KR102379499B1 - Molecular marker for specifically detecting Chikungunya virus and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker capable of specifically detecting Chikungunya virus and its use, and by using the molecular marker according to the present invention, specific and efficient detection of Chikungunya virus is possible Therefore, it may be usefully used in the field of infectious disease diagnosis.

Description

Molecular marker for specifically detecting Chikungunya virus and uses thereof

The present invention relates to molecular markers capable of specifically detecting Chikungunya virus and uses thereof.

Recently, the occurrence of mosquito-borne infectious diseases is increasing worldwide due to rapid climate change, and the possibility of introducing such mosquito-borne infectious diseases into Korea is increasing due to the increase in overseas travel and international exchange in Korea. The number of imported infectious diseases has continued to increase, with 300-400 cases reported every year since 2010. The major imported infectious diseases reported in 2015 were dengue fever (52%), malaria (14%), bacterial shigella and hepatitis A (5% each), and typhoid (4%). The imported countries are mainly distributed in Asia (about 84%) such as the Philippines, Indonesia, Thailand, India, China, Vietnam, Myanmar, and Malaysia, and partly in Africa (about 13%) such as Equatorial Guinea and South Sudan. In general, human infection by mosquito-borne viruses occurs when a female mosquito bites a human. Representative diseases caused by mosquito-borne viruses include malaria, dengue infection (dengue fever), West Nile fever, chikungunya fever, yellow fever, and Japanese encephalitis. there are many

Chikungunya fever is a viral disease transmitted to humans from mosquito-borne mosquitoes infected with Chikungunya virus. Chikungunya fever causes high fever and severe joint pain, and may be accompanied by other symptoms such as muscle aches, headaches, nausea, fatigue, and rashes. Joint pain caused by chikungunya fever is sometimes debilitating, and the duration of the pain varies. To date, no specific treatment for chikungunya fever has been developed.

Chikungunya virus is native to tropical Africa and Asia, belongs to the alpha-virus of the Togaviridae family, and is a virus mediated by arthropods and transmitted to vertebrates (Arthropod-born vertebrate viruses) is an "Arbovirus". It mainly occurs in Africa, Central and South America, etc. In Brazil, although it is decreasing compared to the previous year, the epidemic continues. In Korea, a total of 12 cases were reported in 2018.

On the other hand, Korean Patent No. 2030245 discloses an 'oligonucleotide set for detecting chikungunya virus and its use' targeting a TF (transframe fusion protein) gene, and Korean Patent Publication No. 2011-0118176 discloses 'chikungunya virus' 'Probes and primers for the detection of

The present invention was derived from the above requirements, and the present inventors selected a nucleotide sequence site capable of specifically detecting chikunguniya virus through nucleotide sequence alignment of the chikunguniya virus, and the sequence of the selected site Based on the primer set was prepared. As a result of performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) and RT real time PCR using the prepared primer sets and the synthesized RNA template, the non-specific amplification reaction did not appear, and up to 100 fg concentration of the synthesized RNA template was performed. The present invention was accomplished by identifying a chikunguniya virus-specific primer set that is detectable and does not cross-react with Zika virus, Japanese encephalitis virus and dengue virus RNA samples.

In order to solve the above problems, the present invention provides a primer set for detecting Chikungunya virus comprising an oligonucleotide primer set of SEQ ID NO: 10 and SEQ ID NO: 11.

In addition, the present invention is the primer set; And it provides a kit for detecting chikunguniya virus, including a reagent for performing an amplification reaction.

In addition, the present invention is a method comprising: isolating total RNA from a biological sample isolated from a patient suspected of having chikungunya fever, or a mosquito sample collected in the wild; using the isolated total RNA as a template and performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the primer set of the present invention to amplify a target sequence; and detecting the product of the amplification step; it provides a method for detecting a chikunguniya virus, including.

The use of the molecular marker for chikungunya virus detection according to the present invention enables specific and efficient detection of chikungunya virus within a short period of time. It is thought that it is possible to block the spread of an infectious disease through early diagnosis when an infectious disease occurs.

1 shows the results of converntional RT-PCR using five primer sets (CHIKV1, CHIKV2, CHIKV3, CHIKV4, CHIKV5; see Tables 3 and 4).
2 is a standard curve and Ct value of RT real time PCR using template RNA diluted stepwise to confirm the detection limit of four primer / probe sets (CHIKV1, CHIKV2, CHIKV3, CHIKV4; see Table 3 and Table 4) .
3 is a cross-reaction test result of two primer/probe sets (CHIKV1, CHIKV4).
4 is a comparison experiment result with two primer/probe sets (CHIKV1, CHIKV4) and a commercially available chikunguniya virus diagnostic kit.

In order to achieve the object of the present invention, the present invention provides a primer set for detecting Chikungunya virus comprising an oligonucleotide primer set of SEQ ID NO: 10 and SEQ ID NO: 11.

