Efficient on-site genetic molecular diagnostic method

Abstract

The present invention relates to the most efficient system for RNA gene molecular diagnosis in the field using a Formamide-Ligation-RPA-PK-probe. The Covid-19 diagnostic technology using the Formamide-Ligation-RPA-PK-probe of the present invention enables the extraction of viral RNA in the field without the use of an extraction kit, does not use reverse transcription for the synthesis of cDNA from the extracted RNA, uses RPA (Recombinase Polymerase Amplification) for the amplification of the generated cDNA, and allows for the visual observation of color changes without the use of a detector, thereby enabling rapid and accurate detection of RNA virus-mediated diseases in the field within 30 minutes, making it very useful for point-of-care testing (POCT).

Description

Technology Field

[0001] The present invention relates to the most efficient system for on-site RNA gene molecular diagnosis using a Formamide-Ligation-RPA-PK probe. Background Technology

[0002] The coronavirus (COVID-19) suddenly emerged in 2019, claiming over 5.9 million lives along with 445 million cases, while simultaneously having a devastating impact on global public health that collapsed the world economy. Since the majority of COVID-19 carriers are asymptomatic, continuous testing is necessary, and it is required to prevent the sudden shedding and spread of the infectious virus. Despite the availability of many detection systems, including those based on microfluidic analysis, serology, and nucleic acid detection, there is a need to develop efficient and effective systems for COVID-19 diagnosis.

[0003] Despite the limitations of the test due to viral mutations, the need for sample storage, temperature control during incubation, costs, purification, and long analysis times, reverse transcription polymerase chain reaction (RT-PCR) is a widely used method because it can specifically and accurately detect viral nucleic acids. However, RT-PCR and qPCR are not suitable for point-of-care testing (POCT) because they require standard / advanced PCR machines with skilled personnel.

[0004] Considering the role of vectors in spreading viruses, Point-of-Care Testing (POCT) must prioritize rapid detection, as there is a need to minimize complex diagnostic methods and enable rapid decision-making regarding optimized treatment. Furthermore, POCT must be simple, easy to perform, robust, and portable, and capable of detection using only small sample volumes. For example, isothermal detection of nucleic acids can be utilized through the strand substitution activity of DNA polymerases [e.g., Recombinant Polymerase Amplification (RPA), RCA, and LAMP].

[0005] While many detection systems based on RT-RPA, RT-PCR, RT-LAMP, RT-RPA / CRISPR Cas12b, and qPCR are well-established, RT-RPA, although very simple and requiring no expensive equipment, has significant drawbacks in selectivity when targeting the entire genome of viral RNA, along with the opportunity for primer mis- or self-pairing during reverse transcription (RT). Furthermore, RT-RPA systems can generate a large amount of unwanted cDNA products during reverse transcription, it is difficult to distinguish between non-target sequences and correct target sequences when primers are partially mismatched, and some primers may bind non-specifically to unwanted targets to produce non-specific amplification products. Therefore, designing primers for target sequences is very difficult, and meticulous design is required to obtain accurate results.

[0006] Accordingly, the present invention establishes a system for performing point-of-care testing (POCT) for RNA gene molecular diagnosis, which extracts the virus at the site without an extraction kit, ligates the extracted virus, and then observes the color change with the naked eye. Prior art literature

[0007] (001) MN Aoki, B. de Oliveira Coelho, LGB Goes, ´ P. Minoprio, EL Durigon, LG Morello, FK Marchini, IN Riediger, M. do Carmo Debur, HI Nakaya, L. Blanes, Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load, Sci. Rep. 11 (2021) 1-10,(002) C. Rodriguez Diaz, N. Lafuente-Gomez, ´ C. Coutinho, D. Pardo, H. Alarcon-Iniesta, ´M. Lopez-Valls, ´ R. Coloma, P. Milan-Rois, ´ M. Domenech, M. Abreu, R. Canton, ´ JC Galan, ´ R. Bocanegra, LA Campos, R. Miranda, M. Castellanos, A. ´ Somoza, Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection, Talanta 243 (2022), 123393, The problem to be solved

[0008] The object of the present invention is to provide a composition for detecting a target RNA virus for efficient point-of-care diagnosis using a Formamide-Ligation-RPA-PK probe, a kit including the same, and a method for providing information regarding the detection of a target RNA virus. means of solving the problem

[0009] Accordingly, the present invention provides a composition for detecting a target RNA virus and a kit comprising the same, the composition comprising: an RNA (RiboNucleic Acid) extraction reagent containing formamide; an enzyme for ligating cDNA (complementary Deoxyribo Nucleic Acid) containing a target RNA virus sequence from the extracted RNA; RPA (Recombinase Polymerase Amplification) for amplifying the cDNA containing the target RNA virus sequence; and a PK-probe (pyrophosphate-sensing probe) of the following chemical formula 1.

