Human cytomegalovirus diagnosis method based on recombinase polymerase amplification and crispr-cas12a

Abstract

The present invention relates to a human cytomegalovirus (HCMV) diagnosis method based on recombinase polymerase amplification and CRISPR-Cas12a. It has been identified in the present invention that the HCMV diagnosis method using a CRISPR-Cas12a system and a recombinase polymerase amplification primer, which specifically binds to the genome sequence of the essential envelope glycoprotein H of human cytomegalovirus (HCMV), and can accurately detect only HCMV from among similar viruses. In addition, it has been identified that the present invention can diagnose HCMV infection in an actual clinical sample, and has excellent sensitivity and specificity even in comparison to a conventional molecular biological method, and thus can be used for rapid and accurate point-of-care testing of HCMV.

Description

Recombinant enzyme polymerase amplification method and CRISPR-CAS12A-based human cytomegalovirus diagnostic method

[0001] The present invention relates to a recombinant enzyme polymerase amplification method and a CRISPR-Cas12a-based human cytomegalovirus diagnosis method.

[0002] Human cytomegalovirus (HCMV), also known as human herpesvirus 5, is a virus belonging to the family Herpesviridae and subfamily Betaherpesvirinae. HCMV induces enlargement of infected cells (cytomegaly) and produces characteristic inclusion bodies, hence its name. HCMV infection is widespread worldwide, and most initial infections remain asymptomatic during a lifelong incubation period. However, if the virus becomes active in an immunocompromised patient, it can cause a variety of diseases. HCMV disease can present as a viral syndrome or as a tissue-invasive disease, and HCMV infection in an immunocompromised host can be life-threatening, with the severity of the disease varying depending on the host's immune system.

[0003] HCMV infection is diagnosed by collecting samples from the patient's throat, urine, blood, or other body tissues or fluids. Current diagnostic methods include virus culture, antibody testing, antigenemia, and quantitative polymerase chain reaction (qPCR). The most accurate method for diagnosing HCMV is to isolate and culture the virus from an infected patient's body fluids or tissues. However, due to technical difficulties in virus isolation, a positive diagnosis can occur within one or two days, and a negative diagnosis can take three weeks or more. Although HCMV infection can occur in other immunocompromised states, its prevalence is not high, so it often takes a long time from the clinical suspicion stage to the diagnosis after testing.

[0004] HCMV antibody-based diagnosis detects the presence or absence of IgM and IgG antibodies that increase after infection to determine HCMV infection. It can distinguish primary infection from reactivation, but it takes time for detectable antibodies to be produced after infection, so the diagnosis time is long. Antigenemia test is a diagnostic method that detects the pp65 antigen, a substrate of HCMV, using a monoclonal antibody. It is the most widely used method for diagnosing HCMV. However, it has several limitations, such as manual testing, the need to process within 6 to 8 hours of blood collection, and the requirement of highly specialized skills for interpretation of the diagnostic results.

[0005] qPCR technology, capable of high sensitivity and accuracy, is being used to diagnose various diseases, including HCMV. While PCR's exponential DNA amplification allows for earlier virus detection compared to other diagnostic methods, its technical complexity and expensive equipment requirements limit its use to laboratory-scale studies.

[0006] Meanwhile, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a precise DNA editing technology. One of the CRISPR proteins, Cas12a (Cpf-1), is a gene editing technology that utilizes the property of cleaving both ends of DNA when binding to the target DNA, while randomly degrading the surrounding single DNA strands. It is an innovative technology with enormous potential across biotechnology, medicine, and agriculture. SHERLOCK (Specific High-Sensitivity Enzyme Reporter UnLOCK) developed by Kellner et al. is known as a representative CRISPR protein-based diagnostic method, and the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) technology developed by Chen et al. is also attracting attention in the field of diagnostic medicine. These CRISPR-based diagnostic technologies can utilize the specific gene editing properties of CRISPR to select and detect specific genetic sequences or small pieces of genetic information, and the incidental activities of the CRISPR system that unintentionally affect adjacent nucleic acids can be detected simply and accurately by methods such as fluorescence or lateral flow assays (LFA) that indicate the presence or absence of the target sequence.

[0007] Meanwhile, Recombinase Polymerase Amplification (RPA) is a method that can rapidly amplify specific DNA at an isothermal temperature, and is being applied to detect various pathogens by utilizing the amplification of specific base sequences possessed by specific pathogens.

