KR20230131324A - Method of providing information for Sarcoptes scabiei diagnosis using nucleic acid-based lateral flow analysis - Google Patents

Abstract

The present invention relates to a method of providing information for diagnosis and treatment monitoring of sarcoptes sacbiei infection using nucleic acid-based lateral flow analysis. More specifically, the present invention can provide the method of providing information for diagnosis and treatment monitoring of scabies mite infection that the results can be confirmed without additional probes or expensive equipment after performing PCR to detect the sarcoptes sacbiei genome.

Description

Method of providing information for Sarcoptes scabiei diagnosis using nucleic acid-based lateral flow analysis}

The present invention, using nucleic acid-based lateral flow analysis, relates to a method of providing information for diagnosis and treatment monitoring of scabies mite infection. More specifically, after performing PCR to detect the scabies mite genome, additional probes and expensive It can provide an information-providing method for diagnosis and treatment monitoring of scabies mite infection where results can be confirmed without any equipment.

Scabies, which causes symptoms such as itching at night, is a highly contagious infectious disease caused by skin parasites of the scabies mite (Sarcoptes scabiei), with an incubation period of about 4 to 6 weeks. Scabies is generally a widespread disease in low- and middle-income countries, but its incidence is increasing in elderly care facilities in high-income countries. In Korea, the incidence of scabies has decreased significantly since the late 1980s, but recently, the incidence of scabies has been increasing in hospitals and nursing homes. On the other hand, scabies may present unusual clinical features in immunocompromised patients, infants or the elderly, and in certain cases, including crystalline scabies, so if most doctors rely on clinical symptoms when diagnosing scabies, they should consider scabies as another skin disease. It may be difficult to distinguish between Additionally, because scabies is highly contagious, delayed diagnosis can result in the spread to family members, and in the case of hospitals, scabies can spread to other patients, caregivers, and medical staff, which can lead to temporary hospital closures.

Because scabies mites are very small (approximately 200㎛ to 450㎛ in size), they cannot be observed with the naked eye. To test for scabies mite infection, the patient's skin is generally scraped with mineral oil, transferred to a slide glass, and then examined for mites under a microscope. Alternatively, it is performed by identifying eggs. However, since the reliability of the test is very low at 0.46 (95% confidence interval 0.31 to 0.62), there is a problem that scabies mite infection cannot be ruled out even if the microscopic examination is diagnosed as negative (Arch Dermatol 2011; 147:468-73 ).

Dermoscopy (DS) has generally been used as a useful tool for the differential diagnosis of benign pigmented lesions such as naevi, seborrhoeic keratosis, or melanoma, and has been used in the diagnosis of scabies. It is known to be effective and convenient to use. Dermoscopy-guided skin scraping with microscopic examination (DSGSS-ME) can be used not only to diagnose scabies but also to evaluate treatment efficacy. However, the method using dermoscopy has the problem of requiring long training. Meanwhile, several existing PCR methods have been introduced as alternative diagnostic tools, but they require more time, cost, and effort because they require gel preparation, loading of PCR products, and electrophoresis.

Meanwhile, lateral flow assay (LFA) is widely used for rapid detection of specific antigens or antibodies, but it is mainly based on protein detection and has a problem with low sensitivity and the risk of false negative results. The development of a low-cost scabies mite detection method is urgently needed.

Korean Patent Publication No. 10-2010-0035644 Korean Patent Publication No. 10-2007-0074626

Therefore, the present invention is intended to solve various shortcomings and problems arising in the prior art in consideration of the above-mentioned situation, and provides diagnosis and treatment monitoring of scabies mite infection, which can diagnose scabies mite infection and monitor after treatment at the same time. The purpose is to provide a method of providing information for.

In addition, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

In order to achieve the above object, according to one embodiment of the present invention, a method of providing information for diagnosing scabies mites includes obtaining a labeled PCR product from a biological sample isolated from an individual; Obtaining a conjugate solution by adding detection particles to the PCR product; and treating the conjugate solution on a support, wherein the PCR product includes a first label and a second label, and the detection particle includes a third label, and the first label includes a third label. capable of specifically binding to, and the support includes a first detection unit capable of specifically binding to the second labeling unit; and a second detection unit capable of specifically binding to the third label. A method of providing information is provided, including a.

