KR102076341B1 - Composition for detecting severe fever with thrombocytopenia syndrome virus using LAMP and uses thereof - Google Patents

Abstract

The present invention relates to a primer set for the loop-mediated isothermal amplification for detecting severe fever with thrombocytopenia syndrome virus (SFTSV), and a composition for detecting SFTSV comprising the primer set. In the present invention, the presence or absence of SFTSV in a sample can be quickly confirmed only by turbidity or sediment generation, and fluorescence without a gene amplification device, thus the primer set and composition can be useful for in-situ diagnostic inspections.

Description

Composition for detecting severe fever with thrombocytopenia syndrome virus using LAMP and uses approximation

The present invention relates to a composition for detecting Severe Fever with Thrombocytopenia Syndrome Virus using LAMP (Loop-mediated isothermal amplification) and its use.

Severe fever with thrombocytopenia syndrome (SFTS) is a serious disease with symptoms such as high fever, vomiting, diarrhea, platelet reduction, leukopenia, and multiple organ failure. The virus that causes SFTS is caused by an RNA virus belonging to the genus Bunyaviridae , Phlebovirus , and was first reported in China in 2011. SFTS viruses have a spherical virus form of 80-100 nm in diameter and have a single stranded RNA genome consisting of a total of three segments. Each of these three segments is a S segment (nucleocapsid protein, non-structural protein) of 1,744 bp in total length, an M segment of 3,378 bp in length (encoding two glycoproteins), and an L segment of 6,368 bp in total ( RNA-dependent RNA encoding enzymes.

The SFTS virus is mediated by Haemaphysalis longicornis , which is widely known in Korea. Small cattle ticks and wild animals, which are the carriers of the SFTS virus, are spreading throughout the country, and the risk of SFTS virus infection is increasing as outdoor activities such as climbing and walking around the trail increase. In Korea, since the first case of SFTS was reported in Chuncheon, Gangwon-do in August 2012, the number of patients has been continuously increasing since 2013, and in September 2013, it was designated as the fourth group of infectious diseases by the Ministry of Health and Welfare. Was urgently admitted. Accordingly, it is necessary to develop a diagnostic method for confirming the presence and frequency of SFTS virus in small cattle ticks and animals in the wild, and in particular, the development of a diagnostic kit capable of diagnosing a large number of samples in a short time is urgently needed. It is true.

Meanwhile, Korean Laid-Open Patent No. 2017-0075043 discloses a 'severe febrile thrombocytopenia syndrome virus diagnostic kit', and Korean Laid-open Patent No. 2017-0107720 describes a primer set for detecting SFTS virus and a severe febrile thrombocytopenia syndrome using the same. Diagnostic kit 'is disclosed, but there is no description of a composition for detecting severe febrile thrombocytopenia virus using LAMP of the present invention and its use.

The present invention was derived from the above requirements, and the present inventors fabricated a primer set for isothermal amplification specific to Orthobunyavirus S segment, and reverse transcription loop-mediated isothermal amplification. The present invention was completed by confirming that the nucleic acid of Severe Fever with Thrombocytopenia Syndrome Virus in a sample can be accurately detected without using a non-specific reaction.

In order to solve the above problems, the present invention is Loop-mediated isothermal amplification for detecting Severe Fever with Thrombocytopenia Syndrome Virus comprising oligonucleotide primers of SEQ ID NOS: 1-6 A primer set is provided.

In another aspect, the present invention provides a composition for detecting a severe febrile thrombocytopenia virus comprising the ring-mediated isothermal amplification primer set.

In another aspect, the present invention provides a kit for detecting a severe febrile thrombocytopenia syndrome virus comprising a primer set for the ring-mediated isothermal amplification and a reagent for carrying out the ring-mediated isothermal amplification reaction.

In addition, the present invention comprises the steps of separating the total RNA from the sample; Amplifying a target sequence by a reverse transcription loop-mediated isothermal amplification reaction using the isolated total RNA as a template and using the primer set of the present invention; And detecting the product of the amplification step.

The composition for detecting severe febrile thrombocytopenic syndrome virus according to the present invention and the composition for detecting the severe febrile thrombocytopenia syndrome virus comprising the primer set are shown to have turbidity or precipitate generation without a gene amplification device, and to reduce the severity of thrombocytopenia in the sample only by fluorescence coloration. The presence of the syndrome virus can be quickly identified. Therefore, the primer set and the composition may be provided as a kit for detecting Severe thrombocytopenia syndrome virus or a method for diagnosing Severe thrombocytopenia syndrome virus infection.