[0008] CRISPR diagnostics combined with isothermal amplification can detect a variety of pathogens with high sensitivity and specificity, and can be performed at a standard temperature of 37°C, enabling rapid and accurate pathogen diagnosis even under typical temperature conditions.

[0009] An object of the present invention is to provide a primer set for detecting human cytomegalovirus (HCMV), comprising a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; or a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4.

[0010] Another object of the present invention is to provide a composition for detecting human cytomegalovirus (HCMV) comprising the above primer set.

[0011] Another object of the present invention is to provide a kit for detecting human cytomegalovirus (HCMV) comprising the above composition.

[0012] Another object of the present invention is to provide (1) a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; or a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4;

[0013] (2) crRNA comprising the base sequence of SEQ ID NO: 5 or crRNA comprising the base sequence of SEQ ID NO: 6; and

[0014] (3) A kit for detecting human cytomegalovirus (HCMV) comprising a Cas12a protein is provided.

[0015] Another object of the present invention is to provide a step of isolating a biological sample from a patient;

[0016] A step of extracting DNA from the separated biological sample;

[0017] A step of amplifying the extracted DNA using the above kit using recombinase polymerase amplification (RPA);

[0018] A step of cutting the amplified DNA using the CRISPR-Cas12a system using the above kit; and

[0019] A method for detecting human cytomegalovirus (HCMV) is provided, comprising a step of confirming the above-mentioned cleaved DNA product.

[0020] To achieve the above purpose, the present invention provides a primer set for detecting human cytomegalovirus (HCMV), comprising a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; or a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4.

[0021] In addition, the present invention provides a composition for detecting human cytomegalovirus (HCMV) comprising the above primer set.

[0022] In addition, the present invention provides a kit for detecting human cytomegalovirus (HCMV) comprising the above composition.

[0023] In addition, the present invention provides (1) a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; or a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4;

[0024] (2) crRNA comprising the base sequence of SEQ ID NO: 5 or crRNA comprising the base sequence of SEQ ID NO: 6; and

[0025] (3) A kit for detecting human cytomegalovirus (HCMV) comprising a Cas12a protein is provided.

[0026] In addition, the present invention comprises a step of isolating a biological sample from a patient;

[0027] A step of extracting DNA from the separated biological sample;

[0028] A step of amplifying the extracted DNA using the above kit using recombinase polymerase amplification (RPA);

[0029] A step of cutting the amplified DNA using the CRISPR-Cas12a system using the above kit; and

[0030] A method for detecting human cytomegalovirus (HCMV) is provided, comprising a step of identifying the above-mentioned cleaved DNA product.

[0031] The present invention confirms that a method for diagnosing human cytomegalovirus (HCMV) using a recombinant polymerase amplification reaction primer that specifically binds to the genomic sequence of the essential envelope glycoprotein H of HCMV and a CRISPR-Cas12a system can accurately detect only HCMV among similar viruses. In addition, it is possible to diagnose HCMV infection in actual clinical samples, and it is confirmed that the sensitivity and specificity are superior compared to existing molecular biological methods, so it can be used for rapid and accurate on-site diagnosis of HCMV, and thus can be usefully utilized in related industries.

[0032] Figure 1 is a diagram illustrating a diagnostic method of the RPA-CRISPR-based HCMV diagnostic platform of the present invention.

[0033] Figure 2 is a diagram showing the design process and RPA amplification of primer sets 1 and 2 for HCMV-specific RPA of the present invention (A: target region diagram, B: primer and guide RNA design diagram, C: RPA amplification confirmation).

[0034] Figure 3 is a diagram comparing diagnostic efficiency according to primer sets and crRNA in the RPA-CRISPR-based HCMV diagnostic platform of the present invention (A: confirmation of fluorescence expression, B: quantification of fluorescence expression, C: lateral flow analysis results, D: quantification of lateral flow analysis results).

[0035] FIG. 4 is a diagram confirming the HCMV detection sensitivity of the RPA-CRISPR-based HCMV diagnostic platform using primer set 2 and crRNA 2 of the present invention (A: fluorescence expression analysis results, B: fluorescence expression quantification, C: lateral flow analysis results, D: lateral flow analysis results quantification).

[0036] Figure 5 is a diagram showing the HCMV detection specificity of the RPA-CRISPR-based HCMV diagnostic platform using primer set 2 and crRNA 2 of the present invention confirmed by lateral flow analysis (A: lateral flow analysis results, B: quantification of lateral flow analysis results).