Additionally, in the present invention, the first label may be fluorescein isothiocyanate (FITC).

Additionally, in the present invention, the second label may be biotin or a derivative thereof.

Additionally, in the present invention, the first detection unit may include one or more selected from the group consisting of avidin, streptavidin, neutravidin, and captavidin.

Additionally, in the present invention, the PCR product may be obtained by performing 32 to 34 PCR cycles.

Additionally, in the present invention, the PCR cycle includes a denaturation process performed at 92°C to 97°C for 30 to 90 seconds; Annealing process performed at 63°C to 66°C for 30 to 90 seconds; and an extension process performed at 70°C to 74°C for 30 to 90 seconds.

Additionally, in the present invention, the PCR product may be for amplifying the cox1 gene.

Additionally, in the present invention, the PCR product may be obtained using the primer of SEQ ID NO: 6 and the primer of SEQ ID NO: 7.

Additionally, in the present invention, when the first detection unit does not react and the second detection unit reacts, it is determined that the scabies mite is infected, and when both the first detection unit and the second detection unit react, it is determined that the scabies mite is infected. It may be.

Additionally, in the present invention, the cox1 gene may be derived from the human scabies mite (Sarcoptes scabiei var hominis).

According to another embodiment of the present invention, a first primer labeled with a first label; a second primer labeled with a second label; A detection particle comprising a third label and capable of specifically binding to the first label; and a support including a first detection unit and a second detection unit, wherein the support includes a first detection unit capable of specifically binding to the second labeling unit; And it is possible to provide a composition for diagnosing scabies mites, including a second detection unit capable of specifically binding to the third labeling unit.

Additionally, in the present invention, the first labeling moiety may be fluorescein isothiocyanate (FITC).

Additionally, in the present invention, the second label may be biotin or a derivative thereof.

Additionally, in the present invention, the first primer and the second primer may be used to amplify the cox1 gene.

Additionally, in the present invention, the first primer may include the base sequence of SEQ ID NO: 6, and the second primer may include the base sequence of SEQ ID NO: 7.

According to another embodiment of the present invention, a kit for diagnosing scabies mites containing the composition of the present invention can be provided.

According to another embodiment of the present invention, a diagnostic device for diagnosing scabies mites comprising the composition of the present invention can be provided.

The scabies mite diagnosis method, diagnostic composition, diagnostic kit, and diagnostic device according to the present invention can be used for diagnosis by omitting the process of making a gel in conventional PCR, loading the PCR product, performing electrophoresis, and visualizing the results under UV light. The time required may be reduced. Additionally, costs for diagnostic thermal cyclers, electrophoresis chambers, gel makers, power supplies, UV illuminators and camera systems can be reduced. Additionally, compared to real-time PCR, additional probe preparation and expensive equipment are unnecessary.

If the cost of each diagnostic method is estimated based on the exchange rate of 1,112 won per dollar (excluding the cost of plastic consumables), the cost of conventional PCR is estimated to be approximately $23,843, and the cost of real-time PCR is estimated to be $34,935. Compared to the estimate, the cost required to perform NALFA of the present invention is estimated at $23,843, which can be expressed as 1 (NALFA): 2.46 (conventional PCR): 3.61 (nRT-qPCR). Additionally, the ratio of time required (minutes) was estimated to be 5 (NALFA):90 (conventional PCR):1 (nRT-qPCR).

Therefore, the present invention appears to be more useful in resource-poor settings such as nursing homes where there are no dermatologists skilled in DS and MS, even in developing or developed countries. Additionally, when NALFA is performed in combination with nRT-qPCR, a dermatologist with at least 10 years of experience diagnosing scabies is required. The same or more sensitive results can be obtained as DS and MS performed by .

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned may be clearly understood by those skilled in the art from the remaining description of the present specification.

Figure 1 shows a schematic diagram of a diagnostic kit according to an embodiment of the present invention.
Figure 2 shows the sensitivity of the kit according to an embodiment of the present invention for detecting scabies mites.