Figure 1 shows the location of the nucleotide sequence and LAMP primer used in the fabrication of LAMP primer specific for the Orthobunyavirus S segment gene used in the present invention to detect severe febrile thrombocytopenia syndrome virus.
Figure 2 is a photograph confirming the naked eye detection limit of the amplified product by treating the fluorescent sample (a) or indicator (b) to the obtained LAMP reaction after the LAMP reaction using the LAMP primer set of the present invention.
Figure 3 is a result of confirming the detection limit of the amplification product by performing a real-time LAMP reaction using the LAMP primer set of the present invention.
Figure 4 is a result of performing a real-time LAMP reaction using the LAMP primer set of the present invention on a clinical sample.

In order to achieve the object of the present invention, the present invention provides a loop-mediated amplification for the detection of Severe Fever with Thrombocytopenia Syndrome Virus comprising oligonucleotide primers of SEQ ID NOS: 1-6 A primer set for isothermal amplification is provided.

Primer set for LAMP for detecting severe febrile thrombocytopenia syndrome virus according to the present invention is GenBank accession no. Oligonucleotide primer sets of SEQ ID NOS: 1-6, specific for the Orthobunyavirus S segment gene, which is HQ642768.1 .

Primer set of the present invention is preferably for use in Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP), using a cDNA prepared by performing reverse transcription on RNA 2 Alternatively, the LAMP method may be performed, and the reverse transcriptase and the LAMP reaction may be simultaneously performed by mixing the reverse transcriptase and the polymerase into one tube using RNA as a template, but is not limited thereto.

In the present invention, the term 'loop-mediated isothermal amplification (LAMP)' is a method capable of performing an amplification reaction under isothermal conditions unlike the conventional PCR (polymerase chain reaction) method. Four kinds of primers (F3, B3, FIP, BIP) are basically required for the LAMP reaction, and two kinds of primers (LF, LB) are added to the final six different sequences to improve the reaction rate. Oligonucleotide primers made are required for the reaction.

The four basic primers are composed of two outer primers and two inner primers, and the outer primers are a forward outer (F3) primer and a backward outer (B3) primer. It is composed of and acts to release DNA double strands during the non-cyclic step of the reaction. The inner primer is composed of two types of forward inner primer (FIP) and backward inner primer (BIP), and is composed of forward and reverse sequences to form an essential loop for ring-mediated isothermal amplification reaction. It consists of the corresponding nucleotides. The additional two primers consist of two forward loop (LF) primers and two backward loop (LB) primers, and attach to the base sequence to which the inner primer does not bind to accelerate the ring-mediated isothermal amplification reaction. Let's do it.

In the primer set consisting of oligonucleotide primers of SEQ ID NOS: 1 to 6 of the present invention, the primer of SEQ ID NO: 1 is F3, which is a forward outer primer, the primer of SEQ ID NO: 2 is B3, which is a reverse outer primer, and the primer of SEQ ID NO: 3 Is FIP, which is a forward inner primer, primer of SEQ ID NO: 4 is BIP, which is a reverse internal primer, primer of SEQ ID NO: 5 is LF (LoopF) which is a forward ring primer, and primer of SEQ ID NO: 6 is LB (LoopB, a reverse ring primer) )to be.

The primer of the present invention may include oligonucleotides consisting of fragments of at least 15, at least 16, at least 17 consecutive nucleotides in SEQ ID NOs: 1, 2, 5, and 6, depending on the sequence length of each primer, Oligonucleotides consisting of segments of at least 32, at least 33, and at least 34 contiguous nucleotides in SEQ ID NOs: 3 and 4. For example, a primer of SEQ ID NO: 1 (20 oligonucleotides) comprises an oligonucleotide consisting of fragments of at least 15, 16, 17, 18, 19, or 19 nucleotides in the sequence of SEQ ID NO: 1 can do.

