KR102555045B1 - Molecular marker for specifically detecting Machupo virus and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker capable of specifically detecting Machupo virus and its use, and more specifically, to an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and an oligonucleotide of SEQ ID NO: 3 nucleotide probe; The oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8 and the oligonucleotide probe of SEQ ID NO: 9; And an oligonucleotide primer set of SEQ ID NO: 13 and SEQ ID NO: 14 and an oligonucleotide probe of SEQ ID NO: 15; .

Description

Molecular marker for specifically detecting Machupo virus and uses thereof {Molecular marker for specifically detecting Machupo virus and uses thereof}

The present invention relates to a molecular marker capable of specifically detecting Machupo virus and its use.

With the development of antibiotics and vaccination, deaths from infectious diseases have decreased rapidly, but since the 1970s, more than 30 major new infectious diseases have occurred, and infectious diseases that seemed to disappear have newly spread or re-emerged. From the mid-1980s, new and re-emerging infectious diseases emerged as a major public health problem, and from the 1990s, preparations and countermeasures for this problem began in earnest, centering on developed countries.

Looking at the status of emerging infectious diseases in Korea, after the first case was reported in 2001, dengue fever is the most common and has been increasing every year. Most of the reported patients were infected while staying abroad, and the infected areas are mainly Southeast Asian regions such as the Philippines, Indonesia, Thailand, and Malaysia.

The military is a group organized with a certain discipline and order, and is characterized by high-density group life and many outdoor activities. If an infectious disease occurs in a military unit, it is important to prevent the spread through early diagnosis and treatment of the infectious disease because the speed of transmission is very fast due to group living.

Arenaviridae viruses include Mammarenavirus, Reptarenavirus, and Hartmanivirus genera, Reptarenavirus and Hartmanivirus are reptiles as hosts, and Mammarenavirus are mammals as hosts. Mammarenavirus contains 41 distinct virus species, which are divided into two groups based on genetic, regional and epidemiological relationships: Old World (referring to Europe, Asia and Africa) and New World (referring to North and South America), of which The New World group is further divided into 4 clades (A, B, C and A/Rec(D)), Guanarito virus, Junin virus, Machupo virus, and Sabia ( South American hemorrhagic fever viruses, including Sabia virus and Chapare virus, belong to clade B. In nature, each arenavirus is transmitted by one or a limited number of rodent species and cycles as a regional endemic disease.

Arenavirus is a virus with a bi-segmented RNA genome consisting of negative sense, large (L, about 7.2 kb) and small (S, about 3.4 kb) segments, each segment It consists of two open reading frames (ORFs) and a non-coding intergenic region (hairpin structure). Arenavirus can be spread from person to person through aerosols.

Machupo virus is an arenavirus discovered in 1963 as the causative agent of Bolivian hemorrhagic fever (BHF). The mortality rate is 5-30%, and it is classified as biosafety level 4 due to its pathogenicity. Symptoms of infection are very similar to those of malaria, starting with fever, malaise, headache and muscle aches. Petechial hemorrhage occurs in the upper part of the body, and bleeding from the nose and gums is usually observed within 7 days of onset. One-third of patients show severe bleeding or neurologic symptoms. Neurological symptoms include tremors, delirium, and convulsions. The mortality rate is about 25%.

Meanwhile, Korean Patent Publication No. 2020-0056348 discloses 'primers and probes for simultaneously detecting subtypes of Crimean Congo hemorrhagic fever virus, and method for detecting Crimean Congo hemorrhagic fever virus using the same', and Korean Patent Publication No. 2010 -0028017 discloses 'sulfonyl semicarbazide, carbonyl semicarbazide, semicarbazide and urea, pharmaceutical compositions thereof, and methods for treating hemorrhagic fever virus including infections related to arenavirus' However, the molecular marker capable of specifically detecting Machupo virus of the present invention and its use have not been described.