[0037] Figure 6 is a diagram confirming the HCMV diagnostic effect in clinical samples using the RPA-CRISPR-based HCMV diagnostic platform of the present invention (A: fluorescence analysis results, B: fluorescence analysis quantification, C: lateral flow analysis results and quantification, D: linear regression analysis of fluorescence expression and flow analysis results).

[0038] Figure 7 is a diagram confirming the correlation between the lateral flow analysis results of the RPA-CRISPR-based HCMV diagnostic platform of the present invention and quantitative PCR using linear regression analysis.

[0039] A: Virus copy number calculated using quantitative PCR

[0040] B: Linear regression analysis of viral copy number calculated using flow analysis results and quantitative PCR

[0041] Figure 8 is a diagram showing the diagnostic effect of HCMV in a clinical sample derived from a newborn using lateral flow analysis of the RPA-CRISPR-based HCMV diagnostic platform of the present invention.

[0042] A: Sample information and qPCR preamplification results

[0043] B: Flow analysis results

[0044] C: Quantification of flow analysis results

[0045] Figure 9 is a diagram showing the HCMV diagnostic effect confirmed in clinical samples of patients suspected of and positive for HCMV-associated glaucoma infection using lateral flow analysis of the RPA-CRISPR-based HCMV diagnostic platform of the present invention.

[0046] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0047] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0048] The present invention provides a primer set for detecting human cytomegalovirus (HCMV), comprising a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; or a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4.

[0049] The term "primer" as used herein refers to a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary nucleic acid template and serves as a starting point for strand copying of the nucleic acid template. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

[0050] When designing the above primers, there are several restrictions, such as the ratio of A, G, C, and T contents of the primers, prevention of primer dimer formation, and prohibition of repeating the same base sequence more than three times. In addition, for the single PCR reaction conditions, conditions such as the amount of template DNA, concentration of primers, concentration of dNTP, concentration of Mg2+, reaction temperature, and reaction time must be appropriate.

[0051] The above primers can incorporate additional features that do not alter their basic properties. That is, the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more nucleotides with homologs, and modification of nucleotides with uncharged linkers such as phosphonates, phosphotriesters, phosphoramidates, or carbamates, or with charged linkers such as phosphorothioates or phosphorodithioates. The nucleic acids can also have one or more additional covalently bonded moieties, such as nucleases, toxins, antibodies, signal peptides, proteins such as poly-L-lysine, intercalators such as acridine or psoralens, chelating agents such as metals, radioactive metals, iron oxides, and alkylating agents.

[0052] Additionally, the primer sequence of the present invention can be modified using a label that can directly or indirectly provide a detectable signal. The primer can include a label that can be detected using spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include: 32 P, fluorescent dyes, electron-dense reagents, enzymes (typically used in ELISA), biotin or haptens, and proteins for which antiserum or monoclonal antibodies are available.

[0053] The primers of the present invention can be chemically synthesized using any other well-known method, including cloning and restriction enzyme digestion of appropriate sequences and direct chemical synthesis methods such as the phosphotriester method of Narang et al. (1979, Meth. Enzymol. 68:90-99), the diethylphosphoramidite method of Beaucage et al. (1981, Tetrahedron Lett. 22: 1859-1862), and the solid support method of U.S. Pat. No. 4,458,066.

[0054] According to one embodiment of the present invention, the primer pair of sequence number 1 and sequence number 2; or the primer pair of sequence number 3 and sequence number 4; may be used in recombinase polymerase amplification (RPA).

[0055] The "recombinase polymerase amplification (RPA)" of the present invention is a technology that can confirm DNA and RNA amplification based on PCR technology. Unlike conventional PCR methods, the RPA method can rapidly and accurately amplify target sequences using DNA binding proteins and recombinases. In addition, it can be amplified under isothermal conditions using an isothermal polymerase, so it has the advantage of allowing rapid virus detection and diagnosis with only an isothermal device, without any special equipment. However, since nonspecific reactions often occur in the RPA method, the production of suitable primers is important to prevent this.

[0056] According to one embodiment of the present invention, the amplification product of the recombinant polymerase amplification method may be detected by a CRISPR-Cas12a system, and the CRISPR-Cas12a system may be performed with a crRNA including a base sequence of SEQ ID NO: 5 or a crRNA including a base sequence of SEQ ID NO: 6.