In the present invention, the term “primer” refers to a single strand of oligonucleotide sequence that is complementary to the nucleic acid strand to be replicated and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primers should allow to start the synthesis of the extension product. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

In the present invention, oligonucleotides used as primers may also comprise nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

Each primer of the present invention may be modeled as a label for detection in order to increase the convenience of detection. The detection label may be a compound, a biomolecule, or a biomolecule analog which can be linked, coupled, or attached to a primer to determine the density, concentration, amount, etc. of the amplification product in a conventional manner, but is not limited thereto. FAM, VIC , TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, TYE563, BIOTIN, DIGOXIGENIN, and NED can be used.

The present invention also provides a composition for detecting a severe febrile thrombocytopenia virus comprising the ring-mediated isothermal amplification primer set.

The present invention also provides a kit for detecting a severe febrile thrombocytopenia syndrome virus comprising a primer set for ring-mediated isothermal amplification and a reagent for performing the ring-mediated isothermal amplification reaction.

Kit for detecting severe febrile thrombocytopenia syndrome virus according to the present invention is GenBank accession no. A primer set consisting of oligonucleotide primers of SEQ ID NOS: 1-6, specific for the Orthobunyavirus S segment gene, HQ642768.1 .

The kit of the present invention may preferably be a kit for reverse transcription loop-mediated isothermal amplification, but is not limited thereto.

In the kit of the present invention, the reagent for performing the ring-mediated isothermal amplification reaction may include DNA polymerase, dNTPs and buffers. Since severe febrile thrombocytopenia virus is an RNA virus, reverse transcription-mediated isothermal amplification (RT-LAMP) with the addition of reverse transcription is required for amplification. Thus, the kits of the present invention may further comprise reverse transcriptase.

DNA polymerase according to an embodiment of the present invention may be a Bst polymerase, but is not limited thereto, and is mainly used in conventional ring-mediated isothermal amplification (LAMP) for reverse transcription-mediated isothermal amplification (RT-LAMP). It may be preferable to include GspSSD DNA polymerase having a faster amplification ability and a strong reverse transcriptase ability instead of the prepared Bst polymerase, but is not limited thereto.

In addition, the kits of the present invention may further comprise a user guide describing the optimal reaction performance conditions. The instructions are printed materials that explain how to use the kit, such as how to prepare a buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

The present invention also provides

Separating total RNA from the sample;

Amplifying a target sequence by a reverse transcription loop-mediated isothermal amplification reaction using the isolated total RNA as a template and using the primer set of the present invention; And

It provides a method for detecting a severe febrile thrombocytopenia virus comprising the step of detecting the product of the amplification step.

Severe febrile thrombocytopenia syndrome virus detection method of the present invention comprises the step of separating the total RNA from the sample. As a method for separating total RNA from the sample, any method known in the art may be used, for example, a phenol extraction method may be used. Using the isolated total RNA as a template, it is possible to amplify the target sequence by performing RT-LAMP using a primer set according to an embodiment of the present invention as a primer.

In the method for detecting a severe febrile thrombocytopenia syndrome virus according to an embodiment of the present invention, the isothermal amplification reaction may be performed at 62 ° C. to 68 ° C. for 20 minutes to 50 minutes, and preferably at 65 ° C. for 30 minutes. But may be performed for 45 minutes, but is not limited thereto.

In addition, the sample may be a biological sample isolated from a patient suspected of severe febrile thrombocytopenic syndrome, or a small cattle mite collected in the wild, but is not limited thereto, and the sample is suspected of being infected with severe febrile thrombocytopenia syndrome virus. Do not.

The method for detecting a severe febrile thrombocytopenia virus of the present invention includes detecting a product of the isothermal amplification step. Detection of the amplification products is turbidity, precipitate formation, fluorescence measurement using SYBR Green I, color change of indicator such as phenol red, DNA laddering, capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence It may be carried out through the measurement, preferably turbidity, fluorescence measurement using SYBR Green I, DNA laddering, but is not limited thereto.

In addition, by using a device that can detect the fluorescent dye in the reaction solution in real time, isothermal amplification is confirmed in real time without additional electrophoresis or SYBR Green I after the reaction, and amplification of the target sequence through an anneal peak You can also check.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1.LAMP primer set design for detection of severe febrile thrombocytopenia virus

To prepare a primer set for Loop-mediated isothermal amplification (LAMP) for the detection of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), GenBank accession no. Information on the Orthobunyavirus S segment gene, HQ642768.1 , was obtained. Primers to be used for the LAMP was designed primers using OptiGene's LAMP Design software. The location of the LAMP primer sequence for SFTSV detection used in the present invention is as shown in FIG.