The present invention has been derived from the above needs, and the present inventors have L of South American hemorrhagic fever viruses (Guanarito virus, Junin virus, Machupo virus, Sabia virus) Base sequence sites capable of specifically detecting Machupo virus were selected through nucleotide sequence alignment for the S segment or S segment, and primer sets and probes were prepared based on the sequences of the selected regions. As a result of real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the prepared primer set and probes and the synthesized Machupo virus RNA template, there was no non-specific amplification reaction, and 1 fg concentration of the synthesized RNA template The present invention was completed by identifying three Machupo virus-specific primer sets and probes that can detect up to and have no cross-reactivity with other South American hemorrhagic fever viruses, such as Guanarito virus, Junin virus and Sabia virus RNA samples. did

In order to solve the above object, the present invention is an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and an oligonucleotide probe of SEQ ID NO: 3; The oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8 and the oligonucleotide probe of SEQ ID NO: 9; And a marker for detecting Machupo virus, comprising one or more primer sets and probes selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 oligonucleotide primer sets and SEQ ID NO: 15 oligonucleotide probes. composition is provided.

In addition, the present invention is the marker composition; And it provides a kit for detecting Machupo virus, including a reagent for performing an amplification reaction.

In addition, the present invention comprises the steps of isolating total RNA from a biological sample isolated from a patient suspected of South American hemorrhagic fever; Amplifying a target sequence by using the separated total RNA as a template and performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the marker composition of the present invention; And detecting the product of the amplification step; it provides a method for detecting Machupo virus, including.

Using the molecular marker for detecting Machupo virus according to the present invention, it is possible to specifically and efficiently detect Machupo virus or diagnose Bolivian hemorrhagic fever in a short time. It is considered that it is possible to block the spread of an infectious disease through early diagnosis when an infectious disease occurs in a place where a special purpose group lives.

1 is a result of conventional PCR of a primer/probe set using synthetic DNA of machupo virus.
Figure 2 shows the results of RT real time PCR of a primer/probe set using Machupo virus RNA, the table on the left shows the Ct value and the amount of fluorescence amplification, and the graph on the right shows a real-time amplification curve.
Figure 3 is a real-time amplification curve and standard curve results of RT real time PCR using step-diluted template RNA to confirm the detection limit of the selected primer/probe set.
Figure 4 shows the results of cross-reaction experiments of the selected primer/probe sets. The virus samples used were Sabia virus (SABV), Machupo virus (MACV), Junin virus (JUNV), and Guanarito virus (GTOV).

In order to achieve the object of the present invention, the present invention is an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and an oligonucleotide probe of SEQ ID NO: 3; The oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8 and the oligonucleotide probe of SEQ ID NO: 9; And a marker for detecting Machupo virus, comprising one or more primer sets and probes selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 oligonucleotide primer sets and SEQ ID NO: 15 oligonucleotide probes. composition is provided.

Machupo virus of the present invention is a pathogen that causes Bolivian hemorrhagic fever, one of South American hemorrhagic fevers.

In the marker composition according to the present invention, the marker consisting of the oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and the oligonucleotide probe of SEQ ID NO: 3 specifically amplifies the Z protein coding sequence of the L segment of Machupo virus. The marker consisting of the oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8 and the oligonucleotide probe of SEQ ID NO: 9 can specifically amplify the RNA-dependent RNA polymerase (RdRP) coding sequence of the L segment. The marker consisting of the oligonucleotide primer set of SEQ ID NO: 13 and SEQ ID NO: 14 and the oligonucleotide probe of SEQ ID NO: 15 can specifically amplify the glycoprotein (GP) coding sequence of the S segment is a marker

Arenaviruses, including Machupo virus, have a single-stranded ambisense RNA genome that is segmented into two segments, each RNA segment encoding two viral proteins located in opposite orientations. It serves as a template for transcription of this single mRNA and a template for positive direction replication of the second mRNA. The S-segment RNA is about 3.5 kb in size and encodes the viral nucleocapsid protein (NP) and glycoprotein (GP), and the L-segment RNA is about 7.2 kb in size and encodes the viral RNA-dependent RNA polymerase. (RNA-dependent RNA-polymerase, RdRP) and a small ring (RING) domain-containing protein (Z). The Z protein forms homotypic oligomers and is the structural element of virions.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow for the synthesis of extension products to begin. The specific length and sequence of the primer will depend on the complexity of the DNA or RNA target required, as well as the conditions of use of the primer, such as temperature and ionic strength.

