KR20250021148A - CRISPR-Cas12a-Based Rapid Detection Of Japanese encephalitis virus - Google Patents

Abstract

The present invention relates to a rapid detection method and kit for Japanese encephalitis virus based on gene scissors, and more particularly, to a molecular detection method for rapidly detecting Japanese encephalitis virus using a primer set specific for Japanese encephalitis virus, a specific guide RNA, and Cas12a, and a kit therefor.

Description

CRISPR-Cas12a-Based Rapid Detection Of Japanese Encephalitis Virus

The present invention relates to a method and kit for detecting Japanese encephalitis virus using genetic scissors.

Japanese encephalitis virus (JEV) is a virus that causes Japanese encephalitis, transmitted by Culex mosquitoes. Japanese encephalitis virus is classified into five genotypes, from I to V, based on the E gene.

In 24 countries where Japanese encephalitis occurs, more than 50,000 patients occur each year, and more than 15,000 patients die. Patients develop headaches and high fevers 2 to 4 days after infection, and gradually show symptoms such as nausea and vomiting, including abdominal pain. However, there is still no clear treatment, so it is important to prevent the spread early through rapid and accurate detection of the Japanese encephalitis virus.

The existing methods for detecting Japanese encephalitis virus extract the virus or antibodies from the patient's blood or cerebrospinal fluid and test them using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence assay (IFA), hemagglutination test (HI), and IgM captured enzyme-linked immunoassay (MAC-ELISA). However, detection methods such as ELISA, IFA, and RT-PCR require a relatively long time, and there are technical limitations in developing countries. In addition, MAC ELISA, which uses the patient's serum, has the problem of cross-reactivity with other viruses belonging to the Flaviviridae family, such as dengue virus.

The present invention provides a technology capable of rapidly and accurately detecting Japanese encephalitis virus.

The present invention provides a method for detecting Japanese encephalitis virus using gene scissors, comprising: (a) a step of providing a sample; (b) a step of isothermally amplifying nucleic acid in the provided sample using a primer set specific for Japanese encephalitis virus; and (c) a step of adding a guide RNA, Cas (CRISPR-associated protein) 12a, and a single-stranded DNA probe specific for Japanese encephalitis virus to a product obtained by the isothermal amplification, thereby performing molecular detection using gene scissors.

In addition, the present invention provides a kit for detecting Japanese encephalitis virus, which comprises a primer set specific for each Japanese encephalitis virus, a specific guide RNA, Cas12a, and a single-stranded DNA probe.

The present invention enables rapid and highly sensitive detection of Japanese encephalitis virus in the field using isothermal amplification and CRISPR-Cas12, so that Japanese encephalitis virus infection can be detected without special expertise, technology, equipment, or infrastructure.

In addition, the present invention can detect Japanese encephalitis virus epidemics early, and further, can quickly block Japanese encephalitis virus infection and transmission.

Figure 1 illustrates a schematic diagram of the ONE POT(OP)-DETECTR of the present invention.
Figure 2 shows the results of fluorescence analysis of the trans-cleavage activity of JEV GⅠ guide RNA and other guide RNAs (#2, #3 in Table 4).
Figure 3 shows the results of fluorescence analysis of the trans-cleavage activity of JEV GⅢ guide RNA and other guide RNAs (#2, #3 in Table 5).
Figure 4 shows the results of fluorescence analysis of trans-cleavage activity using JEV GV guide RNA and other guide RNAs (#2, #3 in Table 6).
Figure 5 shows whether the specific guide RNA of each genotype (GI, GIII, GV) of Japanese encephalitis virus reacts with completely different viruses (IAV, IBV, HCV, HEV) using fluorescence analysis and LFA (lateral flow assay).
Figure 6 shows whether the specific guide RNA of each genotype (GI, GIII, GV) of the Japanese encephalitis virus of the present invention reacts with other genotypes of the Japanese encephalitis virus using fluorescence analysis and LFA (lateral flow assay).
Figure 7 shows the sensitivity of the guide RNA of each genotype (GI, GIII, GV) of the Japanese encephalitis virus of the present invention by fluorescence analysis or LFA (lateral flow assay) for each genotype (GI, GIII, GV) of the Japanese encephalitis virus.
Figure 8 shows the sensitivity of the ONE POT(OP) DETECTR of the present invention for each genotype (GI, GIII, GV) of Japanese encephalitis virus using fluorescence analysis or LFA (lateral flow assay).

