TARGET RNA DETECTION METHOD BASED ON DCAS9/GRNA COMPLEX

Abstract

The present invention provides a target RNA detection method based on a dCas9/gRNA complex. A target RNA detection method according to the present invention can detect target RNA with the naked eye and without separate gene isolation and amplification steps, and, in particular, can rapidly and accurately detect target RNA through excellent target specificity and rapidity, and thus can exhibit excellent effects on the detection of various pathogens and/or viruses.

Claims

1. A target RNA detection method comprising: (a) reacting a dCas9/gRNA complex with a PAMmer and a biological sample isolated from the subject, wherein the dCas9/gRNA complex includes inactivated Cas9 (dCas9) and a gRNA (guide RNA) complementary to a target RNA; and wherein the PAMmer is an oligonucleotide in which a labeled ligand indirectly generating a detectable signal is bound to 3′-end, including a 3′-first hybridization region having a hybridization nucleotide sequence complementary to the target RNA, a protospacer-adjacent motif (PAM) sequence, and a 5′-second hybridization region having a hybridization nucleotide sequence complementary to the target RNA, (b) treating a reaction product of step (a) with an anti-ligand that recognizes the detectable signal.

2. The target RNA detection method of claim 1, wherein the labeled ligand capable of indirectly generating a detectable signal at the 3′-end in step (a) is at least one selected from the group consisting of biotin, digoxigenin, aptamers, peptides, fluorescent compounds, oligonucleotides, and polysaccharides.

3. The target RNA detection method of claim 1, wherein the anti-ligand that recognizes a detectable signal in step (b) is at least one selected from the group consisting of avidin or avidin analogs, antibodies, receptors, and lectins.

4. The target RNA detection method of claim 1, wherein the labeled ligand indirectly generating a detectable signal at the 3′-end in step (a) is biotin; and the anti-ligand that recognizes a detectable signal in step (b) is avidin or an avidin analog.

5. The target RNA detection method of claim 1, wherein the gRNA in step (a) is a single chain guide RNA.

6. The target RNA detection method of claim 1, wherein the gRNA in step (a) contains the same sequence as the 5′-second hybridization region of the PAMmer, and the sequence is 5 to 20 nucleotides in length.

7. The target RNA detection method of claim 1, wherein the 5′-second hybridization region of the PAMmer in step (a) is 5 to 20 nucleotides in length in a 3′ to 5′ direction based on the PAM sequence.

8. The target RNA detection method of claim 1, wherein the PAM sequence in step (a) is 5′-NGG or NGGNG, where N is any nucleotide.

9. The target RNA detection method of claim 1, wherein the dCas9/gRNA complex in step (a) is immobilized.

10. The target RNA detection method of claim 4, wherein the avidin analog in step (b) is streptavidin, neutravidin, or captavidin.

11. The target RNA detection method of claim 4, wherein the avidin or avidin analog in step (b) is a horseradish hydrogen peroxide conjugate of avidin or the avidin analog.

12. The target RNA detection method of claim 11, wherein the horseradish hydrogen peroxide substrate in step (b) is any one selected from the group consisting of 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine dihydrochloride (OPD), 3,3′-diaminobenzidine (DAB), and luminol.

13. The target RNA detection method of claim 4, further comprising: (c) confirming a color change of a reaction product obtained in step (b) with the naked eye.

14. The target RNA detection method of claim 1, wherein the target RNA is virus-derived RNA.

15. A target RNA detection kit comprising: (a) a dCas9/gRNA complex immobilized on a substrate surface, wherein the dCas9/gRNA complex includes dCas9 and a guide RNA (gRNA) complementary to a target RNA; (b) PAMmer in which a labeled ligand indirectly generating a detectable signal is bound to 3′-end, including a 3′-first hybridization region having a hybridization nucleotide sequence complementary to the target RNA, a protospacer-adjacent motif (PAM) sequence, and a 5′-second hybridization region having a hybridization nucleotide sequence complementary to the target RNA; and (c) an anti-ligand that recognizes the detectable signal.

16. The target RNA detection kit of claim 15, wherein the labeled ligand indirectly generating a detectable signal at the 3′-end is any one selected from the group consisting of biotin, digoxigenin, aptamers, peptides, fluorescent compounds, oligonucleotides, and polysaccharides.