In the present invention, "probe" is a linear oligomer having a natural or modified monomer or linkage including deoxyribonucleotide and ribonucleotide that hybridizes with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid). it means.

In the marker composition according to one embodiment of the present invention, the probe may be prepared by attaching a fluorescent material. The fluorescent substance is HEX, FAM, JOE, ROX, Texas Red, TET, TRITC, TAMRA, fluorescein, fluorescein chlorotriazinyl, rhodamine green , rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, cyanine-based dyes, or thiadicarbocyanine. It may be a dye, but is not limited thereto.

The primers or probes may have at least 16, at least 17, at least 18, at least 19 in the sequences of SEQ ID NOs: 1, 2, 3, 7, 8, 9, 13, 14 and 15, depending on the sequence length of each primer or probe. oligonucleotides consisting of segments of 20 or more, 20 or more contiguous nucleotides. For example, the primer of SEQ ID NO: 1 (20 oligonucleotides) is an oligonucleotide consisting of fragments of at least 15, at least 16, at least 17, at least 18, at least 19 consecutive nucleotides in the sequence of SEQ ID NO: 1 can include In addition, the primer or probe may also include added, deleted or substituted sequences of the nucleotide sequences of SEQ ID NOs: 1, 2, 3, 7, 8, 9, 13, 14 and 15.

In this specification, oligonucleotides used as primers or probes may also include nucleotide analogs, such as phosphorothioates, alkylphosphorothioates, or peptide nucleic acids. or may contain an intercalating agent. In addition, the primers may incorporate additional features that do not alter the basic properties of the primers that serve as the starting point of DNA synthesis. The suitable length of the primer is determined by the characteristics of the primer to be used, but usually a length of 15 to 30 bp is used. The primer does not have to be exactly complementary to the sequence of the template, but must be complementary enough to form a hybrid-complex with the template.

The present invention also relates to the marker composition; And it provides a kit for detecting Machupo virus, including a reagent for performing an amplification reaction.

In one embodiment of the present invention, the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, and a buffer, and may further include a ribonuclease inhibitor, a thermostable polymerase, and the like. However, it is not limited thereto. The DNA polymerase may be a reverse transcriptase such as RNA dependent DNA polymerase, and may be a reverse transcriptase derived from various sources, such as avian myeloblastosis virus-derived reverse transcriptase. derived virus reverse transcriptase (AMV RTase), murine leukemia virus-derived virus reverse transcriptase (MuLV RTase) and Rous-associated virus 2 reverse transcriptase (RAV-2 RTase) ), but is not limited thereto. In addition, the buffer may include both a reverse transcription buffer and a PCR buffer, and the thermostable polymerase may be EF Taq polymerase.

In one embodiment of the present invention, the kit may further include a user guide describing optimal reaction performance conditions. The guide is a printout that explains how to use the kit, for example, how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions to be presented. The guide includes a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.

The present invention also

isolating total RNA from a biological sample isolated from a patient suspected of South American hemorrhagic fever;

Amplifying a target sequence by using the separated total RNA as a template and performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the marker composition of the present invention; and

It provides a method for detecting Machupo virus, including; detecting the product of the amplification step.

The method of the present invention includes the step of isolating total RNA from a biological sample isolated from a patient suspected of South American hemorrhagic fever. The biological sample includes a wide range of all biological body fluids obtained from individuals, and is preferably a group consisting of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, urine, and combinations thereof derived from individuals suspected of South American hemorrhagic fever. It may be one or more selected from, but is not limited thereto. As a method of obtaining bodily fluids, tissues, biopsies, etc. from mammals, conventional methods known in the art may be used.

As a method for isolating total RNA from the sample, any method known in the art may be used, for example, a phenol extraction method may be used. The target sequence may be amplified by performing RT-PCR using the isolated total RNA as a template and using one or more oligonucleotide primer sets according to an embodiment of the present invention as primers. Such PCR methods are well known in the art, and commercially available kits may be used.