The present invention provides a method for detecting Japanese encephalitis virus using gene scissors, comprising: (a) a step of providing a sample; (b) a step of isothermally amplifying nucleic acid in the provided sample using a primer set specific for Japanese encephalitis virus; and (c) a step of adding a guide RNA, Cas (CRISPR-associated protein) 12a, and a single-stranded DNA probe specific for Japanese encephalitis virus to a product obtained by the isothermal amplification, thereby performing molecular detection using gene scissors.

In one embodiment of the present invention, a primer specific to each genotype of Japanese encephalitis virus and a specific guide RNA provide a specific sequence.

In one embodiment of the present invention, it was confirmed whether detection was possible by Japanese encephalitis virus genotype (GI, GIII, GV) through DETECTR, and this was confirmed through fluorescence measurement and LFA.

In one embodiment of the present invention, a One pot (OP)-DETECTR method is provided to simultaneously perform two processes in one tube to increase convenience and simplify the process.

In one embodiment of the present invention, the sample may be a sample containing Japanese encephalitis virus, and may be a sample obtained from a subject from whom detection of Japanese encephalitis virus is required. The sample includes, but is not limited to, saliva, blood, serum, plasma, urine, aspirate, and biopsy samples.

In one embodiment of the present invention, the step of providing a sample may further include a step of dissolving virus particles present in the sample and inactivating RNase present in them by heat and chemical treatment, for example, addition of EDTA, without a step of extracting nucleic acids from the sample (HUDSON, Heating Unextracted Diagnostic Samples to Obliterate Nucleases). HUDSON is a method of inhibiting the activity of nuclease by adding Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and EDTA (ethylene-diamine-tetraacetic acid) to body fluids such as saliva, blood, and urine containing viruses and culture media, thereby easily extracting the genome and performing a detection process using recombinase polymerase amplification (RPA) and Cas12a protein.

In one embodiment of the present invention, the step of isothermal amplification may include the step of reverse transcribing a sample of the nucleic acid and the step of performing an isothermal amplification reaction using the product obtained by the reverse transcription as a template.

In one embodiment of the present invention, the method for detecting Japanese encephalitis virus may be characterized in that the specific primer set consists of sequence number 1 and sequence number 2, and the Japanese encephalitis virus is Japanese encephalitis virus genotype GI.

In one embodiment of the present invention, the method for detecting Japanese encephalitis virus may be characterized in that the specific primer set consists of sequence number 3 and sequence number 4, and the Japanese encephalitis virus is Japanese encephalitis virus genotype GⅢ.

In one embodiment of the present invention, the method may be for detecting Japanese encephalitis virus, characterized in that the specific primer set consists of sequence number 5 and sequence number 6, and the Japanese encephalitis virus is Japanese encephalitis virus genotype GV.

RPA is one of the isothermal amplification techniques, and is used to amplify relatively short genomes of 80-500 bp. It can amplify up to 500 bp in length, but is effective in amplifying 100-200 bp in length. RPA is performed within 1 hour at a constant temperature of 35°C to 45°C, and has the advantage that a mismatch of up to 5-9 bp in a primer of 30-35 bp in length does not affect the RPA process.

In one embodiment of the present invention, RT-RPA may be performed at 35° C. to 45° C., preferably 37° C. to 45° C., and more preferably 42° C.

In one embodiment of the present invention, the step of isothermally amplifying the nucleic acid and the fluorescence analysis or LFA can be performed at the same temperature.

In one embodiment of the present invention, the step of isothermally amplifying the nucleic acid and fluorescence analysis or LFA can be performed simultaneously at 42° C.

In one embodiment of the present invention, the step of performing the molecular detection includes detection of a target site among the isothermal amplification products by the guide RNA, activation of Cas12a, and cleavage of a single-stranded DNA probe by the activated Cas12a, wherein the single-stranded DNA probe may not hybridize with the guide RNA and may be labeled with a labeling substance.