17. The target RNA detection kit of claim 15, wherein the anti-ligand that recognizes a detectable signal is any one selected from the group consisting of avidin or avidin analogs, antibodies, receptors, and lectins.

18. A target RNA detection kit comprising: (a) a dCas9/gRNA complex immobilized on a substrate surface, wherein the dCas9/gRNA complex includes dCas9 and a guide RNA (gRNA) complementary to a target RNA; (b) PAMmer in which biotin is bound to 3′-end, including a 3′-first hybridization region having a hybridization nucleotide sequence complementary to the target RNA, a protospacer-adjacent motif (PAM) sequence, and a 5′-second hybridization region having a hybridization nucleotide sequence complementary to the target RNA; (c) a horseradish hydrogen peroxide conjugate of avidin or an avidin analog; and (d) a horseradish hydrogen peroxide substrate.

Description

DESCRIPTION OF DRAWINGS

[0099] FIG. 1a shows a nucleotide sequence structure of target RNA, biotin-PAMmer, and gRNA, and FIG. 1b shows electrophoresis results confirming a change in a mobile phase of the target RNA/biotin-PAMmer when the target RNA and biotin-PAMmer react with a dCas9/gRNA complex.

[0100] FIG. 2 shows a result of confirming the surface immobilization of the dCas9/gRNA complex.

[0101] (a) of FIG. 3 shows a nucleotide sequence structure of SARS-CoV-2 N1 (target RNA), biotin-PAMmer, and gRNA, (b) of FIG. 3 shows the results of quantitative detection of the SARS-CoV-2 N1 gene with the naked eye using the dCas9/gRNA complex and biotin-PAMmer, (c) of FIG. 3 shows a nucleotide sequence structure of pH1N1 H1 (target RNA), biotin-PAMmer, and gRNA, and (d) of FIG. 3 shows the result of quantitative detection of the pH1N1 H1 gene with the naked eye using the dCas9/gRNA complex and biotin-PAMmer.

[0102] (a) of FIG. 4 shows the results of selective detection of SARS-CoV-2 and pH1N1 H1 genes using dCas9/gRNA complex and biotin-PAMmer, and (b) of FIG. 4 shows the results of selective detection of influenza virus subtype (H1, H3, and H5) genes using the dCas9/gRNA complex and biotin-PAMmer.

[0103] FIG. 5 shows detection results of drug-resistant influenza virus genes based on the dCas9/gRNA complex, wherein (a) and (b) of FIG. 5 show sequence information, and (c) and (d) of FIG. 5 show detection results of drug-resistant influenza virus.

[0104] FIG. 6 shows results of selective detection of SARS-CoV-2 and pH1N1 genes without separate gene isolation and amplification steps from the virus culture medium, wherein (a) of FIG. 6 shows a related schematic diagram, and (b) of FIG. 6 shows experimental results.

[0105] FIG. 7 shows results obtained by treating SARS-CoV-2, pH1N, and drug-resistant pH1N1 in negative nasopharyngeal aspirate and sputum samples, respectively, and then detecting the treated virus without separate gene isolation and amplification, wherein (a) of FIG. 7 shows a schematic diagram and (b) to (d) of FIG. 7 show detection results.

[0106] FIG. 8 shows results of detecting COVID-19 in the nasopharyngeal aspirate and sputum samples of COVID-19 positive patients without separate gene isolation and amplification steps. (samples of 3 negative patients and 5 positive patients were used.)

[0107] FIG. 9 shows results of detecting COVID-19 in the nasopharyngeal aspirate and sputum samples of COVID-19 positive patients without separate gene isolation and amplification steps. (samples of 10 negative patients and 21 positive patients were used.)

BEST MODE

[0108] Hereinafter, the present disclosure will be described in more detail through Examples. However, these Examples are provided to illustrate the present disclosure by way of example, and the scope of the present disclosure is not limited to these Examples.

Example 1: Target Specificity of dCas9/gRNA Through Introduction of Biotin-PAMmer

[0109] In order to demonstrate the target-specific detection effect by the dCas9/gRNA system through introduction of the biotin-PAMmer of the present disclosure, the present inventors confirmed the target specificity of the dCas9/gRNA complex in the presence of target RNA and biotin-PAMmer.