[0057] The "CRISPR-Cas12a system" of the present invention is a CRISPR / Cas system belonging to Type V of CRISPR-Cas12a Class 2, specifically VA, among the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems, which are adaptive immune systems for defending against viral attacks in bacteria and archaea. Compared to the CRISPR / Cas9 system belonging to Type II, the CRISPR / Cas12a system has significantly smaller effector protein sizes and crRNA sizes and lower off-target activity, thereby providing high target specificity. However, the CRISPR / Cas12a system has lower indel efficiency than the CRISPR / Cas9 system, and is therefore mainly used in the field of diagnosis for detecting specific genes rather than gene editing technology.

[0058] According to one embodiment of the present invention, the CRISPR-Cas12a system may detect the reaction product as single-stranded DNA (ssDNA).

[0059] According to one embodiment of the present invention, the single-stranded DNA is a TTATT sequence labeled with 6-carboxyfluorescein (FAM) at the 5' end and biotin (Biorin ssDNA) at the 3' end; or

[0060] It may be a TTATT sequence with FAM labeled at the 5' end and Black Hole Quencher-1 (BHQ-1) labeled at the 3' end.

[0061] According to one embodiment of the present invention, a single-stranded DNA of the TTATT sequence, in which the 5' end is labeled with 6-carboxyfluorescein (FAM) and the 3' end is labeled with biotin (Biorin ssDNA), may be used for detection in lateral flow assay (LFA).

[0062] According to one embodiment of the present invention, a single-stranded DNA of a TTATT sequence labeled with FAM at the 5' end and with Black Hole Quencher-1 (BHQ-1) at the 3' end may be used for fluorescence detection.

[0063] In addition, the present invention provides a composition for detecting human cytomegalovirus (HCMV) comprising the above primer set.

[0064] The composition for detecting a virus of the present invention may include a primer or probe that specifically binds to HCMV, and the composition may further include a reaction amplification mixture. The reaction amplification mixture refers to a solution containing reagents necessary for performing an amplification reaction, a heat-stable DNA polymerase, deoxynucleotides, nuclease-free sterile water, and a divalent metal cation, and may preferably include a reaction buffer, deoxynucleotides, and DNA polymerase. The terminal of the probe may be labeled with a reporter such as a fluorescent substance.

[0065] In addition, the present invention provides a kit for detecting human cytomegalovirus (HCMV) comprising the above composition.

[0066] In addition, the present invention provides (1) a primer pair of SEQ ID NO: 1 and SEQ ID NO: 2; or a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4;

[0067] (2) crRNA comprising the base sequence of SEQ ID NO: 5 or crRNA comprising the base sequence of SEQ ID NO: 6; and

[0068] (3) A kit for detecting human cytomegalovirus (HCMV) comprising a Cas12a protein is provided.

[0069] In addition, the present invention comprises a step of isolating a biological sample from a patient;

[0070] A step of extracting DNA from the separated biological sample;

[0071] A step of amplifying the extracted DNA using the above kit using recombinase polymerase amplification (RPA);

[0072] A step of cutting the amplified DNA using the CRISPR-Cas12a system using the above kit; and

[0073] A method for detecting human cytomegalovirus (HCMV) is provided, comprising a step of identifying the above-mentioned cleaved DNA product.

[0074] The above "amplification reaction" means a reaction for amplifying a nucleic acid molecule, and includes Recombinase polymerase amplification (RPA), polymerase chain reaction (PCR), recombinase aided amplification (RAA), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), Gap-LCR (WO 90 / 01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (WO88 / 10315), self sustained sequence replication (WO90 / 06995), selective amplification of target polynucleotide sequences, consensus sequence priming polymerase chain reaction The amplification method may be, but is not limited to, consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), nucleic acid sequence based amplification (NASBA), strand displacement amplification, and loop-mediated isothermal amplification (LAMP).

[0075] According to one embodiment of the present invention, the step of identifying the DNA product may be identifying it by lateral flow assay (LFA) or fluorescence detection.

