KR20230075186A - Primer Set for Loop mediated Isothermal Amplification Reaction for Detecting Rift valley fever virus and Uses thereof - Google Patents

Abstract

The present invention relates to a primer set for a loop mediated isothermal amplification (LAMP) reaction for detecting a rift valley fever virus rapidly and a use thereof and, more specifically, to a primer set for an LAMP reaction specific to an S segment of a rift valley fever virus gene, a composition for rapid detection of a rift valley fever virus, and a method for detecting the rift valley fever virus using the same. According to the primer set, the composition containing the same and the method for detecting the rift valley fever virus using the same, which are provided according to the present invention, the rift valley fever virus in a sample can be quickly detected with very high sensitivity and specificity according to the LAMP reaction, thereby being very useful in diagnosing rift valley fever virus infection and detecting the virus.

Description

Primer Set for Loop mediated Isothermal Amplification Reaction for Detecting Rift Valley Fever Virus and Uses thereof}

The present invention relates to a primer set for loop mediated isothermal amplification (LAMP) for rapid detection of Rift Valley fever virus and its use, and more particularly, LAMP specific to the S segment of the Rift Valley fever virus gene It relates to a composition for rapid detection of Rift Valley fever virus containing a set of primers for use, and a method for detecting Rift Valley fever virus using the same.

Rift Valley Fever (RVF) is a three-segment single-stranded linear (-)RNA virus belonging to the Bunyaviridae and Phlebovirus genera. Since it was first discovered in livestock in Kenya in the early 1900s, outbreaks have been reported in many people and animals through infected livestock. The incubation period is usually 2-6 days, and usually shows mild symptoms such as fever, dizziness, and extreme weight loss. can 1% of all infected people die, and the mortality rate for patients with hemorrhagic fever symptoms reaches 50%. However, no specific antiviral agent or preventive vaccine has been developed so far, and only public treatment is possible in case of an outbreak. Infection can occur by being bitten by mosquitoes or bloodsucking insects carrying infected blood, or by coming into contact with the blood, body fluids, or organs of infected animals, or by drinking unprocessed milk of infected animals. It occurs mainly in most African regions and the Arabian Peninsula, especially in West Africa, which is the habitat of vector mosquitoes, but there has been no report of outbreaks in Korea so far.

Rift Valley Fever (RVF) is an infectious disease that occurs in most African regions and has a high possibility of entering Korea, so it is necessary to develop a diagnostic method for it. Culex tritaeniorhynchus and Aedes vexans, which are the carriers of the Rift Valley virus, are spread all over the country, and considering the current situation where exchanges of material and human resources are frequent due to the development of transportation, they are viruses that can spread domestically if introduced into Korea, and thus have a great impact on public health. likely to have an impact Accordingly, in 2019, it was selected as a new infectious disease that could be introduced in the future by the Korea Centers for Disease Control and Prevention.

For preemptive control, it is urgent to develop a molecular diagnostic kit that can detect the Rift Valley virus with high accuracy and is easy to detect in the field.

In the case of the Rift Valley fever virus, the possibility of secondary infection through contact with patients and the dead is low, but it is necessary to develop a simple diagnosis method that can be applied to periodic diagnosis for early detection of occupational groups with a high possibility of contact with patients and deaths, such as medical workers and funeral directors. need.

It is necessary to block the inflow of Rift Valley fever virus into Korea at an early stage by promptly screening suspected patients with suspicious symptoms by deploying rapid and accurate Rift Valley fever virus diagnostic agents at airports, ports, and quarantine sites as well as general medical sites. .

In the case of Rift Valley fever virus infection, the fatality rate is about 1% when symptoms are present, but it is a high-risk virus with a mortality rate approaching 50% when it starts to bleed. In this situation, early diagnosis is the only way to lower the mortality rate, so the development of an accurate and simple diagnosis method is urgent.

On the other hand, conventionally, ITS-RFLP, PCR using species-specific primers, and real-time PCR methods have been studied for virus detection, but the development of primers is very difficult in these PCR-based conventional detection methods because DNA modification is sensitive to temperature. It is important, and has the disadvantage that it takes time to change the temperature in the amplification step. In addition, additional analysis time is required through a series of processes in which the amplified reaction product must be confirmed by electrophoresis and finally sequenced, and high cost due to the need for specialized equipment such as a polymerase chain reactor is also emphasized. It is becoming.