In addition, identification, that is, detection of the amplification product according to the present invention may be performed through gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting the amplification product, gel electrophoresis may be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis may be used for gel electrophoresis depending on the size of the amplification product. In addition, capillary electrophoresis can be performed. For capillary electrophoresis, for example, an ABi Sequencer can be used. In addition, in the fluorescence measurement method, when PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer or probe, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorescence meter. can be measured by In addition, the radioactivity measurement method is performed by adding a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product, and then using a radioactivity measurement instrument, for example, a Geiger counter or liquid scintillation Radioactivity can be measured using a liquid scintillation counter.

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

Example 1. Diagnostic gene synthesis and diagnostic primer/probe design and synthesis

The detection site was selected through homology analysis based on the gene sequence of the South American hemorrhagic fever virus. For homology analysis, the MEGA (http://www.megasoftware.net/) 5.1 program was used, and the primers and probes were not homologous to other species and selected base sequence regions that could specifically distinguish Machupo virus. After selection, the size of the product amplified by a combination of forward and reverse primer sets was prepared to be about 150 to 300 bp.

Reference sequences for sequencing GenBank accession no. name NC_005082 Guanarito virus - L segment NC_005080 Junin virus - L segment NC_005079 Machupo virus - L segment NC_006313 Sabia virus - L segment NC_005077 Guanarito virus - S segment NC_005081 Junin virus - S segment NC_005078 Machupo virus - S segment NC_006317 Sabia virus - S segment

Information on primers and probes designed to detect Machupo virus is shown in Table 2 below. The S segment of Machupo virus consists of glycoprotein and nucleocapsid protein coding genes, and two sets (S-GP1/S-GP2) target the glycoprotein gene, nucleocapsid protein Two sets of primers/probes (S-NP1/S-NP2) were designed to target the gene. The L segment is composed of zinc finger protein and L polymerase gene, with one set targeting the zinc finger protein gene (Z) and two sets targeting the L polymerase gene (L1/L2 ) of primers/probes were designed.

Genetic sites to be used as PCR templates for Machupo virus and South American hemorrhagic fever viruses (Guanarito virus, Junin virus, and Sabia virus) were synthesized, and the L segment of each virus was designed for each virus to test cross-reactivity between viruses. Synthesis was performed by aligning primer and probe positions. The synthesized gene was cloned into a pUC57 plasmid (Macrogen), but cloned again into a pGEM-3Zf(+) vector (Promega) having T7 and SP6 promoters to perform in vitro transcription for securing RNA. When the synthesized gene was transformed into E. coli, it was intended to be synthesized within the range of the minimum gene so that E. coli could not express proteins or change its character, and the used E. coli was JM109 (: SP6 RNA polymerase was not present, so the inserted base sequence was DNA gyrase and Proteins such as topoisomerase IV are not expressed and antibiotic resistance is not affected) Competent cells were used.

Template Sequence Donor Information Plasmid name scientific name transgene
(full size) origin of information
(1. GenBank#,
2. synthetic nucleotide sequence) pGEM-GTOV-S Guanarito virus
(GTOV) S segment
(1140 bp) 1.NC_005077,
2. Glycoprotein precursors
nt 301-780 (480);
nt 1201-1440(240)
nucleocapsid protein
nt 1021-1200 (180);
nt 1201-1440(240) pGEM-GTOV-L L segment
(1128 bp) 1.NC_005082,
2. L protein
nt 1801-1980(180),
nt 2701-2940(240);
nt 3601-3780(180);
nt 6061-6300(240)
Z protein
nt 1-288 (288) pGEM-JUNV-S Junin virus
(JUNV) S segment
(1080bp) 1.NC_005081
2. Glycoprotein precursors
nt 1-660 (660);
nucleocapsid protein
nt 121-540(420) pGEM-JUNV-L L segment
(1098 bp) 1.NC_005080,
2. L protein
nt 1741-2040(300),
nt 4381-4620(240),
nt 6001-6633(273)
Z protein
nt 1-285 (285) pGEM-MACV-S Machupo virus
(MACV) S segment
(1140 bp) 1.NC_005078,
2. Glycoprotein precursors
nt 361-720 (360);
nucleocapsid protein
nt 241-1020(780) pGEM-MACV-L L segment
(1005 bp) 1. NC_005079,
2. L protein
nt 361-480(120);
nt 4021-4200(180);
nt 4561-4740(180);
nt 6061-6300(240)
Z protein
nt 1-285 (285) pGEM-SABV-S Sabia virus
(SABV) S segment
(450bp) 1.NC_006317,
2. Glycoprotein precursors
nt 291-560 (270);
nucleocapsid protein
nt 421-600(180) pGEM-SABV-L L segment
(1023 bp) 1. NC_006313,
2. L protein
nt 241-720(480);
nt 3181-3420(240)
Z protein
nt 1-303 (303)