[0010] In addition, the present invention provides a method for providing information regarding the detection of a target RNA virus, comprising the steps of: extracting RNA (Ribo Nucleic Acid) from a biological sample using formamide; synthesizing cDNA (complementary Deoxyribo Nucleic Acid) containing a target RNA virus sequence using ligation from the extracted RNA; amplifying the cDNA using RPA (Recombinase Polymerase Amplification); adding a PK-probe (pyrophosphate-sensing probe) to a sample containing the cDNA; and diagnosing that the sample containing the cDNA contains a target RNA virus sequence when the color of the sample changes from pink to colorless. Effects of the invention

[0011] The COVID-19 diagnostic technology using the Formamide-Ligation-RPA-PK probe of the present invention enables the extraction of viral RNA at the site without the use of an extraction kit, does not use reverse transcription for the synthesis of cDNA from the extracted RNA, uses RPA (Recombinase Polymerase Amplification) for the amplification of the generated cDNA, and allows for the visual observation of color changes without the use of a detector, thereby enabling rapid and accurate detection of RNA virus-mediated diseases at the site within 30 minutes, making it very useful for point-of-care testing (POCT). Brief explanation of the drawing

[0012] Figure 1 is a schematic diagram showing the process of diagnosing Covid-19 using a Formamide-Ligation-RPA-PK probe. Figure 2 is a diagram and graph showing the PK-probe / Lig-RPA analysis process and results: (A) Lig-RPA result using PAGE; Lane M: 25 / 100bp, Lane 1: Whole-genome RNA extracted from SARS-CoV-2 positive patients + 1–7 ligation templates corresponding to the "N gene" of SARS-CoV-2 + RPA reaction mixture containing ligation and PK-probe; Lane 2: Whole-genome RNA extracted from SARS-CoV-2 negative patients + 1–7 ligation templates corresponding to the "N" gene of SARS-CoV-2 + RPA reaction mixture containing ligation and PK-probe, Lane 1 (blurred, very bright) and Lane 2 (clear) near <40mer size positions, (B) Color change mechanism of PK-probe, (C) Optimization of PK-probe / Lig-RPA; a PK-probe concentration of 2.5 μM is required to optimize the noticeably significant color change between positive and negative reactions, (D) Absorbance of negative and positive PK-probe / Lig-RPA assays (measured at 575 nm). Figure 3 shows a photograph and graph representing the results of colorimetric analysis over time to optimize PK-probe / Lig-RPA analysis: (A) Colorimetric analysis of sensitivity over time, Lane 1: 0 min - 0 min Lig+0 min RPA; Lane 2: 20 min - 10 min Lig+10 min RPA; Lane 3: 25 min - 10 min Lig+15 min RPA; Lane 4: 30 min - 15 min Lig+15 min RPA; Lane 5: 35 min - 15 min Lig+20 min RPA; Lane 6: 40 min - 15 min Lig+25 min RPA; Lane 7: 50 min - 20 min Lig+30 min RPA; Lane 8: 1 hour - 20 min Lig+40 min RPA, (B) Absorbance spectrum according to the colorimetric test of (A), (C) Colorimetric analysis of temperature-dependent studies, Lane 1: Lig-RPA (negative) at 37℃; Lane 2: Lig-RPA at 20℃; Lane 3: Lig-RPA at 25℃; Lane 4: Lig-RPA at 30℃; Lane 5: Lig-RPA (positive) at 37℃, (D) Absorbance spectrum of the PK-probe / Lig-RPA system according to the temperature-dependent study of (C) (measured at 575 nm). Figure 4 shows a photograph and graph representing the sensitivity of the reaction using the PK-probe / Lig-RPA assay: (A) PAGE analysis of Lig-RPA sensitivity tests using different copy numbers. Lane M: 100-bp ; Lane 1: 50 copies; Lane 2: 20 copies; Lane 3: 10 copies; Lane 4: 5 copies; Lane 5: 2 copies; Lane 6: 1 copy; Lane 7: 0 copies, (B) Colorimetric analysis of the sensitivity test of Lig-RPA using a PK-probe with different copy numbers. Lane 1: 0 copy; Lane 2: 1 copy; Lane 3: 2 copies; Lane 4: 5 copies; Lane 5: 10 copies; Lane 6: 20 copies; Lane 7: 50 copies, (C) Sensitivity study performed using whole-genome SARS-CoV-2 at concentrations of 0 to 50 copies / reaction (copies / rxn), A: Absorbance in the presence of target; A0: Absorbance in the absence of target; y-axis: absolute value. The obtained LOD was 1,160 copies / ml (using 1 μl of target RNA) using the 3σ method [LOD = 3 × (SD / S), where SD is the standard deviation and S is the slope of the plot]. Figure 5 is a pictorial graph showing the results of the PK-probe / Lig-RPA selectivity experiment: (A) PAGE analysis of PK-probe / Lig-RPA selectivity test, Lane M: 25 / 100-bp ladder; Lane 1: PK-probe / Lig-RPA with 1 middle misalignment; Lane 2: PK-probe / Lig-RPA; Lane 3: PK-probe / Lig-RPA (including 1) misalignment (head / ligation site); Lane 4: PK-probe / Lig RPA with 2 misalignments (head and middle); Lane 5: PK-probe / Lig-RPA with 3 misalignments (head 1 and middle 2), (B) PAGE analysis of PK-probe / RT-RPA selectivity test using different mismatched reverse primers during cDNA synthesis, Lane M: 25 / 100-bp ladder; Lane 1: PK-probe / RT-RPA positive; Lane 2: PK-probe / RT-RPA with 1 discrepancy; Lane 3: PK-probe / RT-RPA with 2 discrepancies; Lane 4: PK-probe / RT-RPA with 3 discrepancies, (C) Colorimetric analysis of PK-probe / Lig-RPA selectivity test Lane 1: PK-probe / Lig-RPA; Lane 2: PK-probe / Lig-RPA with 1 middle mismatch; Lane 3: PK-probe / Lig-RPA with 1 mismatch (head / ligation site); Lane 4: PK-probe / Lig-RPA with 2 mismatches (head and middle); Lane 5: PK-probe / Lig-RPA with 3 mismatches (head 1 and middle 2), (D) Colorimetric analysis of the selectivity test of PK-probe / RT-RPA using different mismatched reverse primers during cDNA synthesis, Lane 1: PK-probe / RT-RPA positive; Lane 2: PK-probe / RT-RPA with 1 mismatch; Lane 3: PK-probe / RT-RPA with 2 mismatches; Lane 4: PK-probe / RT-RPA with 3 mismatches, (E) Bar diagram of the selectivity of the PK-probe / Lig-RPA system operating in the presence of various bacterial genomes. Figure 6 is a graph showing the results of measuring the absorbance spectrum (575 nm) indicating the selectivity of PK-probe / Lig-RPA operating in the presence of 9 bacterial genomes. Figure 7 shows a photograph and graph of the results of validation using PK-probe / Lig-RPA analysis with inactivated recombinant SARS-Cov-2 virus using universal transport media (QC material) and samples taken from the nasopharynx: (A) Colorimetric test; Lane 1: Negative (extraction at 25℃ + PK-probe / Lig-RPA, 25℃), Lane 2: Positive (extraction at 65℃ + PK-probe / Lig-RPA, 25℃), Lane 3: Positive (extraction at 37℃ + PK-probe / Lig-RPA at 37℃), Lane 4: Positive (extraction at 25℃ + PK-probe / Lig-RPA, 25℃), (B) Absorbance reading corresponding to the colorimetric test. Specific details for implementing the invention