[0076] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0077] <Example 1> Preparation for RPA and CRISPR-Cas12a Diagnosis

[0078] <1-1> Preparation of virus samples

[0079] To establish a human cytomegalovirus (HCMV) diagnostic method based on the recombinase polymerase amplification (RPA) and CRISPR-Cas12a of the present invention, virus samples were prepared. Specifically, cloned UL75 plasmid DNA was used as a standard template, and the UL75 plasmid DNA was provided by the Laboratory of Molecular Cell Biology, Department of Microbiology, Sungkyunkwan University School of Medicine. The UL75 full-length genome was amplified from the HCMV Toledo strain and cloned into an expression vector. Then, to determine the detection limit, 10 UL75 plasmid DNA stocks were used. 5copies / μl. Then, for detection comparison, HFF, COS-7, Vero, and Mewo cell lines were infected with HCMV, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), and varicella-zoster virus (VZV) to propagate the viruses. The cell lines were cultured in Dulbecco's modified eagle medium (DMEM, Welgene, Korea) supplemented with 10% Cosmic calf serum (CCS, Hyclone, USA). Viral genomic DNA was extracted from virus-infected cell cultures (HSV-1, HSV-2, and HCMV) or cultured cells (VZV), and DNA extraction and purification were performed using a DNA extraction kit (SGD41-C100, SolGent, Korea).

[0080] <1-2> Primer design for RPA and RPA reaction

[0081] The HCMV genome sequence of the Toledo strain was obtained from the National Center for Biotechnology Information (NCBI) and used as a template for RPA primer design. The RPA primer set was manually designed according to the TwistAmp guide, and the constructed primer sequences are shown in Table 1. Primer characteristics were then confirmed using BLAST (blastn), and synthesis was performed at Bioneer (Korea).

[0082] The RPA reaction was performed using the TwistAmp Basic Kit (TwistDX, UK). 13.2 μl of template DNA, 2.4 μl of forward and reverse primers, and a mixture of 29.5 μL of rehydration buffer and 2.5 μL of magnesium acetate (MgOAc) were added prior to the start of the reaction. The RPA reaction was performed at 37°C using the ProFlex PCR system (Thermo Fisher, USA). To visualize the RPA amplification products, the amplification products were purified using the Monarch PCR & DNA Cleanup Kit (NEB, USA). The efficiency and characteristics of the RPA primers were confirmed by gel electrophoresis, and the DNA amplification products were visualized using the ChemiDoc system (Azure Biosystems, USA) after electrophoresis.

[0083] [Table 1]

[0084]

[0085] <1-3> CRISPR-Cas12a reaction

[0086] Two CRISPR RNAs (crRNAs) targeting RPA amplicons were designed using the web tool CRISPOR (30). To detect Cas12a-mediated trans-cleavage activity using lateral flow assay (LFA), single-stranded DNA (ssDNA) was labeled with 6-carboxyfluorescein (FAM) at the 5' end and biotin at the 3' end (Biotin ssDNA). Furthermore, to detect Cas12a-mediated trans-cleavage activity using fluorescence detection, ssDNA was labeled with FAM at the 5' end and Black Hole Quencher-1 (BHQ-1) at the 3' end (FAM ssDNA). crRNA and ssDNA were synthesized by Bioneer (Korea), and information on crRNA and ssDNA is shown in Table 2 below.

[0087] To detect the reaction of CRISPR-Cas12a by LFA, the Cas12a reaction mixture containing 1 μl of Lba Cas12a (1 μM), 2 μl of NEBuffer r2.1 (NEB), 1 μl of crRNA (1 μM), 2 μl of RPA amplicon, and 1 μl of ssDNA (10 μM) was mixed with 13 μl of distilled water to make a total volume of 20 μl, and the reaction was performed at 37°C for 30 min using a ProFlex PCR system (Thermo Fisher, USA). After the reaction was completed, 20 μl of the reaction mixture was mixed with 100 μL of assay buffer from the HybriDetect - Universal Lateral Flow Assay Kit (Milenia Biotec, Germany) in a 1.5 ml tube. The LFA strip was then reacted with the above tube at room temperature.

[0088] To quantify the CRISPR-Cas12a reaction by fluorescence in a fluorescence assay, the Cas12a reaction mixture containing 5 μl of Lba Cas12a (1 μM; NEB, #M0653S, USA), 10 μl of NEBuffer r2.1 (NEB), 5 μl of crRNA (1 μM), 10 μl of RPA amplicon, and 10 μl of ssDNA (10 μM) was mixed with 65 μl of distilled water to prepare the Cas12a mixture. The reaction was performed in a 96-well black polystyrene microplate (SPL, Korea), and fluorescence expression was analyzed at wavelengths of 526 and 566 nm using a SpectraMax iD3 microplate reader (Molecular Devices, USA). The emitted fluorescence was then quantified as relative fluorescence units (RFU) for comparison between each sample.