Hereinafter, the present invention will be described in detail. Prior to this, the terms or words used in this specification and patent claims should not be construed as limited to their usual or dictionary meanings, and the inventor must appropriately use the concept of the term to explain his or her invention in the best way. It must be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined clearly. Accordingly, the configuration described in the embodiments described in this specification is only one of the most preferred embodiments of the present invention and does not represent the entire technical idea of the present invention, so at the time of filing this application, various equivalents and It should be understood that variations may exist.

Meanwhile, each description and embodiment disclosed in this specification may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present application. Additionally, the scope of the present invention cannot be considered to be limited by the specific description described below.

As used herein, a “derivative” is a similar compound obtained by chemically changing part of a certain compound. Usually, it refers to a compound in which a hydrogen atom or a specific atomic group in a compound is replaced by another atom or atomic group.

Additionally, in this specification, when a component is referred to as being “connected,” “labeled,” or “connected” to another component, it may be directly connected or connected to the other component. It should be understood that other components may exist in the middle.

Additionally, the terms used in this specification are merely used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

The term "Sarcoptes scabiei" in the present invention is a parasite that lives outside the body in the skin of animals, etc., and is also called "scabies" and is known to cause scabies, an infectious skin disease. The human scabies mites (Sarcoptes scabiei var hominis), dog mites (Sarcoptes scabiei var canis), and pig mites (Sarcoptes scabiei var suis) are known.

The composition for diagnosing scabies mite infection of the present invention may be used to diagnose infection with human scabies mites (Sarcoptes scabiei var hominis), but is not limited thereto.

The term "scabies" in the present invention is a common infectious skin disease that affects a large number of people around the world and can occur regardless of race, age, or income. Because the symptoms of scabies are similar to eczema, atopic dermatitis, contact dermatitis, insect bites, urticaria, and impetigo, there is a possibility of misdiagnosis when diagnosing scabies. In addition, symptoms of scabies can only be felt about a month after the scabies mite infestation, so it is very important to get treatment at the same time as family members or people living with you can continue to be reinfected if you do not receive treatment just because they do not have symptoms.

In the present invention, the term "individual" refers to any living organism that is suspected of being infected with scabies mites or for which scabies mite infections are being treated, and specific examples include mammals including mice, monkeys, cows, pigs, mini-pigs, livestock, humans, etc. , farmed fish, etc., but is not limited thereto.

In the present invention, “scabies mite infection” and “scabies outbreak” can be used interchangeably.

In the present invention, the terms “sample” and “biological sample” refer to any material, biological fluid, tissue or cell derived from an individual that has or is suspected of having developed scabies mite infection, for example, whole blood. blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus. , nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cyst fluid. (cystic fluid), meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, May include bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract, or cerebrospinal fluid. For example, it may be a sample collected from the skin keratin of an individual.

In the present invention, the term "control group" may refer to a general individual who did not develop scabies mite infection or an individual who recovered after scabies mite infection occurred.

In the present invention, the terms “marker” or “biomarker” are those that can detect changes in a living organism and objectively measure the normal or pathological state of the living organism, the degree of response to a drug, etc. As an example, the marker of the present invention may be the cox1 gene of scabies mite mitochondria.

In the present invention, the term “diagnosis” refers to determining the susceptibility of a subject to a specific disease or condition, determining whether the subject currently has a specific disease or condition, and determining whether the subject currently has a specific disease or condition. Predicting or determining the prognosis of an affected subject, or therametrics (e.g., monitoring the condition of a subject to provide information about treatment efficacy). For the purpose of the present invention, the diagnosis is to determine the severity or prognosis of the above-mentioned scabies mite infection.

In the present invention, the term “detection” may be used to measure and compare the presence or absence of a biomarker to be detected, concentration in a biological sample, expression level, etc., and an increase in the level of the biomarker as a result of the detection encodes the biomarker. increasing the number of genes that are activated, increasing gene transcription (e.g., by placing genes under the control of a constitutive promoter), increasing translation of genes, knocking out competing genes, or combinations of these and/or other methods. There are a number of ways to do this, which can lead to an increase in concentration in the biological sample. On the other hand, a decrease in the level of a biomarker may be due to a decrease in the number of genes, a decrease in gene transcription, or the expression of competing genes, which can lead to a decrease in concentration in the biological sample. As an example, the level of copeptin in an individual is higher than that in the control group, meaning that the copeptin level contained in the sample derived from the subject is higher in concentration than the copeptin level contained in the sample derived from the control group. , it may mean that the amount is large.

