DETECTION METHOD USING PAPER CHIP CAPABLE OF MULTI-NUCLEIC ACID COLORIMETRIC DETECTION WITH ONE-STEP

Abstract

The present disclosure relates to a structure capable of simultaneously purifying and detecting a nucleic acid by directly applying a sample, and more particularly, to a structure capable of performing sample preparation, loop-mediated isothermal amplification, detection and analysis steps on a single chip by applying lab-on-paper technology, and capable of finally determining whether the disease or bacterial is infected by moving the sample in a lateral flow method and immediately being connected to genetic big data related to disease and bacterial infection.

Claims

1. A multi-nucleic acid detection structure, comprising: a sample pad for receiving a biological sample; a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer; a first connection pad disposed on the sample pad and connecting the sample pad and a reaction pad; an opener pad disposed on the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed below the first connection pad and the opener pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for loop-mediated isothermal amplification (LAMP), and in which loop-mediated isothermal amplification occurs; a blocking pad disposed on the reaction pad and configured to maintain a reaction temperature and block evaporation of the sample; a second connection pad disposed on the reaction pad and having gold nanoparticles fixed thereon; a detection pad disposed below the second connection pad and configured to obtain a target nucleic acid amplified from the loop-mediated isothermal amplification reactant bound to the gold nanoparticles; an absorbent pad disposed laterally of the detection pad and absorbing the remaining sample; and a heating pad disposed below the sample pad, the opener pad, the reaction pad, and the second connection pad.

2. The multi-nucleic acid detection structure of claim 1, wherein the sample pad and the first connection pad, the buffer pad and the opener pad; the reaction pad; the second connection pad; the detection pad; and the absorbent pad are sequentially disposed laterally at least in partial contact with each other.

3. The multi-nucleic acid detection structure of claim 1, wherein the detection pad includes a plurality of divided detection zones.

4. The multi-nucleic acid detection structure of claim 1, wherein the gold nanoparticles include streptavidin on their surface.

5. The multi-nucleic acid detection structure of claim 1, wherein the reaction pad includes a set of forward and backward primers, and one of the forward and backward primers is biotin-bound, and the other primer is labeled with one or more fluorescent markers selected from the group consisting of Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ, Iowa Black RQ, and IRDye.

6. The multi-nucleic acid detection structure of claim 1, wherein the sample applied to the sample pad moves laterally to the absorbent pad.

7. The multi-nucleic acid detection structure of claim 1, wherein the sample is mixed with a cell lysis buffer containing 5 mM to 80 mM of Tris-HCl (pH 8.0 to 9.0), 5 mM to 50 mM of potassium chloride, 1 mM to 30 mM of magnesium sulfate, 5 mM to 50 mM of ammonium sulfate, 0.01 mg/ml to 0.1 mg/ml of protease, and 0.01 w/w % to 0.2 w/w % of TritonX-100 or Tween20 as a surfactant.

8. A kit for diagnosing a disease, viral or bacterial infection, comprising the multi-nucleic acid detection structure of claim 1.

9. A method of providing information for diagnosing a disease, viral or bacterial infection, the method comprising: applying a biological sample to a sample pad of the multi-nucleic acid detection structure of claim 1, and amplifying a target nucleic acid; and detecting the nucleic acid amplification product on a detection pad.

10. The method of claim 9, further comprising adding an additional buffer dropwise to the buffer pad after applying the biological sample.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0076] FIG. 1 is an illustration of an overall structure of a multi-nucleic acid detection structure according to the present disclosure.

[0077] FIG. 2 is a schematic diagram of the principle of obtaining a target nucleic acid on the detection pad 160.

[0078] FIG. 3 is a side structural view of a portion of the multi-nucleic acid detection structure according to the present disclosure.

[0079] FIG. 4 is a perspective view of a portion of the multi-nucleic acid detection structure according to the present disclosure.

[0080] FIG. 5 is an enlarged structural diagram of the detection pad 160.

[0081] FIG. 6 is an illustration showing the appearance of the multi-nucleic acid detection structure according to the present disclosure surrounded by a case.

[0082] FIG. 7 is an illustration showing a result of detecting SARS-CoV-2 from a blood sample.

BEST MODE

[0083] Hereinafter, the present disclosure will be described in more detail with reference to the following examples, but these are only for explaining the present disclosure, and the scope of the present disclosure is not limited in any way by these examples.