[0089] [Table 2]

[0090]

[0091] <1-4> Preparation of clinical samples

[0092] To determine whether the recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based human cytomegalovirus (HCMV) diagnostic method of the present invention can detect HCMV in clinical samples, clinical samples were prepared. Peripheral blood was obtained from critically ill patients who gave written consent. To collect plasma samples, 3 ml of blood sample obtained from each subject was transferred to a sterile EDTA tube using a sterile syringe and centrifuged at 10,000 rpm for 5 minutes at room temperature to obtain plasma. The plasma sample was then transferred to a sterile centrifuge tube for storage. The clinical trial was conducted after approval by the Institutional Review Board (IRB) of Jeju National University Hospital.

[0093] <Example 2> Construction of an HCMV RPA-CRISPR-Cas12a diagnostic platform

[0094] The present invention has established a platform for diagnosing human cytomegalovirus (HCMV) based on the recombinase polymerase amplification (RPA) method and CRISPR-Cas12a. Specifically, DNA extracted from a patient's blood was subjected to an RPA reaction at 37°C for 20 minutes using two RPA primer sets of the present invention. The RPA product was then combined with a CRISPR-Cas12a mixture containing crRNA and ssDNA for fluorescence analysis or LFA and reacted at 37°C for 30 minutes. After the reaction was completed, a fluorescent signal was generated by cleavage of ssDNA in the fluorescence analysis, and in the LFA reaction, HCMV DNA can be detected within 3 minutes on a diagnostic strip labeled with biotin and FAM, and a negative result can be determined by detection of a C (control) line on the strip, and a positive result can be determined by detection of a T (test) line (Fig. 1).

[0095] <Example 3> RPA primer design and screening

[0096] Based on the accumulated sequence information from various HCMV strains, we confirmed that the HCMV genome has a high degree of polymorphism, and that the essential envelope glycoprotein H (gH, UL75) of HCMV is a conserved gene region in HCMV. Then, based on the sequence information of UL75 obtained from NCBI, a primer set of SEQ ID NOs: 1 and 2 (RPA set1) and a primer set of SEQ ID NOs: 3 and 4 (RPA set2), which are HCMV detection primers for RPA, were constructed (Fig. 2A). In addition, crRNA was constructed to enable CRISPR-Cas12a recognition in the RPA amplification region (SEQ ID NOs: 5 and 6, Fig. 2B).

[0097] Afterwards, to confirm the detection specificity of the RPA primer set, 1 × 10 5 As a result of performing RPA reaction at 37°C for 0, 5, 10, 15, 20, and 25 minutes using UL75 plasmid DNA at a concentration of copies / μl as a template, as shown in Fig. 2C, no non-specific band was observed in RPA set1 and RPA set2 of the present invention, and it was confirmed that the sizes of the amplification products were 118 bp and 187 bp, respectively. It was confirmed that the amplification products were observed from 10 minutes after the RPA reaction, and the expression of the band was strong from 20 minutes after the reaction.

[0098] <Example 4> crRNA screening

[0099] Among the crRNAs of the present invention, crRNAs that are efficient in target detection were screened. Specifically, 1 x 10 2 and 1 x 10 3 To evaluate the sensitivity of Cas12a-based detection of the RPA-amplified HCMV UL75 sequence using the UL75 plasmid at a concentration of 10 copies / μl, real-time fluorescence readouts were used. After performing the RPA reaction for 20 minutes under the same conditions as in Example 3, the CRISPR-Cas12a reaction was performed using UL75 crRNA1 for the products amplified with each RPA set1 and UL75 crRNA2 for the products amplified with each RPA set2. The reaction products were then measured every minute using a fluorescence plate reader. As a result, it was confirmed that the RPA-CRISPR-based detection method of the present invention can accurately detect HCMV (Fig. 3A). In addition, it was confirmed that the fluorescence value of the reaction using RPA set2 and UL75 crRNA2 was higher than the fluorescence value of the reaction using RPA set1 and UL75 crRNA1 (Fig. 3B).

[0100] Furthermore, under the same conditions as above, LFA was used to confirm the diagnostic results, confirming that the T line band was expressed in all samples except the negative control group (Figures 3C and 3D). Based on these results, the optimal reaction conditions using RPA set2 and UL75 crRNA2 were confirmed.