In the present invention, the agent for detecting the biomarkers is not particularly limited, but includes, for example, antibodies that specifically bind to the biomarkers, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers. It may include one or more types selected from the group.

In the present invention, the “antigen” and the “antibody” refer to substances that specifically bind to each other and cause an antigen-antibody reaction. As an example, the antigen may be labeled or tagged on a DNA strand to be detected, such as a first label or a second label, or may be linked to a detection part, such as a third label, and the antibody may include a first label. Detection particle that recognizes the label as an antigen; a first detection unit that recognizes the second label as an antigen; and a second detection unit that recognizes the third label as an antigen.

The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibody can be easily produced using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art, which include injecting the protein antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. These polyclonal antibodies can be produced from any animal, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, etc. In addition, monoclonal antibodies can be prepared using the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), which is well known in the art, or phage antibody library technology (Clackson et al, Nature, 352 :624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). Antibodies prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. Additionally, antibodies of the invention include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

In the present invention, "Peptide Nucleic Acid (PNA)" refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced by Professors Nielsen, Egholm, Berg and Buchardt at the University of Copenhagen, Denmark in 1991. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases its binding force and stability to DNA or RNA and is used in molecular biology. , is used in diagnostic analysis and antisense therapy. PNA was described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". It is disclosed in detail in Science 254 (5037): 1497-1500.

In the present invention, the “aptamer” is an oligonucleic acid or peptide molecule, and general details of aptamers are described in Bock LC et al., Nature 355(6360):5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. “An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24): 142727 (1998)].

In the present invention, the agent for measuring the expression level of genes encoding the biomarkers may include one or more selected from the group consisting of primers and antisense nucleotides that specifically bind to the genes.

In the present invention, the “primer” is a fragment that recognizes the target gene sequence and includes forward and reverse primer pairs, but is preferably a primer pair that provides analysis results with specificity and sensitivity. High specificity can be granted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that the primer amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification. .

The nucleotide sequence used in the present invention is interpreted to include sequences showing substantial identity with the sequences listed in the sequence listing, considering mutations with biologically equivalent activity. The term 'substantial identity' means that when the sequence of the present invention and any other sequence are aligned to the maximum extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, the minimum It refers to a sequence that exhibits 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

In the present invention, the "antisense" refers to a sequence of nucleotide bases in which an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA:oligomer heteroduplex within the target sequence. and oligomers having an intersubunit backbone. Oligomers may have exact or approximate sequence complementarity to the target sequence.

According to one embodiment of the present invention,

A method of providing information for diagnosing scabies mites, comprising: obtaining a labeled PCR product from a biological sample isolated from an individual; Obtaining a conjugate solution by adding detection particles to the PCR product; and treating the conjugate solution on a support, wherein the PCR product includes a first label and a second label, and the detection particle includes a third label, and the first label includes a third label. capable of specifically binding to, and the support includes a first detection unit capable of specifically binding to the second labeling unit; and a second detection unit capable of specifically binding to the third label. A method of providing information is provided, including a.

In the present invention, the first label may be a fluorescent label such as FITC, rhodamine, or lanthanide phosphor, or an enzyme label of horseradish peroxidase, luciferase, or alkaline phosphatase. For example, the first label may be a fluorescent label such as a fluorescent label. It may be resin isothiocyanate (FITC). The FITC includes 5-FITC and 6-FITC, and is a pigment with green excitation and emission spectrum peak wavelengths of about 495 nm and 519 nm.

In the present invention, the second label may be biotin or a derivative thereof, and the first detection unit may be avidin, streptavidin, neutravidin, and captavidin. It may include one or more selected from the group consisting of, and they may utilize the binding activity between avidin and biotin.

In the present invention, the PCR product may be obtained by performing 32 to 34 PCR cycles. For example, the PCR product may be obtained by performing 33 PCR cycles.