[0013] The present invention will be described in detail below.

[0015] The present invention provides a composition for detecting a target RNA virus comprising: an RNA (RiboNucleic Acid) extraction reagent containing formamide; an enzyme for ligating cDNA (complementary Deoxyribo Nucleic Acid) containing a target RNA virus sequence from the extracted RNA; RPA (Recombinase Polymerase Amplification) for amplifying the cDNA containing the target RNA virus sequence; and a PK-probe (pyrophosphate-sensing probe) of the following chemical formula 1.

[0016] [Chemical Formula 1]

[0017]

[0018] According to one embodiment of the present invention, the target RNA virus is a coronavirus, but the target RNA is not limited to viral RNA and may include RNA of human and various animal and plant diseases.

[0019] According to one embodiment of the present invention, the PK-probe is a Cu of a thienyl-hydrazone rhodamine derivative. 2+ As it is a complex, the above PK-probe is Cu2+ It is a probe that makes the rhodamine substance appear pink due to ring-opening complexed with ions, and when this probe comes into contact with PPi, the corresponding ion is Cu previously coordinated to the rhodamine entity 2+ It forms a complex with ions. The PK-probe is Cu 2+ After releasing ions, the rhodamine ring automatically closes, causing the pink color to disappear and become colorless.

[0020] A kit for detecting target RNA viruses comprising the above composition is provided.

[0021] The present invention provides a method for providing information regarding the detection of a target RNA virus, comprising the steps of: extracting RNA (Ribo Nucleic Acid) from a biological sample using formamide; synthesizing cDNA (complementary Deoxyribo Nucleic Acid) containing a target RNA virus sequence from the extracted RNA using ligation; amplifying the cDNA using RPA (Recombinase Polymerase Amplification); adding a PK-probe (pyrophosphate-sensing probe) to a sample containing the cDNA; and diagnosing that the sample containing the cDNA contains a target RNA virus sequence when the color of the sample changes from pink to colorless.

[0022] According to one embodiment of the present invention, the biological sample may be any one sample selected from the group consisting of blood, sputum, bacteria, semen, cell tissue, saliva, hair, and urine.

[0023] According to one embodiment of the present invention, the target RNA virus is a coronavirus, but the target RNA is not limited to viral RNA and may include RNA of human and various animal and plant diseases.

[0024] According to one embodiment of the present invention, the ligation may be performed by an enzyme that binds nucleic acid molecules.