In one embodiment of the present invention, the method may be a method for detecting Japanese encephalitis virus, characterized in that the specific guide RNA consists of sequence number 7, and the Japanese encephalitis virus is Japanese encephalitis virus genotype GI.

In one embodiment of the present invention, the method may be a method for detecting Japanese encephalitis virus, characterized in that the specific guide RNA consists of sequence number 8, and the Japanese encephalitis virus is Japanese encephalitis virus genotype GⅢ.

In one embodiment of the present invention, the method may be a method for detecting Japanese encephalitis virus, characterized in that the specific guide RNA consists of sequence number 9, and the Japanese encephalitis virus is Japanese encephalitis virus genotype GV.

Single-stranded DNA probes can be dual-labeled with a fluorescent agent and a quencher.

Single-stranded DNA probes can be dual-labeled with biotin and a fluorescent substance.

The fluorescent material may be a fluorescent dye including but not limited to FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, etc.

In one embodiment of the present invention, the step of determining whether Japanese encephalitis virus is detected may be detecting whether a single-stranded DNA probe is cut by fluorescence analysis or LFA.

In one embodiment of the present invention, LFA may be performed on a strip.

In addition, the present invention provides a kit for detecting Japanese encephalitis virus, comprising a primer set specific for each Japanese encephalitis virus, a specific guide RNA, Cas12a, and a single-stranded DNA probe.

In one embodiment of the present invention, the kit may further comprise a reagent for detection by fluorescence or LFA.

Unless otherwise defined, the terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

As used herein, the term "isothermal amplification" refers to a method of amplifying nucleic acids by incubation at a single temperature, without a thermocycler, and is used interchangeably with "isothermal nucleic acid amplification." Isothermal amplification is nucleic acid amplification that does not rely on heat denaturation of target nucleic acids during the amplification reaction and does not require rapid changes in temperature, and thus can be performed inside and outside of a laboratory environment. Isothermal amplification methods include, but are not limited to, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and the like.

As used herein, the term "gene scissors" refers to CRISPR (Clustered Interspaced Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) protein. Genome scissors-based molecular detection consists of signal amplification, signal conversion, and signal generation. Isothermal amplification such as RPA and LAMP is mainly used for signal amplification, CRISPR gene scissors technology is used for signal conversion, and various fluorescent substances or proteins are used for signal generation.

As used herein, the term "Cas12a" refers to a type of CRISPR-Cas protein, which has an activity of randomly cleaving non-targeted single-stranded DNA (ssDNA) when activated by detection of target DNA, and is used interchangeably with "CRISPR-Cas12a" or "CRISPR/Cas12a". Cas12a from the bacterium Lachnospiraceae recognizes its target DNA using a guide RNA complementary to the target DNA sequence and a T nucleotide-rich PAM (protospacer-adjacent motif) in the target, and after recognition, the activated LbCas12a not only catalyzes target DNA cleavage, but also promotes non-specific ssDNA cleavage (Chen et al., 2018, Science 360, 436-439). Using this activity of Cas12a, a molecular detection method based on genome-editing scissors is being developed.

The term "molecular detection" as used herein refers to DETECTR, a molecular detection technology based on CRISPR-Cas12a. Using the trans-cleavage activity of LbCas12a and an isothermal nucleic acid amplification technology such as RPA, a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay has been developed for the rapid and specific detection of human papillomavirus (HPV) in clinical samples (Chen et al., 2018, Science 360, 436-439). DETECTR is a method to detect the presence or absence of target DNA by the generation of a easily measurable fluorescence signal using a guide RNA that specifically binds to the target site and a single-stranded DNA probe or reporter that does not bind to the target site, by utilizing the activity of Cas12a to detect target DNA and randomly cleave single-stranded DNA.

As used herein, the term "single-stranded DNA probe" refers to a reporter used to detect the presence of a target nucleic acid by DETECTR, and may be a single-stranded DNA of a sequence that does not hybridize with the target site. It may be labeled with a fluorophore-quencher pair or the like so that it can generate a detectable signal when cleaved by activated Cas12a.