In the present invention, the PCR cycle includes a denaturation process performed at 92°C to 97°C for 30 to 90 seconds; Annealing process performed at 63°C to 66°C for 30 to 90 seconds; and an extension process performed at 70°C to 74°C for 30 to 90 seconds.

In the present invention, the PCR product may be for amplifying the cox1 gene. The cox1 gene is a gene encoding subunit 1 of cytochrome c oxidiase, which is oxidative phosphorylation (OXPHOS) complex IV, is a gene present in mitochondrial DNA, and is a key enzyme in aerobic metabolism, It may be a base sequence including SEQ ID NOs: 8 to 14 in Table 1 below.

sequence number order 8 ATAATTTCTCACATTATTACCTATTCAAGTAATAAAAGAGAACCTTTTGGATCTTTAGGTATAATTTATGCTATGATTTCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGTAAAAATTTTTAGTTGATTATCTACAATATTAGGGGG 9 ATGATTTCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGT 10 ATTTCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGT 11 GCTATGATTTCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGT 12 TATGCTATGATTTCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGT 13 TCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGT 14 TTTGGATCTTTAGGTATAATTTATGCTATGATTTCTATTGCAACCTTAGGTTTTATTGTATGAGCTCATCATATATTTACTGTAGGTTTAGATGTTGATACCCGAGCTTATTTTACTTCAGCTACTATAATTATTGCTGTTCCTACAGGAGTAAAAATTTTTAGTTGATTATCTACAATATTAGGGGGAAAATTAG

The cox1 gene detected by the composition for diagnosing scabies mite infection of the present invention may be derived from the scabies mite (Sarcoptes scabiei), and specifically, the cox1 gene may be derived from the human scabies mite (Sarcoptes scabiei var hominis). , but is not limited to this.

In the present invention, the PCR product may be obtained using the primer of SEQ ID NO: 6 and the primer of SEQ ID NO: 7. As an example, when the forward primer of SEQ ID NO: 6 and the reverse primer of SEQ ID NO: 7 are subjected to the denaturation-binding-extension process 33 times according to the conditions of the present invention, the sensitivity for diagnosing scabies mites can be maximized.

In the present invention, when the first detection unit does not react and the second detection unit reacts, it is determined that the scabies mite is infected, and when both the first detection unit and the second detection unit react, it is determined that the scabies mite is infected. It may be.

In the present invention, the cox1 gene may be derived from the human scabies mite (Sarcoptes scabiei var hominis).

In the present invention, the remaining omitted descriptions may be interpreted similarly to the remaining descriptions in the specification.

According to another embodiment of the present invention,

A first primer labeled with a first label; a second primer labeled with a second label; A detection particle comprising a third label and capable of specifically binding to the first label; and a support including a first detection unit and a second detection unit, wherein the support includes a first detection unit capable of specifically binding to the second labeling unit; And it is possible to provide a composition for diagnosing scabies mites, including a second detection unit capable of specifically binding to the third labeling unit.

In the present invention, the first label may be fluorescein isothiocyanate (FITC).

In the present invention, the second label may be biotin or a derivative thereof.

In the present invention, the first primer and the second primer may be used to amplify the cox1 gene.

In the present invention, the first primer may include the base sequence of SEQ ID NO: 6, and the second primer may include the base sequence of SEQ ID NO: 7.

In the present invention, the remaining omitted descriptions may be interpreted similarly to the remaining descriptions in the specification.

According to another embodiment of the present invention,

A kit for diagnosing scabies mites containing the composition of the present invention can be provided.

In the present invention, the term “kit” refers to a tool that can evaluate the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. It includes not only direct labeling of a detectable substance related to a probe or antibody by reaction with a substrate, but also indirect labeling in which a label that develops color through reactivity with another directly labeled reagent is conjugated. It may include a chromogenic substrate solution, a washing solution, and other solutions that will undergo a color reaction with the label, and may be prepared including reagent components to be used. In the present invention, the kit may be a kit containing the essential elements required to perform RT-PCR, including a test tube, reaction buffer, deoxynucleotides (dNTPs), and Taq-polymerization, in addition to each primer pair specific for the marker gene. It may contain enzymes, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. Additionally, the kit may be a kit for detecting prognosis prediction genes that contain essential elements required to perform a DNA chip. The DNA chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, as long as it is known in the art.