PREPARATION EXAMPLE 1

Preparation of Lab-on-Paper Nucleic Acid Detection Structure

[0084] In order to prepare the Lab-on-paper nucleic acid detection structure according to the present disclosure, the sample pad 120 and the buffer pad 121 were made of 0.5 μm polysulfone, the first connection pad 131 or the opener pad 132 was made of 0.01 μm cellulose, the reaction pad 140 was made of 0.005 μm cellulose acetate, the second connection pad 150 was made of 0.05 μm cellulose, the detection pad 160 was made of 0.005 μm nitrocellulose, and the absorbent pad 170 was made of 0.5 μm glass fiber materials

[0085] The reaction pad 140 was prepared by overlapping a cellulose acetate membrane to make a pad, immersing the pad in a solution containing 45 mM of sucrose, 0.005 w/w % of TritonX-100, and 0.2 w/w % of glycerol, and then drying the pad. And, a well was formed in the pad with a fine drill, and a hydrogel layer including a primer set was formed on the bottom of the well. To form the hydrogel layer, first, 20% v/v of UV-photocrosslinkable poly(ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700), 40% v/v of poly(ethylene glycol) (PEG, Sigma-Aldrich, MW600) and 5% v/v of 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) as a photoinitiator, and 35% of buffer (PBS buffer, pH 7.5) were mixed based on the total volume of the hydrogel solution, and each primer set was mixed thereto to prepare a hydrogel solution. The poly(ethylene glycol) is preferably included in order to increase porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV for 1 minute (360 nm wavelength, 35 mJ/cm.sup.2) to form a hydrogel coating layer.

[0086] Next, dNTP (1.4 mM, dATP, dCTP, dGTP, and dTTP), powder containing loop-mediated isothermal amplification buffer (1×, 20 mM Tris-HCl, 10 mM (NH.sub.4)2SO.sub.4, 50 mM KCl, 2 mM MgSO.sub.4, and 0.1% Tween-20, pH 7.5), and Bst 3.0 DNA polymerase (320 U/ml) were applied to the surface of the reaction pad 140, and the reaction pad 140 was heated in an oven at about 38° C. for about 30 minutes and fixed.

[0087] The gold nanoparticles fixed to the second connection pad 150 were colloidal particles, and were prepared as follows. When a 0.1% HAuCl.sub.4 solution starts to boil while stirring and heating, 0.5% sodium citric acid solution was added to reduce the solution to make gold particles. The resulting gold particles were condensed by adding 1 mg each of streptavidin per 100 ml of the gold particle solution. The condensate was precipitated by centrifugation at 10,000 g, dissolved in physiological saline (PBS) containing 0.1% BSA, and stored so that an OD450 value became 10.

[0088] The second connection pad 150 was manufactured as follows. Specifically, several layers of cellulose membranes were prepared and cut, and soaked in a solution containing 0.4 M of Tris (pH 6.5), 0.2% of Tween-20, 1% of sodium caseinate, 0.1% of sodium azide, and 0.05% of Proclin 300. The prepared gold condensate was prepared by dialysis into a solution having the same composition as the solution. Then, the cellulose membrane was treated with the dialyzed gold condensate and dried to complete the second connection pad 150.

[0089] The detection pad 160 was designated by stamping a detection zone with polyethylene phthalate ink, stamped again with a solution containing an antibody capable of binding to fluorescent markers such as FAM, HEX, and Cy5 thereon, and then an N-hydroxysulfosuccinimide (NHS) solution was applied and reacted to immobilize the antibodies.

[0090] A portion of the second connection pad 150 in contact with the reaction pad was coated with a 5% low-melting-point agarose (Lonza, NuSieve GTG Agarose) solution.

[0091] Then, the components of each structure were arranged as shown in FIG. 3, wherein a copper plate to which a hot wire is connected to a heating pad 141 was disposed at the lower portion of the sample pad 120, the opener pad 132, the reaction pad 140, and the second connection pad 150, and a polyacrylic film was disposed on the reaction pad as a blocking pad 142.

EXPERIMENTAL EXAMPLE 1

Virus Detection Using Blood Samples

[0092] It was possible to easily measure whether it was infected with a virus such as SARS-CoV-2 by extracting nucleic acids from blood samples, amplifying extracted nucleic acids, and measuring fluorescence, by using the Lab-on-paper nucleic acid detection structure according to the present disclosure.