The term "LFA" used in this specification is also called lateral flow assay, and refers to an analytical method that enables rapid detection without skilled personnel or expensive equipment, which is a well-known technology for pregnancy detection kits. By simply dropping a sample solution such as blood onto a device such as a strip, bacteria or viruses in the sample can be detected in a short period of time using lateral flow and specific target binding such as antigen-antibody reaction, and mainly fluorescent/chromogenic labels or nanoparticles are used in a sandwich manner.

Below, the present invention is further explained through experimental examples with reference to the attached drawings. However, the experimental examples are only for explaining the present invention and are not to be construed as limiting the present invention.

<Materials and Experimental Methods>

In order to explain the experimental examples of the present invention, the necessary common materials and common experimental methods are described below.

1. Viral RNA extraction

(1) Preparation of guide RNA and primers

In this experiment, based on the National Center for Biotechnology Information (NCBI) information, the RPA primers (Table 1, SEQ ID NOs. 1 to 6) and gRNA (Table 2, SEQ ID NOs. 7 to 9) were designed for each Japanese encephalitis virus genotype by designating the sequences in the glycoprotein of GⅠ, NS4B of GⅢ, and NS1 of GⅤ genes of each Japanese encephalitis virus. The primers used for RT-RPA were designed according to the TwistAmp® DNA Amplification Kits Assay Design manual.

<RPA primer sequence> Type Genotype Primer 5' - Sequence - 3' RPA
primer Ⅰ RPA-JEV-GⅠ-F GACAGCAGCTACGTGTGCAAACAAGGCTTT
(sequence number 1) RPA-JEV-GⅠ-R CCGAGGTGGTGGTTCCGTGCACGAATATGC
(sequence number 2) Ⅲ RPA-JEV-GⅢ-F TGCCACGAGGCGTGCCTTTTACCGACCTAG
(sequence number 3) RPA-JEV-GⅢ-R TGTCCTTCTCTGGGCAGCCCTGAGTGCTTC
(sequence number 4) V RPA-JEV-GⅤ-F GGAAGGCATTTGTGGAGTGAGATCAGTCAC
(sequence number 5) RPA-JEV-GⅤ-R AATGGAGCTGGTCGGTATCTTCCTGATGGT
(sequence number 6)

Genotype gRNA 5' - Sequence - 3' PAM Ⅰ JEV-GⅠ-gRNA CCGAAAAGUCCACAUCCAUU (SEQ ID NO: 7) TTTC Ⅲ JEV-GⅢ-gRNA ACCCCAACAGCCAAGGAAGA (SEQ ID NO: 8) TTTG V JEV-GⅤ-gRNA UCCACCACCACACUAAGAUC(SEQ ID NO: 9) TTTG

(2) Sample preparation

To culture Japanese encephalitis virus for use in the experiment, BHK-21 cells (Korea Cell Line Bank, Korea) were provided and cultured in Dulbecco's modified Eagle's media (DMEM) (Hyclone, USA) containing 10% fetal bovine serum (FBS) (Capricorn, USA) and 1% penicillin and streptomycin (P/S) (Genomicbase, Seoul, Korea).

Japanese encephalitis virus GⅠ (JEV GⅠ) (NCCP43133, National Culture Collection for Pathogens, Cheongju, Korea) was inoculated onto 20th generation BHK-21 cells, and the culture medium was changed to DMEM medium containing 2% FBS and 1% P/S. The virus titer was confirmed using a 6-well culture plate.

(3) HUDSON

The HUDSON process was performed using a virus culture medium. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and ethylenediaminetetraacetic acid (EDTA) were added to the virus culture medium to make 100 mM and 1 mM, respectively, and incubated at 50°C for 20 minutes and then at 95°C for 5 minutes. Completed HUDSON samples were stored at -80°C.

(4) cDNA synthesis

JEV GⅠ RNA was extracted using AccuPrep® Viral RNA Extraction Kit (Bioneer, Korea). Complementary DNA (cDNA) was synthesized using the extracted RNA using TOPscript™ RT-PCR Kit (Enzynomics, Korea). The JEV GⅢ NS4B gene (GenBank: EF623989.1, nucleotides 6912 to 7649) and JEV GⅤ NS1 gene (GenBank: JF915894.1, nucleotides 2481 to 3542) were synthesized as DNA according to their respective sequences (Macrogen, Seoul, Korea).