[0025] According to one embodiment of the present invention, the PK-probe may be a compound of the following chemical formula 1.

[0026] [Chemical Formula 1]

[0027] .

[0029] The present invention developed a diagnostic system combining ligation-mediated recombinant polymerase isothermal amplification (Lig-RPA) and a PK-probe (PPi-sensing colorimetric probe) for the detection of the target "N gene" (nucleocapsid) of the SARS-CoV-2 genome. Compared to existing diagnostic systems such as RT-RPA, the system of the present invention demonstrated high selectivity for SARS-CoV-2 in the presence of various bacterial genomes, had a very low risk of falsification, and was very simple to use. Furthermore, the PK-probe / Lig-RPA system is highly suitable for application in point-of-care testing (POCT), as the isothermal amplification reaction occurs at room temperature and the diagnosis time is very fast at 30 minutes. It has very high sensitivity (1,160 copies / ml) and can be visually confirmed using colorimetric detection.

[0030] In the present invention, a PK-probe (PPi detection colorimetric probe) is combined with a Lig-RPA reaction for diagnosis targeting SARS-CoV-2, particularly the "N gene" region, and as a result, the PK-probe / Lig-RPA colorimetric isothermal nucleic acid detection system is an analytical technique suitable for point-of-care testing (POCT) because it operates with a simple process and high selectivity and sensitivity (see Fig. 1).

[0031] The term "colorimetric analysis" in this invention refers to a method of measuring the concentration of a solution by comparing the concentration of the color with the color of a standard solution when a substance is dissolved in a solvent to form a colored solution. Generally, the fact that a solution is red means that the remaining color is absorbed in the wavelength range of visible light (400 to 700 nm). By measuring the change in light intensity when the corresponding color light passes through the solution using a photocell, the amount of solute can be accurately measured. In the case where the solution is colorless, a reagent that produces color is added to combine with the solute through a chemical reaction to produce color, and it is determined whether light absorption is occurring in the invisible ultraviolet or infrared wavelength range. The absorption intensity is then measured using light of a wavelength with strong absorption.

[0033] Hereinafter, preferred embodiments are presented to aid in understanding the present invention.

[0034] However, the following examples are provided merely to facilitate a better understanding of the present invention and do not limit the scope of the invention.

[0036] <Example 1> Experimental Materials and Methods

[0037] <1-1> Experimental Materials

[0038] All DNA oligonucleotides were purchased from Bioneer. SplintR ligase and the Isothermal Amplification Buffer Pack were obtained from New England Biolabs (USA). The RPA TwistAmp® Basic Kit was purchased from TwistDx, TABAS03KIT (USA). Formamide (F7503-100 mL) was purchased from Sigma-Aldrich (USA). UV-Vis absorption spectra were analyzed using a Cary Series UV-Vis spectrophotometer (Agilent Technologies, USA). All optical measurements were performed at room temperature using quartz cuvettes (path length: 1 cm). PK probes were prepared according to previously reported procedures. Polyacrylamide gel electrophoresis (PAGE) was performed on a 20% polyacrylamide gel. 40% Acrylamide / Bis solution (BIO-RAD, USA; 2.5 mL), 10X Tris borate EDTA buffer (Enzynomics, South Korea; 0.5 mL), and 20% ammonium persulfate (in H2O) were mixed in a tube and distilled water, and then added to make the total volume 5 mL. Tetramethylethylenediamine (TEMED; Sigma-Aldrich, USA) was added to obtain a 20% polyacrylamide gel. The gel was loaded into an electrophoresis apparatus (Mini-PROTEAN Tetra Cell; BIO-RAD, USA) and processed at 80 V for 6 hours. The gel was stained with ethidium bromide (EtBr) solution for 10 minutes. The stained gel was washed with water for 10 minutes, and gel photographs and colorimetric detection images were captured using a mobile device under a transilluminator.

[0040] <1-2> Ligation and RPA Reaction Conditions

[0041] The total ligation reaction volume was 10 μL, including the solution. In addition to 7 ligation oligonucleotide templates (SEQ NOs. 1 to 7, 10 nM, 5 μL), spike SARS-CoV-2 RNA (2 copies, 1 μL), 10X SplintR ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP; pH 7.5 at 25°C; 1 μL), 10X bovine serum albumin (BSA, 1 μL), SplintR ligase (25 U / μL, 1 μL), PK-probe (0.1 μL), and dH2O were added to provide a total volume of 10 μL, and incubated at 37°C for about 10 minutes, according to the standard procedure of the Twist Dx RPA kit, primer-free rehydration buffer (29.5 μL), forward and reverse primers (50 pmol / μL each, 5 μL), 100 mM DTT (3 μL) and ligated target product (10 μL); treated with a basic reaction kit mixture containing freeze-dried enzyme and then mixed using a pipette. 280 mM Mg(OAc)2 (2.5 μL) was added and mixed well. The reaction mixture was incubated at 37°C for about 20 minutes.