Loop mediated isothermal amplification (LAMP), developed to overcome these disadvantages, uses six primers to synthesize both ends in a loop shape to amplify a specific region of a gene indefinitely, and amplify the DNA It is a method of confirming the presence or absence of amplification by using a reagent for fluorescence detection or by precipitation reaction of the product. Unlike conventional PCR methods, LAMP uses Bst polymerase, which does not require a PCR cycle, to carry out the reaction under isothermal conditions, so the target substance can be quickly detected within 60 minutes. In addition, since the amplification proceeds at isotherm, the amplification device can be configured simply and can be manufactured in a form that is easy to carry, so it is suitable for rapid detection in the field.

Accordingly, as a result of intensive research to develop a detection method that can detect Rift Valley fever virus very quickly and conveniently through the LAMP method, the present inventors have made S segment of the Rift Valley fever virus gene produced in one embodiment of the present invention. It was found that Rift Valley fever virus can be detected with very high sensitivity and specificity using a primer set specific to , and the present invention was completed.

Accordingly, an object of the present invention is to provide a primer set for detecting Rift valley fever virus consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.

Another object of the present invention is to provide a composition for detecting Rift Valley fever virus comprising the primer set.

Another object of the present invention is to provide a kit for detecting Rift valley fever virus, including the primer set.

Another object of the present invention is to provide a composition for diagnosing a Rift valley fever virus infection, including the primer set.

Another object of the present invention is (a) isolating nucleic acids from a specimen; (b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; And (c) to provide a Rift Valley fever virus detection method comprising the step of detecting the amplification product.

Another object of the present invention is (a) isolating nucleic acids from biological samples isolated from mammals other than humans; (b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; And (c) to provide a method for diagnosing Rift Valley fever virus infection, comprising the step of detecting the amplification product.

In order to achieve the above object of the present invention, the present invention provides a primer set for detecting Rift valley fever virus consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.

In order to achieve another object of the present invention, the present invention provides a composition for detecting Rift Valley fever virus comprising the primer set.

In order to achieve another object of the present invention, the present invention provides a kit for detecting Rift valley fever virus, including the primer set.

In order to achieve another object of the present invention, the present invention provides a composition for diagnosing Rift valley fever virus infection, including the primer set.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) isolating nucleic acids from a specimen; (b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; And (c) it provides a Rift Valley fever virus detection method comprising the step of detecting the amplification product.

In order to achieve another object of the present invention, the present invention provides (a) isolating nucleic acids from biological samples isolated from mammals other than humans; (b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; And (c) it provides a method for diagnosing Rift Valley fever virus infection, comprising the step of detecting the amplification product.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set for detecting Rift valley fever virus consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.

In the present invention, the "primer" is an oligonucleotide, which is a short sequence of nucleotides, and is specifically attached to the complementary position of the opposite strand of the target DNA or RNA to initiate gene amplification. As an oligonucleotide that does, it may preferably be an oligonucleotide that specifically binds to the S segment region of the Rift Valley fever virus gene, more preferably a primer consisting of the nucleotide sequence of SEQ ID NOs: 1 to 6, each of these 80 Primers with sequences that are at least %, at least 90%, at least 95%, or at least 99% similar may also be included.

The primer pair may be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These primers can be modified using many means known in the art, as long as they exhibit an effect of amplifying the S segment of the Rift Valley fever virus gene. Examples of such modifications include methylation, capping, substitution of one or more homologs of the natural nucleotide, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or charged linkages (eg, phosphorothioates, phosphorodithioates, etc.). A nucleic acid can contain one or more additional covalently linked moieties, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalants (eg, acridine). , proralene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. In addition, the primer of the present invention, if necessary, may include a label that can be directly or indirectly detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioactive isotopes (eg 32P), fluorescent molecules and chemical groups (eg biotin), and the like. there is.

The primers may also include added, deleted or substituted sequences of the nucleotide sequences of SEQ ID NOs: 1 to 6.

In the primer set, each primer may consist of or include one of the nucleotide sequences of SEQ ID NOs: 1 to 6, and as long as it maintains specific binding to the genetic material of the Rift Valley fever virus, SEQ ID NO: 1 It may have 80% or more, 90% or more, 95% or more, 97% or more or 99% or more homology with the base sequence described in 6 to 6. 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” means DNA amplification under one temperature condition. Conventional polymerase chain reaction (PCR) has denaturation, annealing, and extension steps, respectively. Unlike the special PCR equipment that requires different temperatures and conditions, the isothermal amplification reaction has the advantage of not requiring a separate special equipment.In general, the isothermal amplification method is a target nucleic acid within an hour within a specific temperature range. Can amplify up to 10 ^ 9, 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 the result 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)" 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 viral RNA, but can be designed to further include specific bases, 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, internal primers are first bound to the template and extended, and then external primers are 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.

In the present invention, the "detection" means confirming the presence or absence of the Rift Valley fever virus gene in a specific target sample or individual, or analyzing the amount if present.