2. In vitro transcription

To perform in vitro transcription using the synthesized cDNA, PCR was performed using a primer set including the T7 promoter (Table 3, SEQ ID NOs. 10 to 15), and in vitro transcription (IVT) was performed with the generated PCR products using the mMessage mMachine T7 Transcription Kit (Invitrogen, USA). The synthesized RNA was purified using the GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Scientific™, USA).

< IVT primer sequence > Type Genotype Primer 5' - Sequence - 3' IVT
primer Ⅰ T7-JEV-GⅠ-F TAATACGACTCACTATAGGGAGAGACAGCAGCTACGTGTGCAAACAAGGCTTT
(sequence number 10) T7-JEV-GⅠ-R CCGAGGTGGTGGTTCCGTGCACGAATATGCCAACCT
(Sequence number 11) Ⅲ T7-JEV-GⅢ-F TAATACGACTCACTATAGGGAGACTGACTGGATTGCCAAGCAT
(sequence number 12) T7-JEV-GⅢ-R GAATTCCAAACGGCACTGGCTCCATTGTCCC
(Sequence number 13) V T7-JEV-GⅤ-F TAATACGACTCACTATAGGGAGAATGACGTGGAGGCTTG
(Sequence number 14) T7-JEV-GⅤ-R GCAAAGAGAATGCTCTTCCCCCATGC
(Sequence number 15)

3. RT-RPA

The RT-RPA process was performed using the TwistAmp® Basic Kit (TwistDx, Cambridge, UK) to amplify RNA. Primers used in RT-RPA were designed according to the TwistAmp® DNA Amplification Kits Assay Design manual. The RT-RPA complex was prepared by adding 29.5 μL of rehydration buffer, 2.4 μL of each 10 μM forward and reverse primer (Table 1), 1 μL of SuperScript IV Reverse Transcriptase (Invitrogen, Waltham, Massachusetts, USA), 1 μL of RNase inhibitor (Enzynomics, Korea), and 2.5 μL of 280 mM magnesium acetate. RNA sample and nuclease-free water were added to a final volume of 50 μL. The prepared complex was incubated at 42 °C for 40 minutes.

4. ONE POT(OP) DETECTR

For ONE POT DETECTR (Fig. 1), 29.5 μL of rehydration buffer, 2.4 μL of each of 10 μM forward and reverse primers, 1 μL of SuperScript IV Reverse Transcriptase, and 1 μL of RNase inhibitor were added to a 1.5 mL tube and 18.15 μL of these were divided into two new tubes. 5 μL of RNA sample was added, 13 μL of RPA powder was resuspended, and 6.5 μL was transferred to each tube, and 1.25 μL of 280 mM magnesium acetate was added last. 2.5 μL of 10 X NEB 2.1 buffer, 1 μL of 1 μM Cas12a, 0.5 μL of 1 μM gRNA, and 1 μL of 10 μM FQ-labeled reporter were added inside the tube cap and the cap was closed to prevent the solution from spilling. The above tube was incubated at 42°C for 15 minutes, then gently spun down and the 520 nm wavelength was observed in real time (fluorescence measurement method).

For LFA, 29 μL of 1 X NEB 2.1 buffer, 8 μL of 1 μM Cas12a, 1 μL of 10 μM gRNA, and 1 μL of 10 μM lateral flow cleavage reporter were added to the lid. Similarly, after incubation at 42 °C for 15 minutes, the tube was gently spun down and incubated again for 5 minutes. After 5 minutes, Milenia HybriDetect 1 lateral flow strips were immersed in the solution in the tube, and the results were confirmed after 2 minutes.

5. Fluorescence measurement

For fluorescence measurement, 80 μL of 1 X NEB 2.1 buffer, 18 μL of LbCas12a-gRNA complex, 2 μL of RT-RPA amplicons, and 2 μL of 10 μM FQ-labeled reporter (/56-FAM/TTATT/3IABkfQ; Integrated DNA Technologies, Coralville, IA, USA) were added to each well of a 96-well plate and incubated at 37 °C for 10 minutes. The fluorescence value was measured after 10 minutes (λex, 485 nm; λem, 535 nm).