[0043] <1-3> Sensitivity and Selectivity Measurement

[0044] Simulated sample during SARS-CoV-2 spike RNA preparation; 200 μL (1,000 copies) (5,000 copies / mL; AccuPlex SARS-CoV-2 Molecular Controls Kit- Full Genome, Material Number 0505-0129, SeraCare, Milford, MA) of recombinant SARS-CoV-2 whole genome control material was spiked into 1.8 mL of buffer (eNAT, Copan Italia, Brescia, Italy) of a negative human sample prepared according to standard sample acquisition. Then, a total of 2 mL of solution (1,000 copies) was extracted using an eMAG system (bioMerieux, Marcyltoile, France), yielding an elution volume of 50 μL (100 copies) with an input volume of 200 μL (100 copies) according to the extraction protocol provided by the manufacturer. The replication concentration of the extracted RNA was approximately 2 copies / μL = 2,000 copies / mL. For sensitivity studies, RNA samples were diluted in distilled water and freeze-dried to provide varying concentrations ranging from 0 to 50 copies / μL (0 to 50,000 copies / ml). All samples at each concentration were tested using the PK-probe / Lig-RPA system. Absorbance was measured to calculate the limit of detection (LOD). Selectivity studies included nine bacterial species known to be part of the normal flora of the upper respiratory tract (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus feacium, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, and Acineitobacter bau). All bacterial DNA was extracted using the boiling method with DNA extraction buffer (Seegene, Seoul, South Korea).Viral RNA was extracted using the same validation method as clinical samples. The extracted bacterial DNA and viral RNA were tested with the PK-probe / Lig-RPA system and compared with the results for SARS-CoV-2 detection. In the sensitivity and selectivity study, ddH2O (150 μL) was added to a 250 μL reaction tube along with 25 mM PK-probe (0.1 μL) to check for a color change, and later diluted with ddH2O to make a 1 mL solution; the absorbance of the mixture for each reaction was measured in the presence of the PK-probe.

[0046] <Example 2> Experimental Results

[0047] <2-1> Target Site Selection and Primer Design

[0048] In the present invention, seven templates (Lig T1, Lig T2, Lig T4, Lig T5, Lig T6, Lig T7) were designed from SplintR ligase and are shown in Table 1 below.

[0050] Sequence number name Sequence (5'→3') 1 Lig-T1 GAA ATT TGG ATC TTT GTC ATC CAA TTT GAT GGC 2 Lig-T2 pho ACC TGT GTA GGT CAA CCA CGT TCC CGA AGG TGT 3 Lig-T3 pho GAC TTC CAT GCC AAT GCG CGA CAT TCC GAA GAA 4 Lig-T4 pho CGC TGA AGC GCT GGG GGC AAA TTG TGC AAT TTG 5 Lig-T5 pho CGG CCA ATG TTT GTA ATC AGT TCC TTG TCT 6 Lig-T6 pho GAT CGG TAG TTC CTG GTC CCC AAA ATT TCC TTG GGT TTG 7 Lig-T7 pho TTC TGG ACC ACG TCT GCC GAA AGC TTG TGT T 8 Lig-T3 tail 1mm pho GAC TTC CAT GCC AAT GCG CGA CAT TCC GAA T AA 9 Lig-T4 mid 1mm pho CGC TGA AGC GCT G C G GGC AAA TTG TGC AAT TTG 10 Lig-T4 2mm pho CG T TGA AGC GCT GGG A GC AAA TTG TGC AAT TTG 11 Target RNA (~220 mers) aacacaag ctttcggcag acgtggtcca gaacaaaccc aaggaaattt tggggaccag gaactaatca gacaaggaac tgattacaaa cattggccgc aaattgcaca atttgccccc agcgcttcag cgttcttcgg aatgtcgcgc attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc catcaaattg gatgacaaag atccaaattt c 12 Lig-RPA fwd pri AAC ACA AGC TTT CGG CAG ACG TGG TCC AGA ACA AAC 13 Lig-RPA rev pri / RT primer GAA ATT TGG ATC TTT GTC ATC CAA TTT GAT GGC AC 14 Lig-RPA rev pri / RT primer-1 mm GAA ATT TGG ATC TTT GTC ATC CAA TTT GAT GGC A G 15 Lig-RPA rev pri / RT primer-2 mm GAA ATT TGG ATC TTT GTC ATC CAA TTT GAT GG G A G 16 Lig-RPA rev pri / RT primer-3 mm GAA ATT TGG ATC TTT GTC ATC CAA TTT G C TG C CA G

[0052] Among them, the LT2 (Lig-T2) to LT7 (Lig-T7) templates exhibited a monophosphate unit at the 5'-terminus for ligation. Ligation by SplintR ligase was induced in the presence of target RNA that perfectly matched the template containing the phosphate modification at the 5'-terminus, and PPi was not released as a byproduct during the ligation reaction. RNA target ligation occurred without any opportunity for PPi release during RT. Two primers (Lig-RPA fwd pri, Lig-RPA rev pri) were designed through the RPA amplification process. To investigate selectivity, templates (Lig-T3 tail 1 mm, Lig-T4 mid 1 mm, Lig-T4 2 mm) with one and two bases mismatched were designed. The mismatched bases are from Table 1. It is written in bold.