Example 2 SFTSV Detection limit analysis

The sample used for the detection limit synthesized the S-segment nucleocapsid portion (980 ~ 1,717 bp) of GenBank: HQ642768.1, calculated the base pair and the nucleic acid amount, and used the converted copy number. Nucleic acid pre-cultured at was used. Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) reaction showed 2 μl template DNA, 1 μl Bst polymerase (8U), 10 μm buffer 2.5 μl, 25 mM dNTP 1.5 μl, 10 mM MgSO ₄ 1 μl, 5 M betaine 4 μl. , RocketScript ™ Reverse Transcriptase (Bionia, Korea) 20U, each of 6 primers (5 pmol for F3 and B3, 20 pmol for FIP and BIP respectively, 10 pmol for LoopF and LoopB respectively) Was performed. The LAMP reaction was performed at 65 ° C. for 45 minutes by selecting one of MiniT-100H (ALLSHENG, China) heat block heaters and a water bath. In addition, in order to immediately confirm the result of the isothermal amplification product in real time, an isothermal amplification reaction was performed at 65 ° C. for 30 minutes using Isothermal Fluorescence PCR and Gene-8C (ALLSHENG).

The detection limit of the reacted LAMP reactant was analyzed. For visual observation, a reagent of SYBR Green I (500X) or phenol red was used. In the visual observation, 10 copies were identified as the observation limit value (Fig. 2), and the analysis of the detection limit on the real-time LAMP device using the low concentration SYBR Green I of 1X was performed. It was confirmed that the amplification product (fluorescence value) can be detected by copying (FIG. 3).

In addition, as a result of analyzing positive and negative SFTSV infection samples using real-time LAMP equipment, distinct fluorescence was generated only in positive samples from 20 minutes of isothermal amplification reaction and until the end of isothermal amplification reaction in negative samples. It was confirmed that no fluorescence signal appeared (FIG. 4). Through this result, it was found that the primer set for LAMP of the present invention can accurately detect Severe Fever with Thrombocytopenia Syndrome Virus.

<110> The Armed Forces Medical Command <120> Composition for detecting severe fever with thrombocytopenia          syndrome virus using LAMP and uses specifically <130> PN19221 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F3 primer <400> 1 cgcatcttca cattgatagt 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> B3 primer <400> 2 agcagcctta aaggagtat 19 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> FIP primer <400> 3 ctgaagccac gaccaagacc ttggtgaagg catcttg 37 <210> 4 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> BIP primer <400> 4 gcagttggaa tcagggacca cctgaaggtc gagaatt 37 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LoopF primer <400> 5 cctgatggag gcttactct 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LoopB primer <400> 6 gccatgcaca tcatctca 18

Claims (5)

GenBank accession no. Comprising oligonucleotide primers of SEQ ID NOs: 1-6. Primer for Reverse Transcription Loop-mediated isothermal amplification to detect Severe Fever with Thrombocytopenia Syndrome Virus with Orthobunyavirus S segment gene, HQ642768.1 set. GenBank accession no. Comprising a primer set for reverse transcription-mediated isothermal amplification according to claim 1. A composition for detecting severe febrile thrombocytopenia syndrome virus having an Orthobunyavirus S segment gene of HQ642768.1 . GenBank accession no. Comprising a primer set for reverse transcription-mediated isothermal amplification according to claim 1 and a reagent for performing reverse transcription-mediated isothermal amplification reaction. Kit for detection of severe febrile thrombocytopenia syndrome virus with Orthobunyavirus S segment gene of HQ642768.1 . Isolating total RNA from a biological sample isolated from a suspicious patient with severe febrile thrombocytopenia, or from a small bovine tick sample collected in the wild;
Amplifying a target sequence by using a reverse transcription loop-mediated isothermal amplification reaction using the isolated total RNA as a template and using the primer set of claim 1; And
Detecting a product of the amplifying step; comprising GenBank accession no. A method for detecting severe febrile thrombocytopenia syndrome virus with the Orthobunyavirus S segment gene of HQ642768.1 . The GenBank accession no. According to claim 4, wherein the detection of the amplification product is performed through turbidity, spin down generation, fluorescence or electrophoresis. A method for detecting severe febrile thrombocytopenia syndrome virus with the Orthobunyavirus S segment gene of HQ642768.1 .

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