Chikungunya virus is a (+)-stranded RNA virus with a genome size of about 12 kb and 3' non-structural proteins (nsP1, nsP2, nsP3 and nsP4) at the 5'-end. At -end, structural proteins [capsid protein C, envelope protein E (E3-E2-6K-E1)] are encoded.

The oligonucleotide primer sets of SEQ ID NO: 10 and SEQ ID NO: 11 according to the present invention are primer sets capable of specifically amplifying the structural protein coding gene of chikunguniya virus.

In addition, the primer set of the present invention may further include an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 12.

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

As used herein, the term "probe" refers to a natural or modified monomer comprising deoxyribonucleotides and ribonucleotides that hybridize with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid) or a linear oligomer having a bond. it means.

In the primer set according to an embodiment of the present invention, the probe was prepared by attaching a fluorescent material. The fluorescent material 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 contain 16 or more, 17 or more, 18 or more, 19 or more, 20 or more contiguous nucleotides in the sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, depending on the sequence length of each primer or probe. It may include an oligonucleotide consisting of a fragment of For example, the primer (20 oligonucleotides) of SEQ ID NO: 10 is an oligonucleotide consisting of fragments of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more consecutive nucleotides in the sequence of SEQ ID NO: 10. may include In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

In the present specification, the oligonucleotide used as a primer or probe may also include a nucleotide analogue, for example, phosphorothioate, alkylphosphorothioate or peptide nucleic acid. or may include an intercalating agent. In addition, the primer may incorporate additional features that do not change the basic properties of the primer to 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 is usually used in a length of 15 to 30 bp. The primer need not 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 provides a primer set for detecting the Chikungunya virus; And it provides a kit for detecting Chikungunya virus, including a reagent for performing an amplification reaction.

In one embodiment of the present invention, the primer set for detecting the chikunguniya virus is as described above, and the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, and a buffer, and ribo It may further include a nuclease (ribonuclease) inhibitor, a thermostable polymerase, and the like, but is not limited thereto. The DNA polymerase may be a reverse transcriptase such as an RNA dependent DNA polymerase, a reverse transcriptase from a variety of sources, for example, 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 heat-resistant polymerase may be EF Taq polymerase.

In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include 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 published 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 having chicken gonad fever or a mosquito sample collected in the wild;

using the isolated total RNA as a template and performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the primer set of the present invention to amplify a target sequence; and

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

The method of the present invention comprises isolating total RNA from a biological sample isolated from a patient suspected of having chikungunya fever or a mosquito sample collected in the wild. The biological sample includes a wide range of all biological fluids obtained from an individual, and preferably includes cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, urine, and combinations thereof derived from a subject suspected of chikungunya fever. It may be one or more selected from the group consisting of, but is not limited thereto. A method for obtaining a body fluid, tissue, biopsy, etc. from a mammal may use a conventional method known in the art.

A method for isolating total RNA from the sample may use any method known in the art, for example, a phenol extraction method may be used. A 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, the identification, ie, detection, of the amplification product according to the present invention may be performed through gel electrophoresis, capillary electrophoresis, DNA chip, radiometric 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 depending on the size of the amplification product. In addition, capillary electrophoresis can be performed. Capillary electrophoresis may use, for example, an ABi Sequencer. In addition, in the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorescence meter. can do. In addition, the radioactive measurement method is a radioactive isotope such as ³² P or ³⁵ S added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation Radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only 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

Based on the gene sequence of the Chikunguniya virus strain, the detection site was selected through homology analysis. For homology analysis, MEGA (http://www.megasoftware.net/) 5.1 program was used. Primers and probes have low homology with other mosquito-borne viruses, and nucleotide sequences with high homology between chikunguniya virus strains. After the site was selected, the size of the product to be amplified by the combination of the forward and reverse primer sets was about 150 to 300 bp.

Reference sequence for sequencing analysis GenBank accession no. NC_004162 Chikungunya virus, complete genome MH229986 Chikungunya virus strain 06113879 KX702402 Chikungunya virus strain Chikungunya virus Homo sapiens/Haiti-2/2014 KX702401 Chikungunya virus strain Chikungunya virus Homo sapiens/Haiti-1/2014

Base sequences with poor homology in each reported strain were designed using the base codes in Table 2 below. Information on primers and probes designed for detection of chikungunya virus is shown in Tables 3 and 4.

Degenerate Base, C, Wobble bases, IUB code IUB symbol R Y M K S Mixed base A, G (Y) C, T (R) A, C (K) G, T (M) C, G comment puRines pYrimidine aMine Keto Strong
(3H-bonds) IUB symbol W H B V D Mixed base A, T A, T, C (D) G, T, C (V) G, A, C (B) G, A, T (H) comment Weak
(2H-bonds) Not G Not A Not T Not C

Example 2. Optimal Primer Set Selection Test

Chikunguniya virus template RNA (GenBank accession no. NC_004162 nucleotide sequence) used in the present invention was amplified by PCR reaction, purified, and used after synthesizing RNA by an in vitro transcription method.