The isothermal amplification reaction may be performed at 60 to 75°C, 62 to 73°C, or 65 to 70°C, but is not limited thereto. Specifically, any one lower limit value and any one upper limit value selected from the group consisting of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 75 ° C. It can be performed at a temperature between

The isothermal amplification reaction may be performed for 10 to 40 minutes, 15 to 35 minutes, and 20 to 30 minutes, but is not limited thereto. Specifically, it may be performed for a time between any one lower limit value and any one upper limit value selected from the group consisting of 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes and 40 minutes.

The present invention also provides a composition and kit for detecting Rift Valley fever virus, including a primer set for detecting Rift valley fever virus consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.

The composition or kit for detecting Rift Valley fever virus 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 mM KCl, 0.1 to 2 mM MgSO ₄ , and 0.01 to 0.1% Tween-20, more specifically It may be a solution containing 20mM Tris-HCl, 10mM (NH ₄ ) ₂ SO ₄ , 50nM KCl, 2mM MgSO4, and 0.1% Tween-20 at pH 7 to 9, preferably pH 8.8.

In one embodiment, the composition or kit for detecting the Rift Valley fever virus may further include a reagent capable of visually confirming the reaction product. As such a reagent, commonly known or commercially available ones may be used, for example, WarmStart® Colorimetric 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 mentioned above to make the reaction occur efficiently.

The composition or kit for detecting Rift Valley fever virus 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 unrelated to the Rift Valley fever virus can be used, and as a positive control material, a biological sample obtained from a patient infected with the Rift Valley fever virus or a template corresponding to a part of the genome of the Rift Valley fever virus strain RNA or DNA may be used. These control substances can be applied to the gene amplification reaction separately or together with the biological sample in one tube, if necessary.

The present invention also provides a composition for diagnosing a Rift valley fever virus infection consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.

(a) isolating nucleic acids from a specimen; (b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; And (c) it provides a Rift Valley fever virus detection method comprising the step of detecting the amplification product.

In one aspect of the present invention, the sample may be a clinical sample.

The clinical sample may be interpreted in the same sense as a biological sample, and may be a sample isolated from a subject suspected of being infected with Rift Valley fever virus, that is, a mammal such as a human. Non-limiting examples thereof may include blood, plasma, serum, bone marrow, tissue, cells, saliva, sputum, fur, urine, and the like.

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, treatment with proteinase K followed by heat treatment, 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 Brij-35 0.1% ~ 0.5w / w%, Tris-HCl 10mM ~ 300mM (pH7.0 ~ 8.0), and CaCl2 1mM ~ 10mM, specifically Brij-35 0.3w / w%, Tris-HCl 100 It may mean treating a solution containing mM (pH 7.5) and CaCl 2 5mM, 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 a specimen and enhance the isothermal amplification reaction efficiency of the primer set of the present invention.

The amplification in step (b) may be an isothermal amplification reaction, preferably LAMP. Specific amplification conditions may be applied in the same manner as described above.

The Rift Valley fever virus detection method includes detecting the amplified gene. Detection of the amplification product is fluorescence measurement using SYBR Green I, color change of an indicator such as phenol red, turbidity, precipitate formation, DNA laddering, capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, or phosphorescence measurement. It may be performed through, but is not limited thereto.

(a) isolating nucleic acids from biological samples isolated from mammals other than humans; (b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; And (c) it provides a method for diagnosing Rift Valley fever virus infection, comprising the step of detecting the amplification product.

According to the primer set provided by the present invention, the composition containing the same, and the Rift Valley fever virus detection method using the same, it is possible to rapidly detect the Rift Valley fever virus in a sample with very high sensitivity and specificity according to an isothermal amplification reaction. It can be very useful for diagnosing fever virus infections and detecting viruses.

Figure 1 is a result showing the region where the lamp primer set was selected with the PrimerExplorer program by comparing S-segment sequences obtained from 23 isolates isolated from countries where Rift Valley occurred.
2 and 3 are results of evaluating the detection sensitivity of Rift Valley fever virus of the primer set according to the present invention.
4 is a result of evaluating the specificity of Rift Valley fever virus detection of the primer set according to the present invention.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1: Preparation of primers for detecting Rift Valley fever virus

In order to prepare lift valley fever virus-specific detection primers, multiple alignment was performed primarily using the genetic information of the virus. The results are shown in Figure 1. Among the genes of Rift Valley fever virus, the S segment was selected. Primers for LAMP for the S segment of Rift Valley Fever virus were designed using PrimerExplorer V5 (FIG. 1).

Five candidate primers were generated, and one primer pair specific to the S segment of the Rift Valley fever virus gene was selected from among them, and a Loop primer was designed and used in further experiments.