Example 2. Optimal Primer Set Selection Test

The Machupo virus is an experiment using the genes of pathogenic microorganisms notified by the Minister of Health and Welfare that state management is necessary for national health reasons, Article 22-2 (1) of the 「Act on Transboundary Movement of LMOs」, and Article 22-2 of the Enforcement Decree of the same Act In accordance with Article 23-6 Paragraph 2 and Article 14-5 Paragraph 1 of the Enforcement Rule of the same Act, approval for development and testing of LMOs is required as above. On May 29, 2020, the present inventors obtained approval for genetically modified organism development experiments from the Centers for Disease Control and Prevention (No. 20-RDM-016).

The template RNA of Machupo virus was synthesized as a DNA fragment from the sequence obtained through homology analysis (see Table 3), then amplified and purified by PCR reaction, and used after synthesizing RNA by in vitro transcription method. .

First, conventional PCR was performed to confirm the Machupo virus specificity of the designed primer and probe set (Table 2). Each reaction was performed using NTC (non template control) and 1 ng of S-segment/L-segment DNA of machupo virus. The reaction solution is shown in Table 4 and the protocol is shown in Table 5. The results of conventional PCR were confirmed by electrophoresis, and the gel concentration was a mixture of 1.5% agarose and 1% low melting agarose.

Conventional PCR reaction solution composition Component volume Template (1 ng/μl) 1 μl Forward primer (10 pmol/μl) 1 μl Reverse primer (10 pmol/μl) 1 μl 2× Master mixture* 10 μl Nuclease-free water up to ~20 μl Total 20 μl * 2× master mixture uses Bioneer’s AccuPower Dual-star qPCR master mix

Conventional PCR conditions reaction Step Temp. (℃) Time Cycle Taq-polymerase activation 95 5min
reaction denaturation 95 20 seconds
40 annealing 55 20 seconds extension 72 30 seconds Final-extension 72 10min

As a result of performing conventional PCR, it was found that non-specific reactions were not confirmed in all of the seven primer sets, and a product of the predicted size was amplified by accurately detecting the target site (FIG. 1). RT real time PCR was performed on the RNA template by applying probes designed together for seven primer sets in which non-specific reactions were not confirmed. The reactant composition and reaction conditions of RT real time PCR are shown in Tables 6 and 7 below. The probe used in the present invention was a TaqMan probe, FAM was used as a fluorescent dye, and BHQ1 was used as a quencher.

Composition of RT real time PCR reaction solution Component volume Template (1 ng/μl) 1 μl Forward primer (10 pmol/μL) 1 μl Reverse primer (10 pmol/μL) 1 μl TaqMan probe (10 pmol/μl) 1 μl 10× Hotstart buffer 2 μl 2× Master mixture* 10 μl Nuclease-free water up to ~20 μl Total 20 μl * 2× master mixture uses AccuPower Dual-Hotstart RT-qPCR master mix from Bioneer Co., Ltd.

One-step RT real time PCR conditions reaction Step Temp. (℃) Time Cycle Reverse Transcription 50 30min Taq-polymerase activation 95 10min reaction denaturation 95 20 seconds 40 annealing 55 1 min

As a result, as shown in FIG. 2, it was confirmed that GP2 of the S segment had lower Ct values than Z and L2 of the L segment, and that L2 had a lower Ct value than Z. In addition, the template RNA of Machupo virus was diluted 10-fold and prepared to 1 ng ~ 0.1 fg/rxn, and then RT real-time PCR was performed. As a result, MACV - It was found that the S-GP2 and MACV-Z markers stably discriminated between the samples (FIG. 3).