[0110] A brief description is as follows: First, a dCas9/gRNA complex was formed by reacting 100 nM of gRNA and 1 μM of dCas9 protein at room temperature for 10 minutes.

[0111] In addition, the PAMmer is a short oligonucleotide designed to contain a PAM sequence while simultaneously containing a labeled ligand to generate a detectable signal so that single-stranded target RNA that does not contain a PAM sequence is able to be recognized by the dCas9/gRNA complex.

[0112] The PAMmer of the present disclosure is an oligonucleotide including a PAM sequence capable of interacting with a guide nucleotide sequence-programmable RNA binding protein,

[0113] which includes nucleotide sequence regions (3′-first hybridization region and 5′-second hybridization region) complementary to the target gene (RNA) and the PAM sequence; and

[0114] includes a labeled ligand indirectly generating a detectable signal at the 3′-end (3′-first hybridization region) of the oligonucleotide;

[0115] wherein the 5′-second hybridization region is a region extended by 8 base pairs in the 5′-end direction from the PAM sequence, and the extension site is designed to match (sequence is the same) with the target gene binding (hybridization) region of the gRNA.

[0116] In the present Example, biotin-PAMmer in a biotin-bound form was used.

[0117] The dCas9/gRNA complex was diluted by concentration (10, 50, 100, and 250 nM), and 1 μM of the target RNA gene, 1 μM of biotin-PAMmer, and 1× reaction buffer were mixed therewith, followed by reaction at 37° C. for 1 hour. The reaction product was subjected to electrophoresis using 8% native PAGE gel, and then the mobility shift of biotin-PAMmer and target RNA was confirmed. The nucleotide sequence structure of gRNA, biotin-PAMmer, and target RNA used in the reaction is shown in FIG. 1a.

[0118] As a result, as shown in FIG. 1b, it was confirmed that in the presence of target RNA and biotin-PAMmer, when the dCas9/gRNA complex was not treated, there was no change in the mobile phase of biotin-PAMmer and target RNA, whereas when the dCas9/gRNA complex was treated, as the concentration of the complex increased, the mobile phase changed, specifically, amounts of biotin-PAMmer and target RNA increased.

[0119] It was confirmed from the above experiment that the dCas9/gRNA complex specifically bound to the biotin-PAMmer and the target RNA.

Example 2: Immobilization of dCas9/gRNA Complex on Solid Phase

[0120] In order to confirm that the biotin-PAMmer-introduced dCas9/gRNA system according to the present disclosure could operate when the dCas9/gRNA complex was immobilized on a solid phase, the present inventors immobilized the dCas9/gRNA complex on the surface of a solid substrate.

[0121] A brief description is as follows: A dCas9/gRNA complex was formed by reacting 600 nM of gRNA and 1 μM of dCas9 at room temperature for 10 minutes and then the dCas9/gRNA complex diluted 10 times with 1× PBS solution was treated in a 96-well plate and reacted at room temperature for 2 hours.

[0122] Then, a surface was washed using a washing buffer containing 1× PBS and 0.05% tween 20. Next, the surface was treated with 0.1 mg/mL of bovine serum albumin (BSA) and reacted at room temperature for 40 minutes, and the surface was washed with a washing buffer. Then, the surface was treated with Cas9 monoclonal antibody diluted in 5% skim milk powder and reacted for 1 hour. After washing the surface using washing buffer, the surface was treated with HRP-conjugated anti-mouse IgG secondary antibody diluted in 5% skim milk and reacted for 1 hour. The surface was washed and sequentially treated with a TMB solution and a 2.5 M sulfuric acid solution to confirm a color change.

[0123] As a result, as shown in FIG. 2, it was confirmed that the color change occurred only when the dCas9/gRNA complex was treated compared to the surface where the dCas9/gRNA complex was not treated. From this confirmation, it could be appreciated that the dCas9/gRNA complex was successfully coated on the surface.

[0124] In other words, it is demonstrated that even if it is not performed under specific immobilization conditions and/or using a dedicated buffer for solid surface immobilization commonly known in the art, it is possible to perform the detection by application on the solid support only including treatment of the diluted dCas9/gRNA complex on the solid support such as the 96-well plate, followed by incubation at room temperature.

