGCGTGGCATATTG SEQ ID NO: 24

※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer

Test Example 2. Serotype specificity test

In order to confirm that each primer set is specific to the target serotype, an isothermal amplification reaction was performed under the conditions of Test Example 1-(1) at 10 copies/rxn, 65° C., and 30 minutes, and Test Example 1-( Fluorescence analysis was performed as in 4) and marked as positive (O) and negative (X). In order to confirm specific amplification, different fluorescent markers (FAM, HEX, Cy3, Texas Red, respectively) were labeled at the 5' end of the F3 primer among the primer sets of Examples 1, 4, 7, and 10. The results are shown in Table 4 below.

template mix Example 1 Example 4 Example 7 Example 10 serotype 1 O O X X X serotype 2 O X O X X serotype 3 O X X O X serotype 4 O X X X O

As a result, when all the primer sets of Examples 1, 4, 7, and 10 were mixed, it was confirmed to be positive for all serotypes 1 to 4, but when a single primer set was used, it was confirmed to be positive in accordance with each targeted serotype type. /showed a negative aspect. Even when serotypes 1 to 4 were mixed, it was confirmed that there was no change in reaction efficiency. Therefore, the primer set of the present invention enables simultaneous detection of each serotype even when all primer sets corresponding to each serotype are mixed and used.

Test Example 3: Confirmation of Efficiency Change with or without 4T

A change in efficiency was confirmed when the nucleotide sequence was the same as in Table 3, but four thymines were not added. Specifically, Examples 1 and 10 were prepared as primer sets, and Comparative Examples 1 and 2 were prepared with four primer sets not containing thymine in Examples 1 and 10, respectively. Then, colorimetric analysis according to time was performed as in Test Example 1-(3) using the serotype genetic material that targets each other as a template using each primer set, and the results are shown in Table 5 below.

Example 1 Example 10 Comparative Example 1 Comparative Example 2 Isothermal expansion reaction time (min) 30 30 50 over 60

As a result, it was confirmed that both Examples 1 and 10 were positive within 30 minutes, whereas Comparative Examples 1 and 2 did not show a positive reaction within 30 minutes, indicating a significant decrease in efficiency.

Test Example 4: Analysis performance evaluation using virtual specimens

In order to evaluate whether analysis can be performed on actual clinical specimens, virtual specimens were prepared to evaluate the analytical ability of the primer set according to the present invention.

Specifically, blood samples from three healthy people who have not been bitten by mosquitoes within the past month are collected and subdivided, and dengue virus genetic material corresponding to serotypes 1 to 4 is mixed at 10 copies/25 μl per blood sample to simulate virtual It was used as a patient specimen. As a negative control group, two types of Zika virus genetic material were prepared, one in which the genetic material was mixed, and one in which no primer set was added. A mixed solution for the amplification reaction was prepared using the above sample and an isothermal amplification reaction was performed.

The results are shown in Table 6 below.

template no primer Example 1 Example 4 Example 7 Example 10 negative control group
(Zika) X X X X X serotype 1 X O X X X serotype 2 X X O X X serotype 3 X X X O X serotype 4 X X X X O

As a result, it was confirmed that the primer set works for each serotype, there is no false positive reaction, and the reaction efficiency is maintained, so that it is possible to diagnose dengue virus infectious diseases using the primer set of the present invention without separately purifying the viral genetic material from the sample. confirmed that it can.