[0053] A 220-bp cDNA targeting the "N gene" in SARS-CoV-2 was prepared by linking seven templates (each approximately 32bp) and then analyzed for RPA reaction. Designing templates to bind to specific regions of viral RNA for the ligation reaction is a critical step for selective detection, and full-length cDNA is formed only when each template binds to the target "N gene" of SARS-CoV-2. In this invention, 100 copies of the spike SARS-CoV-2 whole genome RNA were used, and the 7 templates and SplintR ligase were analyzed via PAGE to confirm cDNA formation (Fig. 2A). The formation of 220-mer cDNA was confirmed after RPA, and a specific band was identified at approximately the 220-mer position when the "N gene" was added to SARS-CoV-2 (lane 1). In the absence of a target, the corresponding band was not observed, so no ligation occurred (Lane 2), confirming that the Lig-RPA system was functioning.

[0055] <2-2> PK-probe combined with Lig-RPA system

[0056] The PK-probe is a Cu of a thienyl-hydrazone rhodamine derivative. 2+ It is a complex. Cu 2+ - The complex probe was used for the colorimetric detection of DNA / RNA amplification, but it was selective for PPi and exhibited cross-reactivity with DTT. Another type of AuNCs-Cu 2+ Although the system is used to detect PPi in human urine, the PK-probe is a probe that exhibits high selectivity for PPi even in the presence of dNTPs (deoxynucleotide triphosphates), DTT, and cysteine. The PK-probe is Cu 2+It is a probe that makes the rhodamine substance appear pink due to ring-opening complexed with ions, and when this probe comes into contact with PPi, the corresponding ion is Cu previously coordinated to the rhodamine entity 2+ It forms a complex with ions. The PK-probe is Cu 2+ After releasing ions, the rhodamine ring automatically closes, causing the pink color to disappear (Fig. 2B). DTT is Cu 2+ Despite being a good competitor in complex formation, the PK-probe is Cu 2+ The selective release of ions to PPi results in a decrease in absorbance at 575 nm in the presence of PPi. It was confirmed that the selective colorimetric sensor for PPi exhibits high resistance to all other biomolecules and buffer components, particularly large amounts of DTT and human serum. Based on the mechanism of action of the PK-probe, we investigated whether SARS-CoV-2 could be detected using a combination of the PK-probe and the Lig-RPA system. To investigate the utility of the PK-probe / Lig-RPA system, negative conditions (absence of the target "N gene" in SARS-CoV-2) and positive conditions (presence of the target "N gene") were tested. The response to SARS-CoV-2 and various PK-probe concentrations was analyzed. Figure 2C confirmed that a significant color change between positive and negative conditions was visually observable. Qualitative absorbance readings also showed a significant difference in values ​​measured at a wavelength of 575 nm (Figure 2D).

[0058] <2-3> Point-of-Care Testing (POCT) Optimization Diagnostic Process

[0059] It was confirmed that the system operates based on temperature and time while optimizing the PK-probe / Lig-RPA detection analysis. Combinations of PK-probe / Lig-RPA were analyzed from 0 minutes to 1 hour.

[0060] As a result, it was proven that 10 minutes is sufficient for ligation by SplintR ligase, and RPA takes less than 20 minutes to amplify one copy of DNA. The PK-probe / Lig-RPA system already confirmed a visually distinguishable color change within a total diagnostic time of 20 minutes (Lig, 10 minutes; RPA, 10 minutes), and after 50 minutes, it was almost perfectly visually distinguishable (Figs. 3A and 3B). Therefore, it was confirmed that the target "N gene" of SARS-CoV-2 can be detected within 30 minutes through a visible color change using the PK-probe / Lig-RPA system.

[0061] In addition, to verify the temperature optimized for point-of-care testing (POCT), SplintR ligase has been reported to exhibit optimized activity at 37°C, whereas RPA has been reported to exhibit optimized activity at 39-42°C. However, since the enzymes can be activated at a low temperature of 16°C and both enzymes function well at room temperature, performing point-of-care testing (POCT) is simpler and more feasible if detection is possible under ambient conditions / room temperature; therefore, Lig-RPA analysis was tested at various temperatures including 20, 25, 30, and 37°C.

[0062] As a result, the system of the present invention operated in the range of 20-37°C, and while it was satisfactory for qualitative analysis at 20°C, the efficiency was higher at 37°C (Figs. 3C and 3D).

[0064] <2-4> Photosensitivity Analysis

[0065] To analyze detection sensitivity when using the PK-probe / Lig-RPA system, simulation samples were prepared for LOD calculation. Spike SARS-CoV-2-positive RNA samples with assigned copy numbers (2 copies / μl) were used, and samples at various concentrations were prepared for the sensitivity study; sensitivity was measured by absorbance at 575 nm. Sensitivity tests were performed using 0 to 50 copies / μl = 0 to 50,000 copies / ml, and verified via PAGE.