Example 3: Detection of Target RNA with Naked Eye

[0125] The present inventors confirmed whether the target RNA could be detected with the naked eye by reacting the target RNA and biotin-PAMmer to the solid surface-immobilized dCas9/gRNA complex of Example 2, Followed by Treatment With Streptavidin-HRP and TMB.

[0126] Specifically, a dCas9/gRNA complex was formed by reacting 600 nM of gRNA and 1 μM of dCas9 at room temperature for 10 minutes and then the dCas9/gRNA complex diluted 10 times with 1× PBS solution was treated in a 96-well plate and reacted at room temperature for 2 hours. Then, a surface was washed using a washing buffer containing 1× PBS and 0.05% tween 20. Next, the surface was treated with 0.1 mg/mL of bovine serum albumin (BSA) and reacted at room temperature for 40 minutes, and the surface was washed with a washing buffer. Then, target RNA (0 to 100 nM) prepared for each concentration was mixed with 1 μM of biotin-PAMmer and 1× reaction buffer, and reacted on the surface at 37° C. for 1 hour. The surface was washed and reacted with 20 μg/mL of streptavidin-HRP for 30 minutes at room temperature. The surface was washed and sequentially treated with a TMB solution and a 2.5 M sulfuric acid solution to confirm a color change, and the absorbance was measured with a microplate machine. The absorbance was observed at 450 nm.

[0127] Here, the nucleotide sequence structures of gRNA, biotin-PAMmer, and target RNA used in the reaction are shown in FIGS. 3a and 3c.

[0128] As a result, as shown in (b) and (d) of FIG. 3, it was confirmed that the measured absorbance increased as the concentration of the target RNA increased (0 to 100 nM).

[0129] It is demonstrated that when the surface-immobilized dCas/gRNA complex, biotin-PAMmer, streptavidin-HRP, and TMB are reacted according to the present disclosure, it is possible to detect target RNA with the naked eye.

Example 4: Specific Detection of Target RNA

[0130] The present inventors confirmed whether multiple genes could be simultaneously detected using dCas9/gRNA-based target RNA detection technology with the naked eye.

[0131] Specifically, as described in Example 3, dCas9/gRNA complexes targeting different genes were formed, immobilized on different surfaces of a 96 well plate, and treated with BSA. Then, samples in which several types of genes were mixed were simultaneously treated on the surfaces on which dCas9/gRNA complexes targeting different genes were immobilized. Then, as in Example 3, the detection reaction with the naked eye was performed through biotin-PAMmer and streptavidin-HRP treatment steps. Here, the biotin-PAMmer was designed to have a different nucleotide sequence for each target RNA, and each targeting gene was treated on the surface on which the corresponding dCas9/gRNA complex was immobilized.

[0132] Hereinafter, the sequence information used in the present Examples is shown in Table 1 below.