Conventional RT-PCR was performed to confirm the specificity of the chikunguniya virus of the designed primer sets (Tables 3 and 4), and the reaction solution was composed of the composition of Table 5, and the protocol is shown in Table 6 below. The results of conventional RT-PCR were confirmed by electrophoresis, and the concentration of the gel was 1.5% agarose and 1% low melting agarose.

Composition of conventional RT-PCR reaction solution component volume Template (1 ng/μl) 1-2 μl Forward primer (10 pmol/μl) 1 μl Reverse primer (10 pmol/μl) 1 μl 2× PCR pre-mixture* 10 μl D.W. up to ~20 μl Total 20 μl * 2× PCR pre-mixture uses Bioneer’s 2× PlusDual Star qPCR Mix

conventional RT-PCR conditions reaction Step Temp. (℃) Time Cycle Reverse Transcription 50 30 min Taq-polymerase activation 95 5 min
reaction denaturation 95 20 sec
40 annealing 60 20 sec extension 72 30 sec final-extension 72 10 min

Conventional RT-PCR results using 5 primer sets (CHIKV1 (189 bp), CHIKV2 (195 bp), CHIKV3 (224 bp), CHIKV4 (100 bp) and CHIKV5 (108 bp)), non-specific reaction in the case of CHIKV5 primer set Products were identified together and the corresponding primer set was excluded (FIG. 1).

RT real time PCR was performed by applying the probes designed for the four primer sets (CHIKV1 to CHIKV4) in which the non-specific reaction was not confirmed. Chikunguniya virus template RNA was used in serial dilutions of 1 ng, 100 pg, 10 pg, 1 pg and 100 fg/rxn, and Ct values for each concentration were compared. Each experiment was performed in triplicate.

Composition of RT real time PCR reaction solution component volume Template (1 ng/μl) 1-2 μl Forward primer (10 pmol/μl) 1 μl Reverse primer (10 pmol/μl) 1 μl Probe (10 pmol/μl) 1 μl 2× PCR pre-mixture* 10 μl D.W. up to ~20 μl Total 20 μl * 2× PCR pre-mixture uses 2× Rapi:Spec Probe Master Mix with RTase for Model UF-150 from Gene Systems Co., Ltd. and 2× PlusDual Star RT-qPCR Master Mix from Bioneer Co., Ltd.

RT real time PCR conditions reaction Step Temp. (℃) Time Cycle Reverse Transcription 50 30 min Taq-polymerase activation 95 10 min reaction denaturation 95 20 sec 40 annealing 60 1 min

As a result, as in FIG. 2, the change (ΔCt) of the Ct value for each concentration of the template RNA was uniformly confirmed for the two primer sets (CHIKV1, CHIKV4), and the results of the amplification amount of fluorescence and the amplification curve for each concentration were obtained. was selected based on

Example 3. Confirmation of cross-reactivity

In order to confirm that the two primer sets (CHIKV1, CHIKV4) selected in Example 2 are molecular markers specific to chikunguniya virus, cross-reactivity with other mosquito-borne viruses was analyzed by RT real-time PCR. As the virus samples used, Zika virus (ZIKA), Japanese encephalitis virus (JEV) and Dengue virus (DENV) serotypes 1, 2, 3 and 4 were used.

As a result, as shown in FIG. 3 , the amplification reaction of the two primer/probe sets (CHIKV1 and CHIKV4) was not confirmed with respect to other virus samples. In addition, a commercially available chikunguniya virus diagnostic kit (Kogen Biotech Co., Ltd., PowerCheck Zika/DENV/CHIKV Real-time PCR) to test the chikunguniya virus diagnostic performance of the two primer/probe sets (CHIKV1, CHIKV4) As a result of comparing Ct values by performing RT real time PCR at the same RNA template concentration using kit Catalog # R5008), it was confirmed that the CHIKV4 primer/probe set can be diagnosed at a level similar to that of a commercially available kit (FIG. 4). From these results, it was found that the CHIKV4 molecular marker of the present invention can be used as an efficient detection marker for chikunguniya virus.

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Claims (6)

A primer set for detecting Chikungunya virus comprising an oligonucleotide primer set of SEQ ID NO: 10 and SEQ ID NO: 11. The primer set for detecting chikunguniya virus according to claim 1, wherein the primer set further comprises an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 12. A primer set according to claim 1 or 2; and a reagent for performing an amplification reaction, a kit for detecting Chikungunya virus. The kit according to claim 3, wherein the 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 having chikungunya fever or a mosquito sample collected in the wild;
using the isolated total RNA as a template, and performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the primer set of claim 1 or 2 to amplify a target sequence; and
A method for detecting Chikungunya virus, including; detecting the product of the amplification step. The method according to claim 5, wherein the detection of the amplification product is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

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