[0101] <Example 5> Confirmation of HCMV diagnostic sensitivity and specificity

[0102] The sensitivity and specificity of the RPA and CRISPR-Cas12a-based HCMV detection method of the present invention were confirmed. Specifically, to determine the limit of detection (LoD), the RPA-CRISPR reaction was performed using serially diluted UL75 plasmid, and the fluorescence generated for 40 minutes was measured to confirm the reaction result. As a result, no increase in fluorescence was observed in negative samples, but an increase in fluorescence expression was observed in all positive samples (Fig. 4A). When the concentration of the DNA template was 1 × 10 1 In samples with more than 10 copies / μl, it was confirmed that fluorescence expression increased rapidly after 10 minutes, and it was confirmed that fluorescence expression was saturated after 30 minutes of reaction (Fig. 4B). In addition, when the concentration of DNA template was 1 × 10 -1 and 1 × 10 0 Fluorescence expression was observed even in samples with relatively low amounts of copies / μl, 1 x 10 -1 It was confirmed that detection was possible starting from a concentration of copies / μl.

[0103] Additionally, after performing the CRISPR-Cas12a reaction for 30 minutes using ssDNA labeled with FAM and biotin, the LFA reaction was confirmed to be up to 1 x 10 0 It was confirmed that detection was possible up to a concentration of copies / μl (Figs. 4C and 4D), and the sensitivity of the RPA and CRISPR-Cas12a-based HCMV detection method of the present invention was 1 × 10 -1It was confirmed that the concentration was .

[0104] In order to confirm the specificity of the RPA and CRISPR-Cas12a-based HCMV detection method of the present invention, viral DNA was extracted from HSV-1, HSV-2, VZV, and HCMV, and the RPA and CRISPR-Cas12a-based detection reaction of the present invention was performed. As a result, it was confirmed that the RPA and CRISPR-Cas12a-based detection reaction specifically detected only HCMV (Fig. 5).

[0105] <Example 6> Validation in clinical samples

[0106] <6-1> Clinical sample verification

[0107] The RPA and CRISPR-Cas12a-based HCMV detection method of the present invention was used to verify the effectiveness of HCMV detection in clinical samples. Blood samples were obtained from 13 patients suspected of HCMV infection, and HCMV detection using the RPA and CRISPR-Cas12a-based HCMV method of the present invention was performed. To compare the diagnostic efficacy, quantitative PCR (qPCR) was performed simultaneously. Four samples (30.7%) tested positive in both qPCR and RPA-CRISPR diagnosis, and nine samples tested negative (69.2%) (Figures 6A to 6D and Figure 7A). These results confirmed that HCMV detection using the RPA-CRISPR method of the present invention was consistent with the qPCR analysis results. In addition, to confirm the correlation of the analyses, a linear regression analysis was performed, and as a result, it was confirmed that the RPA-CRISPR fluorescence analysis and RPA-CRISPR-LFA analysis of the present invention showed a strong positive correlation (Fig. 7B).

[0108] <6-2> Detection of HCMV in biological samples isolated from newborns

[0109] The RPA and CRISPR-Cas12a-based HCMV detection method of the present invention was used to verify the HCMV detection effect in saliva and urine isolated from newborns as clinical samples. Specifically, DNA was isolated from three saliva samples and one urine sample isolated from newborns (B046, B048, and B044) using a nucleic acid purification column (Solg™ Plasmid Mini-prep Kit, SolGent, Daejeon) or a Chelex 100 chelating kit (Chelex 100 Chelating Resin, molecular biology grade, Bio-rad #1421253), and the RPA and CRISPR-Cas12a-based HCMV detection of the present invention was performed. In addition, to verify the CMV detection effect of the LFA detection method of the present invention, each sample was amplified by qPCR, and the amount of HCMV amplification product was quantified.

[0110] For Chelex100 chelating assay, 10% Chelex100 (10 times the volume of the nucleic acid sample) was mixed and heated at 92°C for 10 minutes. After centrifugation, the supernatant was collected and DNA was isolated.

[0111] As a result, as shown in Fig. 8A, the most amplified products were confirmed in the qPCR results from the B046 saliva and urine samples, and it was confirmed that HCMV was detected in the DNA obtained using Column or Chelex100 in the samples isolated from the newborn (Fig. 8B). In addition, similar to the qPCR results, it was confirmed that the detection intensity of the band in the C line in the LFA detection was significantly higher in the B046 saliva and urine samples (Fig. 8C).