TABLE-US-00001 TABLE 1 gRNA Sequence (5′ to 3′) SARS-Cov-2 N1 mA*mA*mA* CGU AAU GCG GGG UGC AUG UUU UAG AGC UAG AAA (SEQ ID NO: 1) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U SARS-Cov-2 N2 mU*mG*mG* GGG CAA AUU GUG CAA UUG UUU UAG AGC UAG AAA (SEQ ID NO: 2) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U SARS-Cov-2 N3 mG*mG*mG* UGC CAA UGU GAU CUU UUG UAG AAA UAG CAA GUU (SEQ ID NO: 3) AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U pH1N1 H1 mC*mC*mA* GCA UUU CUU UCC AUU GCG UUU UAG AGC UAG AAA (SEQ ID NO: 4) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U pH1N1 WT N1 mC*mC*mU* CUU AGU GAU AAU UAG GGG UUU UAG AGC UAG AAA (SEQ ID NO: 5) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U pH1N1/H275Y N1 mC*mC*mU* CUU AGU AAU AAU UAG GGG UUU UAG AGC UAG AAA (SEQ ID NO: 6) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U IFV H3 mC*mU*mU* CCA UUU GGA GUG AUG CAG UUU UAG AGC UAG AAA (SEQ ID NO: 7) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U IFV H5 mC*mA*mA* CCA UCU ACC AUU CCC UGG UUU UAG AGC UAG AAA (SEQ ID NO: 8) UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC mU*mU*mU* U Target Sequence (5′ to 3′) SARS-Cov-2 N1 GAC CCC AAA AUG AGC GAA AUG CAC CCC GCA UUA CGU UUG (SEQ ID NO: 9) G SARS-Cov-2 N2 UUA CAA ACA UUG GCC GCA AAU UGC ACA AUU UGC CCC CA (SEQ ID NO: 10) SARS-Cov-2 N3 GGG AGC CUU GAA UAC ACC AAA AGA UCA CAU UGG CAC CC (SEQ ID NO: 11) pH1N1 H1 GGU ACC GAG AUA UGC AUU CGC AAU GGA AAG AAA UGC UGG (SEQ ID NO: 12) AUG UG pH1N1 WT N1 AUG AGU CGA AAU GAA UGC CCC UAA UUA UCA CUA UGA GGA (SEQ ID NO: 13) AUG CUC CUG pH1N1/H275Y N1 AUG AGU CGA AAU GAA UGC CCC UAA UUA UUA CUA UGA GGA (SEQ ID NO: 14) AUG CUC CUG IFV H3 UUG GCA AGU GCA AGU CUG AAU GCA UCA CUC CAA AUG GAA (SEQ ID NO: 15) GCA UU IFV H5 GGU UUU AUA GAG GGA GGA UGG CAG GGA AUG GUA GAU GGU (SEQ ID NO: 16) UGG UAU G SARS AAC AUG CUU AGG AUA AUG GCC UCU CUU GUU CUU GCU CGC (SEQ ID NO: 17) A Biotin-PAMmer Sequence (5′ to 3′) SARS-Cov-2 N1 GGG TGC ATC GGG CTG ATT TTG GGG TC-Biotin (SEQ ID NO: 18) SARS-Cov-2 N2 GTG CAA TTC GGG GCC AAT GTT TGT AA-Biotin (SEQ ID NO: 19) SARS-CoV-2 N3 GAT CTT TTC GGG TAT TCA AGG CTC CC-Biotin (SEQ ID NO: 20) pH1N1 H1 rUrCrC rATrU GrCC rGGrU GrCA rUArU CrUC rGGrU ArCC (SEQ ID NO: 21) rArAC rUT-Biotin pH1N1 WT N1 and rAArU rUArG GrGC GrGrU rUCA rUrUT CGA CrUG AT-Biotin PH1N1/H275Y N1 (SEQ ID NO: 22) IFV H3 rGrUG rATrG CrArC GrGrA GrAC TrUrG rCAC rUTG rCrCA- (SEQ ID NO: 23) Biotin IFV H5 rArUT rCCrC TrGrC GrGrU CrCT CrCrC rUCT rATA rArAA- (SEQ ID NO: 24) Biotin m_*: 2′-O-methyl/phosphorothioate modification, Seq No. 21~24: Chimeric (r_: chimeric)

[0133] As a result, as shown in FIG. 4, it could be confirmed that the target RNA could be detected very selectively by each dCas9/gRNA complex, and it was shown that highly specific detection with the naked eye could be performed depending on the nucleotide sequence of the target RNA.

[0134] Specifically, (a) of FIG. 4 shows the results for SARS-CoV-2 N1 (CoV-2 N1), SARS-CoV-2 N2 (CoV-2 N2), SARS-CoV-2 N3 (CoV-2 N3), and pH1N1 H1 (H1). As can be appreciated from the results, the target RNA could be clearly detected with the naked eye, confirming that the detection of the coronavirus could be achieved.

[0135] Further, (b) of FIG. 4 shows the results for pH1N1 H1 (H1), IFV H3 (H3), and IFV H5 (H5). As can be appreciated from the results, the target RNA could be clearly detected with the naked eye, confirming that the detection of the influenza virus could be achieved.

[0136] In other words, it was demonstrated that simultaneous detection of multiple target RNAs could be achieved through the dCas9/gRNA-based target RNA detection technology with the naked eye of the present disclosure.

Example 5: Detection of Gene Having Difference in Single Nucleotide Sequence

[0137] Among drug-resistant viruses that are resistant to Oseltamivir, a treatment for swine flu (H1N1 influenza) virus, the H275Y N1 mutant type is known to show a difference in single nucleotide sequence as compared to the drug-susceptible wild type. For proper treatment, it is required to perform a rapid diagnosis of drug-resistant virus infection.