The sequences of the prepared LAMP primer sets are shown in Table 1 below:

Example 2: Production of target gene for Rift Valley Fever Virus S segment RPA

To test the performance of diagnostic probes and primers for RPA, nucleic acids of the Rift Valley fever virus are required, but since nucleic acids for the virus do not exist in Korea, we requested the consensus sequence of 23 Rift Valley viruses to Bioneer. mRNA transcript was produced and used in the experiment.

The mRNA transcript sequence of the Rift Valley S segment is as follows:

Example 3: Detection sensitivity of prepared primers and probes

For the performance test of the primer set prepared in Example 1, the minimum detection limit was measured using the synthetic nucleic acid target gene of the Rift Valley fever virus prepared in Example 2.

Diluted in ten steps from 10 ng to 0.01 pg, the reaction was performed at 67° C. for 25 minutes using an isothermal nucleic acid amplifier. As a result, as shown in FIG. 2, it was possible to detect up to 1 pg within 25 minutes.

In addition, as a result of diluting the detection limit for mRNA copies in ten stages from 1x10 ⁶ to 1x10 ⁰ and reacting with an isothermal nucleic acid amplifier for 25 minutes at 67 ° C, as shown in FIG. 3, it was confirmed that detection was possible within 25 minutes up to 1x10 ³ It was confirmed that the detection sensitivity was very excellent.

Example 4: Detection specificity of prepared primers and probes

In order to examine the specificity of the selected primer set, the reactivity to 17 bacteria and 16 viruses was investigated, and as a result, it reacted only with the Lift Valley positive control and did not react with other nucleic acids, confirming 100% specificity ( Fig. 4).

According to the primer set provided by the present invention, the composition containing the same, and the Rift Valley fever virus detection method using the same, it is possible to rapidly detect the Rift Valley fever virus in a sample with very high sensitivity and specificity according to an isothermal amplification reaction. It can be used very usefully for diagnosing fever virus infection and virus detection, so industrial applicability is very high.

<110> UNIVERSITY INDUSTRY FOUNDATION, YONSEI UNIVERSITY WONJU CAMPUS <120> Primer Set for Loop mediated Isothermal Amplification Reaction for Detecting Rift valley fever virus and Uses its <130> NP21-0078 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> artificial sequence <220> <223> F3 primer <400> 1 tccaccatgc cagcaaag 18 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> B3 primer <400> 2 agctaaagga ggggaatcct 20 <210> 3 <211> 42 <212> DNA <213> artificial sequence <220> <223> FIP <400> 3 cctgtcactg ggacaaccat gggggatgca tcatatgcct cg 42 <210> 4 <211> 40 <212> DNA <213> artificial sequence <220> <223> BIP <400> 4 actcaagacg accaaagcct ggccgggatg agctgactct 40 <210> 5 <211> 17 <212> DNA <213> artificial sequence <220> <223> LoopF <400> 5 ggtctatccc ctgcata 17 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> LoopB <400> 6 cagggcagca gcaactcgtg 20

Claims (15)

A primer set for detecting a Rift valley fever virus consisting of the nucleotide sequences of SEQ ID NOs: 1 to 6.
The primer set according to claim 1, wherein the primer set is for loop mediated isothermal amplification (LAMP).
A composition for detecting Rift Valley fever virus comprising the primer set according to claim 1.
The composition according to claim 3, wherein the composition is for loop mediated isothermal amplification (LAMP).
[Claim 5] The composition according to claim 4, further comprising a LAMP reaction buffer containing DNA polymerase, dNTP and Mg2+ ions.
A kit for detecting Rift valley fever virus, comprising the primer set according to claim 1.
The kit according to claim 6, characterized in that the kit is for loop mediated isothermal amplification (LAMP).
A composition for diagnosing a Rift valley fever virus infection, comprising the primer set according to claim 1.
The composition according to claim 8, wherein the composition is for loop mediated isothermal amplification (LAMP).
(a) isolating nucleic acids from the specimen;
(b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; and
(c) a Rift Valley fever virus detection method comprising the step of detecting the amplification product.
11. The detection method according to claim 10, wherein the amplification is performed by a LAMP method.
The method of claim 10, wherein the sample is a clinical sample.
11. The method of claim 10, wherein the amplification is performed at 60 to 75 °C.
11. The method of claim 10, wherein the amplification is performed for 10 to 40 minutes.
(a) isolating nucleic acids from a biological sample isolated from a non-human mammal;
(b) performing an amplification reaction using the nucleic acid as a template and the primer set according to claim 1; and
(c) a method for diagnosing a Rift Valley fever virus infection, comprising the step of detecting the amplification product.

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