[0112] <6-3> Detection of HCMV in biological samples from patients with high-tension uveitis and glaucoma

[0113] Using the RPA and CRISPR-Cas12a-based HCMV detection method of the present invention, we confirmed whether HCMV could be detected in ocular aqueous humor samples from patients with high-tension uveitis and glaucoma. Specifically, ocular aqueous humor samples were isolated from patients clinically suspected of having CMV-related glaucoma (Clinical CMV; 47, 59, 69, 64, 70), and HCMV was detected using the LFA detection method of the present invention. In addition, HCMV was also detected using the LFA method using ocular aqueous humor samples from patients whose CMV positivity was confirmed by qPCR (High positive; 58, 60, 66, 71, 73), as described above.

[0114] As a result, as shown in Fig. 9, it was confirmed that HCMV could be detected by the LFA method of the present invention in the Clinical CMV group and the High positive group.

[0115] Accordingly, the present invention confirmed that a method for diagnosing human cytomegalovirus (HCMV) using a recombinant polymerase amplification reaction primer that specifically binds to the genomic sequence of the essential envelope glycoprotein H of HCMV and a CRISPR-Cas12a system can accurately detect only HCMV among similar viruses. In addition, it was confirmed that HCMV infection can be diagnosed in actual clinical samples, and that it has superior sensitivity and specificity compared to existing molecular biological methods, confirming that it can be used for rapid and accurate on-site diagnosis of HCMV.

Claims

1. A primer set for detecting human cytomegalovirus (HCMV), comprising a primer pair of sequence numbers 1 and 2; or a primer pair of sequence numbers 3 and 4.

2. In paragraph 1, A primer set, wherein the primer pair of sequence numbers 1 and 2; or the primer pair of sequence numbers 3 and 4; is used in recombinase polymerase amplification (RPA).

3. In paragraph 2, A primer set wherein the amplification product of the above recombinant polymerase amplification method is detected by the CRISPR-Cas12a system.

4. In paragraph 3, The above CRISPR-Cas12a system is performed with a primer set comprising a crRNA comprising a base sequence of SEQ ID NO: 5 or a crRNA comprising a base sequence of SEQ ID NO:

6.

5. In paragraph 3, The above CRISPR-Cas12a system is a primer set that detects the reaction product as single-stranded DNA (ssDNA).

6. In paragraph 5, The single-stranded DNA is a TTATT sequence labeled with 6-carboxyfluorescein (FAM) at the 5' end and biotin at the 3' end; or A primer set comprising a TTATT sequence labeled with FAM at the 5' end and Black Hole Quencher-1 (BHQ-1) at the 3' end.

7. In paragraph 6, A primer set wherein a single-stranded DNA of the TTATT sequence, wherein the 5' end is labeled with 6-carboxyfluorescein (FAM) and the 3' end is labeled with biotin, is used for detection in a lateral flow assay (LFA).

8. In paragraph 6, A primer set comprising a single-stranded DNA of a TTATT sequence labeled with FAM at the 5' end and Black Hole Quencher-1 (BHQ-1) at the 3' end, which is used for fluorescence detection.

9. A composition for detecting human cytomegalovirus (HCMV) comprising the primer set of paragraph 1.

10. A kit for detecting human cytomegalovirus (HCMV) comprising the composition of Article 9. 11.(1) A primer pair of sequence numbers 1 and 2; or a primer pair of sequence numbers 3 and 4; (2) crRNA comprising the base sequence of SEQ ID NO: 5 or crRNA comprising the base sequence of SEQ ID NO: 6; and (3) Kit for detecting human cytomegalovirus (HCMV) comprising Cas12a protein.

12. Step of isolating a biological sample from a patient; A step of extracting DNA from the separated biological sample; A step of amplifying the extracted DNA using the kit of Article 11 using recombinase polymerase amplification (RPA); A step of cutting the amplified DNA using the kit of claim 9 with the CRISPR-Cas12a system; and A method for detecting human cytomegalovirus (HCMV), comprising: a step of identifying the above-mentioned cleaved DNA product.

13. In paragraph 12, A method wherein the step of confirming the above DNA product is performed by lateral flow assay (LFA) or fluorescence detection.

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