[0138] Therefore, in order to confirm whether the dCas9/gRNA-based target RNA detection technology with the naked eye of the present disclosure is able to distinguish a gene having a difference in single nucleotide sequence, the present inventors conducted experiments on the mutant pH1N1/H275Y N1 and the wild type pH1N1 WT N1.

[0139] Specifically, as shown in (a) and (b) of FIG. 5, a gRNA was constructed so that a region having a difference in nucleotide sequence (for example, a single nucleotide mutation) compared to the wild type virus on pH1N1/H275Y RNA, which is the target RNA, was selected as a gRNA binding site to which the gRNA binds, and one nucleotide sequence mismatch is present at a position spaced by 5 base pairs (bp) in the 5′-end direction from the position of the different nucleotide sequence on the gRNA.

[0140] Then, as described in Example 3, the detection with the naked eye was performed by forming dCas9/gRNA complex and immobilizing it on the solid phase surface, followed by treatment with biotin-PAMmer, streptavidin-HRP, and TMB.

[0141] As a result, as shown in (c) and (d) of FIG. 5, color change was observed when the gene of the H275Y N1 mutant type was treated on the surface immobilized with dCas9/gRNA composed of gRNA complementary to the H275Y mutant gene of influenza virus.

[0142] On the other hand, no color change was observed when the wild-type gene having a difference in single nucleotide sequence from the target RNA was treated.

[0143] This suggests that the dCas9/gRNA-based target RNA detection technology with the naked eye of the present disclosure is able to distinguish the gene having a difference in single nucleotide sequence.

Example 6: Virus Detection in Virus Culture Medium

[0144] The present inventors attempted to selectively detect target viral RNA by using the dCas9/gRNA-based target RNA detection technology with the naked eye of the present disclosure, without gene extraction and amplification steps through a separate kit, in culture media in which SARS-CoV-2 and novel influenza virus were cultured ((a) of FIG. 6).

[0145] In more detail, SARS-CoV-2 at a concentration of 10.sup.3 PFU/mL, swine flu virus (pH1N1) at a concentration of 10.sup.4 PFU/mL, and a mixture of the two viruses (SARS-CoV-2 and swine flu) were prepared, and then the respective samples were treated with a TCEP/EDTA (final concentration 100 mM/1 mM) solution. Then, the reactants were sequentially heat-treated at 50° C. for 5 minutes and at 64° C. for 5 minutes and used as samples. Next, as described in Example 3 above, the detection with the naked eye was performed by immobilizing the dCas9/gRNA complexes targeting genes of SARS-CoV-2 and H1N1 influenza virus on the solid surface, followed by treatment with biotin-PAMmer, streptavidin-HRP, and TMB.

[0146] As a result, as shown in (b) of FIG. 6, no color change was observed in the condition where the virus was not treated, but when the SARS-CoV-2 virus solution was treated alone, color change was observed only on the surfaces immobilized with the dCas9/gRNA complexes complementary to the SARS-CoV-2 genes (CoV-2 N1, N2, and N3).

[0147] In addition, when the H1N1 influenza virus solution was treated alone, color change was observed only on the surface on which the dCas9/gRNA complex complementary to the swine flu gene (H1) was immobilized.

[0148] Further, it could be confirmed that in the condition of mixing the two viruses, color changes were observed on all surfaces on which the dCas9/gRNA complex complementary to SARS-CoV-2 and H1 was immobilized.

[0149] Through this observation, it could be confirmed that the virus gene in the virus culture medium was capable of being detected very selectively through the dCas9/gRNA-based target RNA detection technology with the naked eye and without separate gene isolation and amplification steps.

Example 7: Confirmation of Target RNA Detection in Nasopharyngeal Aspirate and Sputum

[0150] Viruses that cause respiratory diseases, such as SARS-CoV-2 and H1N1 influenza virus, are generally extracted from nasopharyngeal aspirate or sputum by a viral RNA isolation kit and detected by RT-PCR.

[0151] Accordingly, the present inventors attempted to detect viral RNA from nasopharyngeal aspirates or sputum by using the dCas9/gRNA-based target RNA detection technology with the naked eye of the present disclosure and without the separate gene extraction step through the kit ((a) of FIG. 7).