The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, in the present invention, the kit may further include essential elements required to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains a pair of primers specific for the gene encoding the marker protein. Primers are nucleotides having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include primers specific to the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffer (pH and magnesium concentration vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, and the RNase inhibitor DEPC. -Can include DEPC-water, sterilized water, etc.

Additionally, the kit of the present invention may include essential elements required to perform DNA chip processing. A DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent label probe. The substrate may also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.

The kit of the present invention may further include a fixture, and the fixture includes a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, and a glass A slide glass, etc. may be used, but is not limited thereto.

In addition, the labeling agent for the secondary antibody in the kit of the present invention is preferably a conventional coloring agent that produces a color reaction, and includes HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, and FITC ( Labels such as fluorescein and dye, such as poly L-lysine-fluorecein isothiocyanate) and RITC (rhodamine-B-isothiocyanate), may be used, but are not limited thereto. .

In addition, the chromogenic substrate for inducing color development in the kit of the present invention is preferably used depending on the label that produces a color reaction, such as TMB (3,3',5,5'-tetramethyl bezidine), ABTS [ 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. can be used. At this time, it is more preferable that the chromogenic substrate is provided dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a marker for the secondary antibody conjugate to produce a chromogenic deposit, and the presence or absence of the marker proteins is detected by visually checking the degree of deposition of the chromogenic deposit.

In the kit of the present invention, the washing solution preferably contains a phosphate buffer solution, NaCl, and Tween 20, and more preferably a buffer solution (PBST) consisting of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20. After the antigen-antibody binding reaction, the washing solution reacts with the secondary antibody to the antigen-antibody conjugate, then adds an appropriate amount to the fixative and washes 3 to 6 times. The reaction stopping solution may preferably be a sulfuric acid solution (H ₂ SO ₄ ).

According to another embodiment of the present invention,

It is possible to provide a diagnostic device for diagnosing scabies mites, including the composition of the present invention.

In the present invention, the remaining omitted descriptions may be interpreted similarly to the remaining descriptions in the specification.

Hereinafter, the present invention will be described in further detail through examples, but it is obvious that the present invention is not limited by the following examples.

Example 1: Subject recruitment and DNA sample extraction

Subject recruitment and sample extraction were conducted at Kangdong Sacred Heart Hospital, Hallym University, Seoul, South Korea from September 2019 to May 2021, and were approved by the Kangdong Sacred Heart Hospital Institutional Review Board (IRB 2021-04-001-002), and all participants Written consent was obtained from the pediatric patients and their guardians.

A total of 111 patients with suspected scabies infection were enrolled in this study. The average age was 51.88±25.36 years (median age 58.0 years, range 0.5~93 years), and the male to female ratio was 1:2.08. Of the 111 patients, 37 had DS, 40 had MS, 42 had NALFA, and 44 were positive for nRT-qPCR. All 40 microscopically confirmed and 4 microscopically negative patients with positive nRT-qPCR cured within 2 to 4 weeks after treatment with 5% permethrin cream. Sixty-seven patients who were negative in all tests did not develop symptoms suggestive of scabies infection during the 2-4 week follow-up period. The sample included (i) healthcare workers working there, (ii) their family members, (iii) patients referred by non-specialists or dermatologists who are not skilled in dermoscopy or microscopy, and (iv) people who had never been treated for scabies. It has been done.

The examination was performed through history taking, clinical examination, dermoscopy and microscopic examination by a senior dermatologist who has been working in this field since 2008. All patients were examined by dermoscopy (DS) at 10X magnification (Dermlite III; 3Gen Inc., San Juan Capistrano, CA, USA). Next, skin samples were scraped and observed under a microscope (MS) and the results were recorded. Scraping specimens from glass slides were transferred to 1.7 ml microcentrifuge tubes and stored at -80°C as soon as possible for further PCR analysis. “Jetliner with its trail” was defined as a positive dermoscopic finding, and mites or eggs observed under MS were defined as positive. To extract total DNA from each sample, the Silica-membrane Wizprep™gDNA Mini Kit (Wizbiosolutions, Seongnam-si, South Korea) was used and the manufacturer's instructions were followed.