[0093] In order to detect SARS-CoV-2, a primer set capable of selectively binding to N protein gene specific for SARS-CoV-2 or Rdrp gene may be used. When such a primer set was used, either the forward primer or the backward primer of each set was, for example, in a form bound to FAM, HEX, or Cy5, and the other primer was bound to biotin. When a target nucleic acid capable of specifically binding to the primer set is present in the sample, biotin amplified in the reaction pad 140 and bound to the amplified nucleic acid while passing through the second connection pad 150 is specifically bound to streptavidin of the gold nanoparticles. The amplified nucleic acid bound to the gold nanoparticles by the lateral flow moved to the detection pad 160, and the detector bound to the other side of the amplified nucleic acid binds to a receptor capable of specifically binding to FAM, HEX, or Cy5 fixed to the detection pad 160 to change the color of the detection area of the detection pad to pink.

[0094] The principle of binding of the target nucleic acid to the detection pad is schematically shown in FIG. 2.

[0095] After an additional detection zone on the detection pad in order to increase reliability was formed, a primer set that selectively binds to a gene specific for the virus with high potential for cross-detection with SARS-CoV-2 was introduced and detected together as a negative control.

EXPERIMENTAL EXAMPLE 2

Nucleic Acid Extraction and Amplification Using Blood Samples

[0096] (1) Preparation of Test Samples and Paper Chips

[0097] It was confirmed whether nucleic acids could be extracted from blood samples using the Lab-on-paper nucleic acid detection structure according to the present disclosure and the extracted nucleic acids could be amplified to exhibit fluorescence.

[0098] First, human blood as whole blood was purchased from Innovative Research (IWB1K2E10ML, USA) and prepared, and 18S rRNA primer as a positive control was purchased from Tocris (#7325, USA). It was confirmed through the data sheet that the whole blood used was not infected with any virus or bacteria. One test sample was prepared by mixing 1 μl of 0.1 pg/μl SARS-CoV-2 positive control from siTOOLs Biotech with 100 μl of human blood.

[0099] A primer set for detection of SARS-CoV-2 mixed in blood is shown in Table 1 below.

TABLE-US-00001 TABLE 1 Primer Gene type Sequence N gene F3 5′-ACCGAAGAGCTACCAGACGA-3′ of  B3 5′-CTGCGTAGAAGCCTTTTGGC-3′ SARS- FIP 5′-TCCAGCTTCTGGCCCAGTTCCTTTTTATTCGTGG CoV-2 TGGTGACGGTAA-3′ BIP 5′-TATGGGTTGCAACTGAGGGAGCCTTTTTTCATTG TTAGCAGGATTGCGGG-3′ FL 5′-ACCATCTTGGACTGAGATCTTTC-3′ BL 5′-TACACCAAAAGATCACATTGGCA-3′) ※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer

[0100] In each of the primer sets, a detector was bound to the 5′ end of the F3 primer, and biotin was bound to the 5′ end of the B3 primer. FAM for Human 18s RNA (A), which is a positive control, and Cy5 for N gene (B) were introduced as a detector.

[0101] (2) Nucleic Acid Detection from Samples

[0102] 50 μl of each of the test sample and the negative control sample not mixed with SARS-CoV-2 was taken, 50 μl of lysis buffer (20 mM Tris.HCl (pH 8.8), 15 mM MgSO.sub.4, 15 mM KCl, 15 mM (NH.sub.4)2SO.sub.4, 0.1 w/w % Tween20, 0.05 mg/ml protease (Protenase K)) was added thereto, the resultant was lightly tapped, and then incubated at room temperature for about 5 minutes. Then, the resultant was slowly added dropwise within 5 minutes to the sample pad 120 of the nucleic acid detection structure manufactured in Preparation Example 1 while the heating pad 141 below the sample pad 120 was operated at 60° C., 250 μl of the addition buffer (20 mM Tris-HCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 50 mM KCl, 2 mM MgSO.sub.4, 0.1% Tween® 20, pH 8.8) was slowly added dropwise to the sample pad for about 2 minutes, and then the heating pad below the reaction pad was heated to 60° C. to react for 30 minutes. Thereafter, the heating pad below the second connection pad 150 and the heating pad below the opener pad 132 were heated to 65° C., 250 μl of the addition buffer was slowly added dropwise to the buffer pad for 2 minutes, and the color change of the detection zone displayed on the detection pad was observed.

[0103] As a result, from FIG. 7 it was confirmed that the structure worked normally by confirming a positive control (A) band in both the test sample and the negative control. Also, it is was confirmed that SARS-CoV-2 (B) was detected in the test sample, whereas only the positive control (A) was observed in the negative control sample, so that the system according to the present disclosure may be used to diagnose whether a number of diseases, viral or bacterial infections are present.