[0066] As a result, when using copy numbers 1 to 50 of the target SARS-CoV-2 RNA for the specific amplification product (ca. 220 mer), no band could be observed at this position in the case of copy 0 (Fig. 4A). While all 0-copy reactions showed a pink color, increasing the target copy number resulted in more colorless characteristics of the PK-probe (Fig. 4B).

[0067] In addition, the average change in absorbance at each specific concentration was measured. The inset in Fig. 4(C) confirmed a linear relationship by showing a linear equation between the data values ​​when calculating the LOD using the 3σ method [LOD = 3 × (SD / S), where SD is the standard deviation and S is the slope of the plot] (Fig. 4C). Since the calculated LOD was 1,160 copies / ml for a target with a Pearson r value of 0.99843, indicating high sensitivity, the PK-probe / Lig-RPA system of the present invention operated sensitively even when the number of SARS-CoV-2 viral RNA copies was low. It was confirmed that the PK-probe / Lig-RPA system of the present invention has an LOD of 1,160 copies / ml, which is similar to the RT-qPCR method reported by Yang M, et al. and Corman et al., and exhibits very high detection sensitivity compared to other colorimetric detection methods mentioned in Table 2 below.

[0069] Detection method LOD References RT-qPCR 1,040 copies / ml VM Corman, O. Landt, M. Kaiser, R. Molenkamp, ​​A. Meijer, DKW Chu, T. Bleicker, S. Brunink, J. Schneider, ML Schmidt, DGJC Mulders, BL Haagmans, B. Van Der Veer, S. Van Den Brink, L. Wijsman, G. Goderski, JL Romette, J. Ellis, M. Zambon, M. Peiris, H. Goossens, C. Reusken, MPG Koopmans, C. Drosten, Detection of 2019 novel Corona virus (2019-nCoV) by real-time RT-PCR, Euro Surveill. 25 (2020) 1; dLig-LAMP (colorimetric) 12,280 copies / ml MH Choi, J. Lee, YJ Seo, Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real ARS-CoV-2, Microchim. Acta 189 (2022) RT-LAMP (colorimetric) 48,000 ~ 4,800 copies / ml MN Aoki, B. de Oliveira Coelho, LGB Goes, ´ P. Minoprio, EL Durigon, L. \. Morello, FK Marchini, IN Riediger, M. do Carmo Debur, HI Nakaya, L. Blanes, Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load, Sci. Rep. 11 (2021) 1?10 AuNP (colorimetric) 10,00,000~100,00,000 copies / ml C. Rodriguez Diaz, N. Lafuente-Gomez, ´ C. Coutinho, D. Pardo, H. Alarcon-Inesta, ´M. Lopez-Valls, ´ R. Coloma, P. Milan-Rois, ´ M. Domenech, M. Abreu, R. Canton, ´ JC Galan, ´ R. Bocanegra, LA Campos, R. Miranda, M. Castellanos, A. ´ Somoza, Development of colorimetric sensors based on gold particles for SARS-2 RdRp, E and S genes detection, Talanta 243 (2022), 123393 PK-probe / Lig-RPA (colorimetric) 1,160 copies / ml 본 발명

[0071] <2-5> 선택성 분석

[0072] Theoretically, conventional RT-RPA systems cannot distinguish between perfectly matched sequences and sequences that differ by one or two bases; furthermore, since reaction primers can amplify multiple mismatched sequences when binding to the target RNA for the RT process, they may produce false positives. On the other hand, because the Lig-RPA system of the present invention was expected to identify mismatch points during the ligation step, it begins forming full-length cDNA only when all ligation templates match the SARS-CoV-2 viral RNA, and the SplintR ligase generates a complementary cDNA sequence. In other words, because cDNA is not synthesized if one of the ligation templates does not match, the selectivity of the analysis using the Lig-RPA of the present invention was expected to be superior to that of conventional RT-RPA. To determine the selectivity of the Lig-RPA detection method, single, double, and triple mismatches were identified on the ligation template plates (LT3, tail region; LT4, head and mid region). To evaluate the selectivity of the PK-probe / Lig-RPA system The selectivity and selectivity of the PK-probe / RT-RPA system were compared. To compare Lig-RPA and RT-RPA, the amplified products were verified using PAGE.

[0073] As a result, when Lig-RPA was used with SARS-CoV-2 target RNA, band patterns appeared differently due to different sequences that did not match, depending on 1 to 3 mismatches (Figs. 5A and 5B). In contrast, RT-RPA analysis showed nearly identical gel band patterns even with 1 to 3 mismatches, indicating that the RT-RPA system could not distinguish incorrect binding to similar sequences.

[0074] In colorimetric tests performed using Lig-RPA, clearly distinguishable color changes were observed between various mismatched systems, whereas RT-RPA-based colorimetric tests showed almost identical color changes, which increased the likelihood of false positive results (Figs. 5C and 5D). Therefore, through PAGE and colorimetric tests, it was confirmed that the PK-probe / Lig-RPA system has higher selectivity for target-based detection.