[0152] Specifically, SARS-CoV-2 (10.sup.3 PFU/mL), swine flu (10.sup.4 PFU/mL), and H275Y drug-resistant swine flu (10.sup.4 PFU/mL) viruses were treated in nasopharyngeal aspirate or sputum. Then, the virus-treated nasopharyngeal aspirate and sputum were treated with a TCEP/EDTA (final concentration: 100 mM/1 mM) solution, sequentially heat-treated at 50° C. for 5 minutes and at 64° C. for 5 minutes, and then used as samples. As described above in Example 3, the detection with the naked eye was performed by immobilizing the dCas9/gRNA complex targeting genes of SARS-CoV-2, swine flu virus (pH1N1) and H275Y drug-resistant swine flu (pH1N1/H275Y) on the surface, followed by treatment with biotin-PAMmer, streptavidin-HRP, and TMB.

[0153] As a result, as shown in (b) to (c) of FIG. 7, no color change was observed in the negative nasopharyngeal aspirate and sputum sample conditions that were not treated with the virus, but it could be confirmed that the positive samples treated with the virus showed selective color change on the surface where each complementary dCas9/gRNA complex was immobilized.

[0154] Through this confirmation, it could be appreciated that target RNA detection could be performed through the dCas9/gRNA-based target RNA detection technology with the naked eye and without separate gene isolation and amplification steps from nasopharyngeal aspirate and sputum samples.

[0155] Specifically, it was confirmed in (b) and (c) of FIG. 7 that it was possible to confirm the presence or absence of SARS-Cov-2, and in (d) of FIG. 7 that it was also possible to identify not only the influenza virus but also variants having single mutations.

Example 8: Confirmation of Detection of Target RNA in Nasopharyngeal Aspirate and Sputum of COVID-19 Positive Patients

[0156] In order to prove that COVID-19 was actually detectable in clinical practice by using the dCas9/gRNA-based target RNA detection technology with the naked eye and without the separate gene extraction step through a kit according to the related art, the present inventors confirmed the target RNA detection from the patient's nasopharyngeal aspirate and sputum.

[0157] Briefly, nasopharyngeal aspirate and sputum from COVID-19 positive and negative patients were treated with TCEP/EDTA (final concentration 100 mM/1 mM) solution, respectively, sequentially heat-treated at 50° C. for 5 minutes and at 64° C. for 5 minutes, and then used as samples. As described in Example 3 above, the detection with the naked eye was performed by immobilizing the dCas9/gRNA complex targeting a gene of SARS-CoV-2 on the surface, followed by treatment with biotin-PAMmer, streptavidin-HRP, and TMB.

[0158] As a result, as shown in FIG. 8, no color change was observed in the nasopharyngeal aspirate and sputum sample conditions of the negative patient, but the samples of the positive patient showed selective color change on the surface where each complementary dCas9/gRNA complex was immobilized.

[0159] The above experiment was conducted with 3 negative patients and 5 positive patients.

[0160] Accordingly, an additional experiment was conducted on more samples using the same experimental method. Specifically, samples of 21 positive patients and 10 negative patients were used in the additional experiment.

[0161] The result was shown in FIG. 9.

[0162] As shown in FIG. 9, no color change was observed in the samples of 10 negative patients, while a color change was observed in the samples of 21 positive patients, so that excellent efficacy of the detection method was confirmed.

[0163] Through this confirmation, it could be appreciated that COVID-19 infection could be diagnosed through the dCas9/gRNA-based target RNA detection technology with the naked eye and without separate gene isolation and amplification steps from nasopharyngeal aspirate and sputum samples.

[0164] From the above results, it was confirmed that the target RNA detection method according to the present disclosure could detect target RNA with the naked eye and without separate gene isolation and amplification steps, and in particular, could quickly and accurately detect the target RNA with excellent target specificity and rapidity. Therefore, it was demonstrated that the target RNA detection method could exhibit excellent effects in detecting various pathogens and/or viruses, in particular, highly prevalent viruses.

[0165] From the above description, those skilled in the art to which the present disclosure pertains will understand that the present disclosure may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. As the scope of the present disclosure, it should be construed that all changes or modifications derived from the meaning and scope of the claims to be described below and equivalents thereof rather than the above detailed description are included in the scope of the present disclosure.