6. LFA

For LFA, 38 μL of 1 X NEB 2.1 buffer, 36 μL of LbCas12a-gRNA complex, 2 μL of 10 μM lateral flow cleavage reporter (/56-FAM/TTATT/3Bio/; Integrated DNA Technologies, Coralville, IA, USA), and 4 μL of RPA products were added to a single tube and incubated at 37 °C for 10 min. Milenia HybriDetect 1 lateral flow strips (Milenia Giesesen, Germany) were immersed in the solution in the tube, and the results were checked after 2 minutes.

Experimental Example 1. Comparison of DETECTR gRNA trans-cleavage activity

1-1. Method

In order to confirm the activity of the specific guide RNA (SEQ ID NOs: 7 to 9) of the present invention, in addition to the specific guide RNA of the present invention, the trans-cleavage activity was compared using Japanese encephalitis virus genotype GⅠ, GⅢ, and GⅤ guide RNA sequences (Tables 4 to 6, #2, #3).

<Specific RPA primer (GⅠ RPA-primer) and guide RNA (Used) of the present invention, and the remaining guide RNA and primer sequences (#2, #3)> Label Type Direction/PAM 5' - Sequence - 3' GI-RPA-primer F GACAGCAGCTACGTGTGCAAACAAGGCTTT R CCGAGGTGGTGGTTCCGTGCACGAATATGC Used gRNA TTTC CCGAAAAGUCCACAUCCAUU #2 gRNA TTTG CGCAUGTGUCAAUGCUUCCU #3 gRNA TTTC GGGAAAGGAAGCAUUGACAC

<Specific RPA primer (GⅢ RPA-primer) and guide RNA (Used) of the present invention, and the remaining guide RNA and primer sequence (#2)> Label Type Direction/PAM 5' - Sequence - 3' GIII-RPA-primer F TGCCACGAGGCGTGCCTTTTACCGACCTAG R TGTCCTTCTCTGGGCAGCCCTGAGTGCTTC Used gRNA TTTC ACCCCAACAGCCAAGGAAGA #2 gRNA TTTTA CCGACCTAGACTTGACCGTT

<Specific RPA primer (GⅤ RPA-primer) and guide RNA (Used) of the present invention, and the remaining guide RNA and primer sequences (#2, #3)> Label Type Direction/PAM 5' - Sequence - 3' GV-RPA-primer F GGAAGGCATTTGTGGAGTGAGATCAGTCAC R AATGGAGCTGGTCGGTATCTTCCTGATGGT Used gRNA TTTG UCCACCACCACACUAAGAUC #2 gRNA TTTTA ACUCGUCCCUGACGGCUUCC #3 gRNA TTTG AUGUUCCAGUCUGGUGACUG

1-2. Results

As a result of fluorescence measurement, the trans-cleavage activity of the specific guide RNA of the present invention was significantly higher than that of other guide RNAs (#2, #3) in Japanese encephalitis virus genotypes GⅠ, GⅢ, and GⅤ (Figs. 2 to 4). Therefore, it was confirmed that the specific guide RNA of the present invention (SEQ ID NOs: 7 to 9) had high gRNA trans-cleavage activity for each of the Japanese encephalitis virus genotypes GⅠ, GⅢ, and GⅤ.

Experimental Example 2. Specific Detection of Japanese Encephalitis Virus Using DETECTR

2-1. Method

It was confirmed whether the method for detecting Japanese encephalitis virus using the DETECTR of the present invention reacts to other viruses. Influenza A virus (IAV) (A/Puerto Rico/8/1934), Influenza B virus (IBV) (B/Brisbane/60/2008), Hepatitis C virus (HCV) (JFH-1), and Hepatitis E virus (HEV) (47832c) were treated to samples with 100 mM TCEP and 1 mM EDTA using 50 μl of culture medium each, incubated at 50 °C for 20 minutes, then incubated at 95 °C for 5 minutes, and then stored at -80 °C (HUDSON).