[0075] In addition, to further investigate selectivity, various types of bacterial genomes were used to examine whether cross-reactivity occurred when using the PK-probe / Lig-RPA system. The genomes of nine pathogens, including the normal flora present in the upper respiratory tract or pathogens prone to respiratory infections, ( S. aureus, S. epidermidis, E. faecalis, E. feacium, E. coli, K. pneumoniae, E. cloacae, P. aeruginosa and A. baumannii A PK-probe / Lig-RPA reaction was performed using ). Since COVID-19 samples are typically collected from the mouth and nose, if a bacterial infection is present, the virus and some of these bacteria may be present, and any diagnostic system that reacts with bacteria diagnoses the patient as a false positive. Therefore, selective detection is essential for practical application.

[0076] As a result, nine bacteria did not react with the PK-probe / Lig-RPA system, and since no amplification occurred, the absorbance values ​​were almost negative and could not produce PPi (Figs. 5E and 6).

[0077] As indicated in the bar diagram, colorimetric detection coincided with changes in absorbance, and based on this selectivity data, it was confirmed that the PK-probe / Lig-RPA system of the present invention exhibits stronger selectivity than the PK-probe / RT-RPA system.

[0079] <2-6> 재조합 SARS-CoV-2의 검증

[0080] Since the spike SARS-CoV-2 RNA also contains human genomic RNA, it was verified as recombinant SARS-CoV-2.

[0081] Specifically, inactive recombinant SARS-Cov-2 whole genome material (fully extractable into actual viral protein coats) was used with nasopharyngeal samples using universal transport medium (T-SWAB TRANSPORT Universal Transport Medium, Noble Biosciences, Hwaseong, Korea). It was used for viral culture, antigen testing, and PCR (via extraction). If a detection system works with the sample, that system must also work with the actual sample. Focusing on practical applicability for point-of-care testing (POCT), 6% formamide was prepared with a 1X isothermal amplification buffer to extract RNA within 10 minutes at 25°C, and recombinant SARS-Cov-2 virus experiments demonstrated that RNA extraction is possible at all temperatures (25°C, 37°C, or 65°C) when using the aforementioned extraction buffer. Since the PK-probe / Lig-RPA system was confirmed to function well in simulated recombinant SARS-Cov-2 virus experiments, it was confirmed that point-of-care testing is possible with actual samples as well (Fig. 7).

Claims

Claim 1 RNA (RiboNucleic Acid) extraction reagent containing formamide; an enzyme for ligating cDNA (complementary Deoxyribo Nucleic Acid) containing a target RNA virus sequence from the extracted RNA; and RPA (Recombinase Polymerase Amplification) for amplifying the cDNA containing the target RNA virus sequence; A composition for detecting a target RNA virus for point-of-care testing (POCT), comprising a PK-probe (pyrophosphate-sensing probe) of the following Chemical Formula 1, wherein the ligation is characterized by synthesizing the cDNA by multiple ligation without reverse transcription (RT) by splintR ligase only when seven ligation templates (adjacent templates include monophosphate modifications at the 5′ ends) designed complementarily to the target RNA virus sequence and hybridized adjacently on the target sequence to form six junctions are all completely matched to the target sequence: [Chemical Formula 1] . Claim 2 A kit for detecting target RNA viruses comprising the composition of claim 1 above. Claim 3 A step of extracting RNA (Ribo Nucleic Acid) from a biological sample using formamide; a step of synthesizing cDNA (complementary Deoxyribo Nucleic Acid) containing a target RNA virus sequence from the extracted RNA using ligation; and a step of amplifying the cDNA using RPA (Recombinase Polymerase Amplification). A method for providing information regarding the detection of a target RNA virus for point-of-care testing (POCT), comprising the steps of: adding a PK-probe (pyrophosphate-sensing probe) to a sample containing the cDNA; and diagnosing that the sample containing the cDNA contains a target RNA virus sequence when the color of the sample changes from pink to colorless; wherein the ligation is characterized by synthesizing the cDNA by performing multiple ligation without reverse transcription (RT) by splintR ligase only when seven ligation templates (adjacent templates containing monophosphate modifications at the 5′ end) designed complementarily to the target RNA virus sequence and hybridized adjacently on the target sequence to form six junctions are all completely matched to the target sequence. Claim 4 A method for providing information regarding the detection of a target RNA virus, wherein, in claim 3, the biological sample is any one sample selected from the group consisting of blood, sputum, bacteria, semen, cell tissue, saliva, hair, and urine. Claim 5 A method for providing information regarding the detection of a target RNA virus, wherein, in claim 3, the ligation is performed by an enzyme that binds nucleic acid molecules. Claim 6 A method for providing information regarding the detection of a target RNA virus according to claim 3, wherein the PK-probe is a compound of the following Chemical Formula 1: [Chemical Formula 1] .

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