Example 3. Confirmation of cross-reactivity

In order to confirm whether the primer/probe set of the present invention is a molecular marker specific to Machupo virus, cross-reactivity with other South American hemorrhagic fever viruses was analyzed by RT real time PCR. The virus samples used were Guanarito virus, Junin virus, and Sabia virus, and the template sequences are shown in Table 3.

As a result, as shown in Table 8 below, in the case of MACV-S-NP1/2, non-specific reactions were confirmed to occur in Sabia virus, Guanarito virus, and Junin virus, and PCR reaction was also confirmed in NTC to detect Machupo virus. It was found that it was not suitable as a marker for . Through the above results, the present inventors found that the three primer/probe sets (MACV-Z, MACV-L2, MACV-S-GP2) could be used as efficient detection markers for Machupo virus (FIG. 4).

RT real-time PCR of 4 South American hemorrhagic fever viruses using a Machupo virus-specific primer/probe set Ct value for each primer/probe set (average value of 3 repetitions) MACV-Z MACV-L2 MACV-S-GP1 MACV-S-GP2 MACV-S-NP1, -NP2 SABV n/a n/a n/a n/a Non-specific amplification confirmed in NTC and other viruses. MACV 13.95 14.21 13.83 13.49 JUNV n/a n/a n/a n/a GTOV n/a n/a n/a n/a NTC n/a n/a n/a n/a

<110> Republic of Korea (Armed Forces Medical Command) <120> Molecular marker for specifically detecting Machupo virus and uses it <130> PN21120 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 1 cgaccactac ttgtgcctga 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 2 acgggaactg tgatggatgt 20 <210> 3 <211> 21 <212> DNA <213> artificial sequence <220> <223> probe <400> 3 tgtcacatat gctggaaacc a 21 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 4 ctgaagcatg acctgcaaaa 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 5 tttcaatcac ccaatcagca 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 6 tgatggcagg agcaattaca 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 7 gctcgagatg atctgcttcc 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 8 tggtgtttct tcacccatga 20 <210> 9 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 9 ccaagagtgt catcgggact 20 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 10 ctggcttatg gatcctccaa 20 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 11 atggaagccc ctgaagagat 20 <210> 12 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 12 caaagctgat gatgccagag 20 <210> 13 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 13 tcaaatgctt tgggacatga 20 <210> 14 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 14 agcacacttt cccttcctca 20 <210> 15 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 15 ctggcttatg gatcctccaa 20 <210> 16 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 16 aaggacgaga gctccatcaa 20 <210> 17 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 17 ccctcttttc gcttgacttg 20 <210> 18 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 18 gtcagctgct gtgaaagctg 20 <210> 19 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 19 ctggctataa cttcagtctg 20 <210> 20 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 20 tccaagcgtt gacatctctg 20 <210> 21 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 21 ttgatggagc tctcgtcctt 20 <210> 22 <211> 20 <212> DNA <213> artificial sequence <220> <223> probe <400> 22 cgaatgcttg cagatcgtaa 20 <210> 23 <211> 22 <212> DNA <213> artificial sequence <220> <223> probe <400> 23 gaccttgaca agctaactct ag 22

Claims (5)

an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and an oligonucleotide probe of SEQ ID NO: 3; The oligonucleotide primer set of SEQ ID NO: 7 and SEQ ID NO: 8 and the oligonucleotide probe of SEQ ID NO: 9; and an oligonucleotide primer set of SEQ ID NO: 13 and SEQ ID NO: 14 and an oligonucleotide probe of SEQ ID NO: 15; containing, a marker composition for detecting Machupo virus. The marker composition of claim 1; And a kit for detecting Machupo virus, comprising reagents for performing an amplification reaction. The kit according to claim 2, wherein reagents for performing the amplification reaction include DNA polymerase, dNTPs, and a buffer. isolating total RNA from a biological sample isolated from a patient suspected of South American hemorrhagic fever;
Amplifying a target sequence by using the separated total RNA as a template and performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the marker composition of claim 1; and
Detecting the product of the amplification step; a method for detecting Machupo virus, including. The method according to claim 4, wherein the detection of the amplification product is performed by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

Images