KR102379508B1 - Molecular marker for specifically detecting Japanese encephalitis virus and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker capable of specifically detecting Japanese encephalitis virus and its use. Using the molecular marker according to the present invention, it is possible to detect Japanese encephalitis virus specifically and efficiently within a short time. Therefore, it is thought that it is possible to block the spread of an infectious disease through early diagnosis when an infectious disease occurs.

Description

Molecular marker for specifically detecting Japanese encephalitis virus and uses thereof

The present invention relates to molecular markers capable of specifically detecting Japanese encephalitis 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 inflow 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

Japanese encephalitis virus is a major cause of encephalitis worldwide with arboviruses (Arthropod-born vertebrate viruses mediated by arthropods), Japan, China, Taiwan, Korea, Philippines, Russia. It is an endemic virus widely distributed throughout Asia, including the Far East of the Far East, Southeast Asia, India, Papua New Guinea, and the Torres Strait of Australia. This species originates from the Indonesia-Malaysia region where all genotypes (I, II, III, IV, V) circulate. There is no evidence of virulence differences between genotypes, all reported to form a single serotype. In mosquitoes, it is maintained through an enzootic cycle, and pigs, poultry (Ardeidae, herons), and vertebrates such as ducks are the main vectors. In particular, pigs cause high viremia and are considered hosts from which the virus is amplified. Human infections occur mainly in rural areas, often associated with vector and host habitats (rice crops and flooded areas), and in some parts of Asia, depending on the ecological conditions of urban areas.

Since the 1980s, incidence rates in Japan, South Korea and Taiwan have been declining with vaccines and other preventive measures. Most of them are asymptomatic, with clinical symptoms appearing in less than 1% of people infected with JE virus, with an incubation period of 5-15 days, usually prodromal (2-3 days), acute (3-4 days), subacute ( 7-10 days) and a recovery phase (4-7 days). Typically, fever symptoms, diarrhea, and convulsions occur for several days, headache, vomiting, loss of consciousness, and mild clinical symptoms such as aseptic meningitis, febrile seizures or respiratory-like fever occur, but mainly encephalitis symptoms. In the case of a poor prognosis, encephalitis develops in the brainstem, leading to changes in breathing patterns, flexor and extensor changes, and impaired papillary and ocular reflexes.

On the other hand, Korean Patent No. 0942388 discloses 'a primer set for detecting Japanese encephalitis virus, internal control ribonucleic acid, a reverse transcription polymerase chain reaction kit including the same, and a polymerase chain reaction method using the same', and a Korean registered patent No. 1236197 discloses 'differential diagnosis of West Nile virus and Japanese encephalitis virus', but there is no description of 'a molecular marker capable of specifically detecting Japanese encephalitis virus and its use' of the present invention.

The present invention was derived in response to the above needs, and the present inventors specifically detected the Japanese encephalitis virus through nucleotide sequence alignment with the E (envelope protein) gene or NS (non-structural protein) gene of the Japanese encephalitis virus. Possible nucleotide sequence sites were selected, and a primer set was prepared based on the sequence of the selected site. As a result of performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the prepared primer sets and the synthesized Japanese encephalitis RNA template, a non-specific amplification reaction did not appear, and it was possible to detect up to 100 fg of the synthesized RNA template. The present invention was completed by identifying two Japanese encephalitis virus-specific primer sets that did not cross-react with Zika virus, chikungunia virus and dengue virus RNA samples.

In order to solve the above problems, the present invention provides an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2; Or SEQ ID NO: 4 and oligonucleotide primer sets of SEQ ID NO: 5; provides a primer set for detecting Japanese encephalitis virus comprising a.

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

In addition, the present invention is a method comprising the steps of isolating total RNA from a biological sample isolated from a patient suspected of Japanese encephalitis 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 Japanese encephalitis virus, including.

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

1 is a result of converntional RT-PCR using four primer sets (JEV1, JEV2, JEV3 and JEV5; see Table 3).
2 is a standard curve, Ct value and real-time amplification curve result of RT real time PCR using template RNA diluted stepwise to confirm the detection limit of two selected primer/probe sets (JEV1, JEV2; see Table 3) .
3 is a cross-reaction test result of two selected primer/probe sets (JEV1, JEV2). The virus samples used are Zika virus (ZIKA), Chikungunia virus (CHIKV) and Dengue virus (DENV).

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2; Or SEQ ID NO: 4 and oligonucleotide primer sets of SEQ ID NO: 5; provides a primer set for detecting Japanese encephalitis virus comprising a.

Japanese encephalitis virus is a dodecahedral (+) single-stranded RNA virus belonging to the genus Flavivirus of the Flaviviridae family. is formed In addition, the virus genome encodes several non-structural proteins such as NS1, NS2a, NS2b, NS3, N4a, NS4b, and NS5 in addition to the structural proteins.