Afterwards, the RNA of IAV, IBV, HCV, and HEV extracted by HUDSON was subjected to RT-RPA under the same conditions, and the results were confirmed using the fluorescence measurement method and LFA described above (Fig. 6). All virus handling processes were performed at biosafety level-2.

C-line represents the control line, T-line represents the test line, and NTC represents the no template control.

2-2. Results

The detection method of the present invention reacted only to Japanese encephalitis virus, both in fluorescence measurement and LFA, and did not react to IAV, IBV, HCV, and HEV viruses (Fig. 5). Therefore, it is suggested that the present invention specifically detects only Japanese encephalitis virus.

Experimental Example 3. Detection of different genotypes of specific guide RNA for each genotype of Japanese encephalitis virus

3-1. Method

To confirm whether there was a response for each genotype of Japanese encephalitis virus through the DETECTR of the guide RNA specific for each genotype of the present invention, an FQ-labeled reporter was used. Other experimental methods are the same as Experimental Example 2.

3-2. Results

The experimental results also showed that the guide RNA specific to each genotype of Japanese encephalitis virus reacted only with each genotype and did not react with the others (Fig. 6). Therefore, this suggests that the guide RNA specific to each genotype of Japanese encephalitis virus of the present invention specifically detects only each genotype of Japanese encephalitis virus.

Experimental Example 4. Confirmation of the sensitivity of the specific guide RNA of the present invention

4-1. Method

To confirm the sensitivity of the specific guide RNA of the present invention, RNA obtained by in vitro transcription was used for confirmation. RNA was diluted from 1×10⁴ copies to 1×10¹ copies using RNase Free Distilled Water and used for RT-RPA.

The results are shown in Fig. 7. C-line represents the control line, T-line represents the test line, and NTC represents the no template control.

4-2. Results

For each genotype, GI reacted identically in both fluorescence measurement and LFA up to 1×10 ¹ RNA copies, and GIII and GV reacted identically in up to 1×10 ² RNA copies (Fig. 7). Therefore, we concluded that GI can be detected up to 1Х10 ¹ copy, and GIII and GV can be detected up to 1Х10 ² RNA copies using the specific guide RNA of the present invention.

Experimental Example 5. Sensitivity Verification of OP-DETECTR

5-1. Method

The present invention was designed to complement the inconvenience of existing DETECTRs that use two different temperatures and require complexes to be transferred, by designing an OP-DETECTR that operates at one temperature and in one tube. Unlike existing DETECTRs, it operates in real time (RT) to confirm the time period when a clear change is shown, and the sensitivity was measured in the same way as in Experimental Example 2.

5-2. Results

As a result of the experiment, the sensitivity was the same for both fluorescence measurement and LFA for GⅠ and GⅢ up to 1 × 10 RNA copies and GⅤ up to 1 × 10⁴ RNA copies by genotype (Fig. 8). Therefore, we concluded that the detection method of the present invention can detect up to 1 × 10 RNA copies for GⅠ and GⅢ and up to 1 × 10⁴ RNA copies for GⅤ.

Additionally, the moment of difference in reactivity was 5 minutes after the reaction (after spin down), and the total time required including the incubation process at 42 ℃ was 21 minutes (Fig. 8).

Attach electronic file of sequence list

Claims (34)