<110> NUTRIGENE <120> A Primer set for Diagnosis of Dengue Virus Infectious Disease based on Multiple Isothermal Amplification, A Composition for Diagnosing Denque Virus Infectious Disease including the same, and A Visual Diagnosis method for Dengue Virus Infectious Disease using the same <130> WP213181_D <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 1, F3 primer (DENV1-F3) <400> 1 tggggtagca gactagtgg 19 <210> 2 <211> 19 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 1, B3 primer (DENV1-B3) <400> 2 tctgtgcctg gaatgatgc 19 <210> 3 <211> 39 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 1, FIP primer (DENV1-FIP) <400> 3 ccaccagggt acattttccc gacccctccc aaaacacaa 39 <210> 4 <211> 41 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 1, BIP primer (DENV1-BIP) <400> 4 agaggttaga ggaggttttcc cagcaggatc tctggtctct c 41 <210> 5 <211> 17 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 1, LF primer (DENV1-LF) <400> 5 tggtgttggg ccccgct 17 <210> 6 <211> 17 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 1, LB primer (DENV1-LB) <400> 6 acagcatatt gacgctg 17 <210> 7 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 2, FIP primer (DENV2-F3) <400> 7 cccgtccaag gacgttaa 18 <210> 8 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 2, B3 primer (DENV2-B3) <400> 8 ctgctgcgtt gtgtcatg 18 <210> 9 <211> 36 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 2, FIP primer (DENV2-FIP) <400> 9 acgacggagc tacattttga agaagtcagg cccaaa 36 <210> 10 <211> 40 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 2, BIP primer (DENV2-BIP) <400> 10 gggacgtaaa gcctggtttt ctctaaccac tagtctgcta 40 <210> 11 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 2, LF primer (DENV2-LF) <400> 11 gtttgctcaa accgtggc 18 <210> 12 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 2, LB primer (DENV2-LB) <400> 12 aaccgtggaa gctgtacg 18 <210> 13 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 3, F3 primer (DENV3-F3) <400> 13 gctgtacgca cggtgtag 18 <210> 14 <211> 20 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 3, B3 primer (DENV3-B3) <400> 14 cctggaatga tgctgaggag 20 <210> 15 <211> 41 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 3, FIP primer (DENV3-FIP) <400> 15 ggtacagctt ccctcagttt tcgtggttag aggagacccc t 41 <210> 16 <211> 41 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 3, BIP primer (DENV3-BIP) <400> 16 agaggttaga ggagaccctt ttgcaggatc tctggtctct c 41 <210> 17 <211> 17 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 3, LF primer (DENV3-LF) <400> 17 ctgctgcgtt gtgtcat 17 <210> 18 <211> 17 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 3, LB primer (DENV3-LB) <400> 18 cagcatattg acgctgg 17 <210> 19 <211> 19 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 4, F3 primer (DENV4-F3) <400> 19 gctcctttcg agagtgaag 19 <210> 20 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 4, B3 primer (DENV4-B3) <400> 20 agtacagctt cctcctgg 18 <210> 21 <211> 40 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 4, FIP primer (DENV4-FIP) <400> 21 cgttatggc ggagctattt tgaggctatt gaagtcaggc 40 <210> 22 <211> 40 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 4, BIP primer (DENV4-BIP) <400> 22 ggaggcgtta aattttcagg ggtctcctct aaccgctagt 40 <210> 23 <211> 18 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 4, LF primer (DENV4-LF) <400> 23 cagcacggtt tgctcaag 18 <210> 24 <211> 19 <212> DNA <213> artificial sequence <220> <223> Dengue virus Class 4, LB primer (DENV4-LB) <400> 24 ctgtacgcgt ggcatattg 19

Claims (8)

Primer sets represented by the nucleotide sequences of SEQ ID NOs: 1 to 6; Primer sets represented by the nucleotide sequences of SEQ ID NOs: 7 to 12; Primer sets represented by the nucleotide sequences of SEQ ID NOs: 13 to 18; And a primer set for diagnosing a dengue virus infectious disease for simultaneously detecting dengue virus serotypes 1 to 4, comprising a primer set represented by the nucleotide sequences of SEQ ID NOs: 19 to 24. The primer set for diagnosing dengue virus infectious diseases according to claim 1, wherein the primer set for diagnosing dengue virus infectious diseases is capable of diagnosing dengue virus infectious diseases by loop-mediated isothermal amplification. delete The primer set for diagnosing a dengue virus infectious disease according to claim 1, wherein the dengue virus infectious disease is dengue fever or dengue hemorrhagic fever. A composition for diagnosing a dengue virus infectious disease, comprising the primer set for diagnosing a dengue virus infectious disease according to claim 1. The composition for diagnosing dengue virus infectious diseases according to claim 5, wherein the limit of detection (LOD) of the composition is 10 copies/rxn. A kit for diagnosing a dengue virus infectious disease, comprising the composition for diagnosing a dengue virus infectious disease according to claim 5. Information provision method for diagnosing dengue virus infectious disease, including the following steps:
Mixing the composition for diagnosing a dengue virus infectious disease according to claim 5 and a biological sample; and
A step of amplifying a gene by isothermal amplification reaction.