The oligonucleotide primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention are primer sets capable of specifically amplifying the NS2b gene of Japanese encephalitis virus, and the oligonucleotide primer sets of SEQ ID NO: 4 and SEQ ID NO: 5 are Japanese encephalitis virus It is a primer set that can specifically amplify the NS1 gene of

In addition, the primer set of the present invention may further include an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 6, and specifically, the oligonucleotide primer set of SEQ ID NO: 1 and SEQ ID NO: 2 and the sequence The oligonucleotide probe of No. 3 can be used together, and the oligonucleotide primer sets of SEQ ID NOs: 4 and 5 and the oligonucleotide probe of SEQ ID NO: 6 can be used together.

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, etc. It may be a dye, but is not limited thereto.

The primer or probe consists of a fragment of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequence of SEQ ID NO: 1 to SEQ ID NO: 6, depending on the sequence length of each primer or probe oligonucleotides. For example, the primer (20 oligonucleotides) of SEQ ID NO: 1 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: 1 may include In addition, the primer may include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 6.

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 includes the primer set; And it provides a kit for detecting Japanese encephalitis 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, etc. However, it 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 Japanese encephalitis 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 Japanese encephalitis virus, including; detecting the product of the amplification step.

The method of the present invention includes isolating total RNA from a biological sample isolated from a patient suspected of Japanese encephalitis or a mosquito sample collected in the wild. The biological sample includes all biological fluids of a wide range obtained from an individual, preferably a group consisting of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, urine, and combinations thereof derived from a subject suspected of Japanese encephalitis. It may be one or more selected from, 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. 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, 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 Japanese encephalitis virus strain, the detection site was selected through homology analysis. For homology analysis, MEGA (http://www.megasoftware.net/) 5.1 program was used, and the primers and probes had poor homology with other species of the genus Flavivirus , and homology between Japanese encephalitis virus strains. After selecting this high nucleotide sequence site, it was prepared so that the size of the product 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_001437 Japanese encephalitis virus KP164498 Japanese encephalitis virus isolate JEV/SW/IVRI/395A/2014 JN381869 Japanese encephalitis virus isolate LYZ EF543861 Japanese encephalitis virus strain SH0601 AB551990 Japanese encephalitis virus RNA strain: JaTAn1/75 KF711994 Japanese encephalitis virus strain Anyang300 KU821122 Japanese encephalitis virus strain SA14

Base sequences with poor homology in each reported strain were designed using the base codes in Table 2 below. Table 3 provides information on primers and probes designed to detect Japanese encephalitis virus.

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

The template RNA of Japanese encephalitis virus was KBPV-VR-27, infected with Vero cells, cultured for several days, and the culture medium in which cell mutation was observed was obtained and RNA was extracted (QIAamp Virtual RNA Mini kit (QIAGEN, Hilden, Germany)) was used.

In order to confirm the specificity of the designed primer set (Table 3), conventional RT-PCR was performed, the reaction solution was composed of the composition of Table 4, and the protocol is shown in Table 5 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 50 20 sec extension 72 30 sec final-extension 72 10 min

As a result of conventional RT-PCR using four primer sets (JEV1 (208 bp), JEV2 (238 bp), JEV3 (204 bp) and JEV5 (157 bp)), non-specific gene amplification in two primer sets (JEV3 and JEV5) This was confirmed (FIG. 1).

RT real time PCR was performed by applying the designed probes (refer to Table 3) together with respect to two primer sets (JEV1, JEV2) in which non-specific reactions were not confirmed. The Japanese encephalitis 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 50 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 (JEV1, JEV2), and no amplification reaction was confirmed for the template RNA of 100 fg. .

Example 3. Confirmation of cross-reactivity

In order to confirm that the two primer sets (JEV1, JEV2) selected in Example 2 are molecular markers specific to the Japanese encephalitis virus, cross-reactivity with other flavivirus genus viruses was analyzed through RT real time PCR. Virus samples used were Zika virus (ZIKA strain AFMC-U), Chikungunia virus (CHIKV vaccine strain) and Dengue virus (DENV) serotypes 1 (Hawaii 11 NA 12), 2 (KBPV-VR-29), 3 ( KBPV-VR-30) and 4 (KBPV-VR-31) were used.

As a result, as shown in FIG. 3, the two primer sets (JEV1, JEV2) did not undergo amplification reaction with respect to virus samples of other flavivirus genus, and it was found that they were very specific to Japanese encephalitis virus. From these results, it was found that the two primer sets (JEV1, JEV2) of the present invention can be used as efficient detection markers for Japanese encephalitis virus.

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Claims (6)

oligonucleotide primer sets of SEQ ID NO: 1 and SEQ ID NO: 2; And SEQ ID NO: 4 and oligonucleotide primer sets of SEQ ID NO: 5; Japanese encephalitis virus (Japanese encephalitis virus) comprising a primer set composition for detecting. The primer set composition for detecting Japanese encephalitis virus according to claim 1, wherein the primer set composition further comprises an oligonucleotide probe consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 6. A primer set composition according to claim 1 or 2; and a reagent for performing an amplification reaction, a kit for detecting Japanese encephalitis 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 Japanese encephalitis 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 composition of claim 1 or 2 to amplify a target sequence; and
A method for detecting Japanese encephalitis 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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