A method for detecting Japanese encephalitis virus using genetic scissors,
(a) a step of providing a sample;
(b) a step of isothermally amplifying nucleic acids from the provided sample using a primer set specific for Japanese encephalitis virus, and
(c) A method for detecting Japanese encephalitis virus, comprising the step of adding a guide RNA, Cas (CRISPR-associated protein) 12a, and a single-stranded DNA probe specific for Japanese encephalitis virus to the product obtained by the above isothermal amplification, and performing molecular detection using gene scissors. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the step of providing the sample further includes a step of heating the sample to remove nuclease activity in the sample and decompose virus particles. In the first paragraph,
The above specific primer set is characterized by consisting of sequence number 1 and sequence number 2,
A method for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is of Japanese encephalitis virus genotype GⅠ. In the first paragraph,
The above specific primer set is characterized by consisting of sequence number 3 and sequence number 4,
A method for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GⅢ. In the first paragraph,
The above specific primer set is characterized by consisting of sequence number 5 and sequence number 6,
A method for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GV. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the above isothermal amplification step is performed by RT-RPA (Reverse Transcription-Recombinase Polymerase Amplification). In the first paragraph,
The above specific guide RNA is characterized by having sequence number 7,
A method for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is of Japanese encephalitis virus genotype GⅠ. In the first paragraph,
The above specific guide RNA is characterized by consisting of sequence number 8,
A method for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GⅢ. In the first paragraph,
The above specific guide RNA is characterized by having sequence number 9,
A method for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GV. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the specific guide RNA targets the glycoprotein gene of Japanese encephalitis virus GI having a PAM sequence rich in T-nucleotides. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the specific guide RNA targets the NS4B gene of Japanese encephalitis virus GⅢ having a PAM sequence rich in T-nucleotides. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the specific guide RNA targets the NS1 gene of Japanese encephalitis virus GV having a PAM sequence rich in T-nucleotides. In the first paragraph,
A method for detecting Japanese encephalitis virus, wherein the step of performing molecular diagnosis using the above-mentioned gene scissors includes detection of a target region among isothermal amplification products using the above-mentioned guide RNA, activation of CRISPR-Cas12a, and cleavage of a single-stranded DNA probe by the activated CRISPR-Cas12a, wherein the single-stranded DNA probe does not hybridize with the above-mentioned guide RNA and is labeled with a labeling substance. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the step of performing molecular detection using the above genetic scissors detects whether a single-stranded DNA probe is cut by fluorescence analysis or LFA. In Article 14,
A method for detecting Japanese encephalitis virus, characterized in that the above LFA is performed on a strip. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the above isothermal amplification step and fluorescence analysis or LFA are performed at the same temperature. In Article 16,
A method for detecting Japanese encephalitis virus, characterized in that the temperature is 37 ℃ to 42 ℃. In the first paragraph,
A method for detecting Japanese encephalitis virus, characterized in that the above Cas12a is LbCas12a derived from Lachnospiraceae bacteria. A kit for detecting Japanese encephalitis virus, comprising a primer set specific for Japanese encephalitis virus, a specific guide RNA, Cas12a, and a single-stranded DNA probe. In Article 19,
The above specific primer set is characterized by consisting of sequence number 1 and sequence number 2,
A kit for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is of the Japanese encephalitis virus genotype GⅠ. In Article 19,
The above specific primer set is characterized by consisting of sequence number 3 and sequence number 4,
A kit for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GⅢ. In Article 19,
The above specific primer set is characterized by consisting of sequence number 5 and sequence number 6,
A kit for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GV. In Article 19,
The above specific guide RNA is characterized by having sequence number 7,
A kit for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is of the Japanese encephalitis virus genotype GⅠ. In Article 19,
The above specific guide RNA is characterized by consisting of sequence number 8,
A kit for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GⅢ. In Article 19,
The above specific guide RNA is characterized by having sequence number 9,
A kit for detecting Japanese encephalitis virus, characterized in that the above Japanese encephalitis virus is Japanese encephalitis virus genotype GV. In Article 19,
A kit for detecting Japanese encephalitis virus, wherein the kit further comprises a reagent for detection by fluorescence or LFA. In Article 19,
A kit for detecting Japanese encephalitis virus, characterized in that the above Cas12a is LbCas12a derived from Lachnospiraceae bacteria. In Article 19,
The above kit is a kit for detecting Japanese encephalitis virus, characterized in that it detects Japanese encephalitis virus by RT-RPA and CRISPR-Cas12a-based detection. A primer set for detecting Japanese encephalitis virus, comprising a forward primer of sequence number 1 and a reverse primer of sequence number 2. A primer set for detecting Japanese encephalitis virus, comprising a forward primer of sequence number 3 and a reverse primer of sequence number 4. A primer set for detecting Japanese encephalitis virus, comprising a forward primer of sequence number 5 and a reverse primer of sequence number 6. Guide RNA for detection of Japanese encephalitis virus, consisting of sequence number 7. Guide RNA for detection of Japanese encephalitis virus, consisting of sequence number 8. Guide RNA for detection of Japanese encephalitis virus, consisting of sequence number 9.