Example 2: Primer, probe design and PCR performance

Primer sequences used in the examples and experimental examples of this specification are listed in Table 2 below, and the PCR method is listed in Table 3 below.

PCR type primer Sequence (5' to 3') sequence number Nest Nest-scab-F2 TTT TTC GGA CAC CCA GAA GT One Nest Nest-scab-R1 CCA ATT GCC CAA TAT ATA GAA GGA 2 Real time q-scab-F GCT ATG ATT TCT ATT GCA ACC T 3 Real time q-scab-R ACT CCT GTA GGA ACA GCA ATA 4 Real time probe 56-FAM /AC TGT AGG T/ZEN/T TAG ATG TTG ATA CCC GAG C/3IABkFQ 5 NALFA FITC-q-scab-F FITC- GCT ATG ATT TCT ATT GCA ACC T 6 NALFA Biotin-q-scab-R Biotin- ACT CCT GTA GGA ACA GCA ATA 7 Conventional q-scab-F TTT TTC GGA CAC CCA GAA GT One Conventional q-scab-R CCA ATT GCC CAA TAT ATA GAA GGA 2

PCR type PCR order and
Program sequence number PCR program by primer Nest 1-1 10 minutes at 95°C; 35 cycles of 95°C for 1 min, 52.9°C for 1 min, and 72°C for 1 min; Final extension at 72℃ for 10 minutes Real time 2-1 10 minutes at 95°C; 40 cycles of 95°C 1 min, 58.3°C 1 min, 72°C 1 min NALFA 2-2 10 minutes at 95°C; 33 cycles of 95°C 1 min, 64.4°C 1 min, 72°C 1 min; Final extension at 72℃ for 10 minutes Conventional 2-3 10 minutes at 95°C; 40 cycles of 95°C for 1 min, 60.2°C for 1 min, and 72°C for 1 min; Final extension at 72℃ for 10 minutes

In the first PCR step, the reaction mixture contained 1ul of sample DNA, 1ul of 10pM nest-scab-F2 and nest-scab-R1 primers, 7ul of distilled water (DW), and 10ul of PCR master mix (H-star Taq PCR Master). Mix 1, BioFact, Daejeon, South Korea), and PCR reactions were performed according to Program 1.

The following secondary PCR steps were performed in three parts. First, for RT-qPCR, each PCR mixture contains 1ul of primary PCR product, 10pM q-scab-F and q-scab-R primers, 0.5ul of 10pM probe, 6.5ul of distilled water (DW), and 10ul of PCR master mix ( Multi-Star Real-time PCR Master Mix, BioFact) was prepared and RT-qPCR process was performed according to program 2-1. Specifically, a 130bp fragment of the S. scabiei cox1 gene (GenBank accession number: MT133635) obtained by q-scab-F and R primers was performed with serially diluted clone DNA observed under a microscope. Confirmed patients were inserted. The cutoff value was defined as Ct ≤ 30.

Second, for NALFA, 1 μl of the primary PCR product, 1 μl each of the mixture prepared by 10 pM fluorescein isothiocyanate (FITC)-tagged q-scab-F and Biotin-tagged q-scab-R (biomers.net, Ulm, Germany) ), PCR was performed according to program 2-2 using 7ul of DW and 10ul of PCR master mix (H-star Taq PCR Master Mix 1, BioFact), and the results were visualized by designing a kit as shown in Figure 1. . Specifically, 100ul of detection buffer was added to the PCR tube and a membrane strip was inserted. As a result, control and test bands were observed on the strip after 2 minutes.

Finally, to confirm the scabies mite DNA of some samples by sequencing, the mixture contained 1ul of primary PCR product, 1ul each of 10pM q-scab-F and q-scab-R primers, 7ul of DW and 10ul of PCR master and mix (H- star Taq PCR Master Mix 1, BioFact) was prepared and performed according to the program in 2-3. Washed DNA was directly sequenced in both directions using the same forward and reverse primers used for DNA amplification by an ABI PRISM 3730xl DNA analyzer (Applied Biosystems).

Example 3: Primer, probe design and PCR performance

To evaluate the test results, McNemar's test and Fisher's exact test were used, and the results of DS, MS, and NALFA were compared with the results of nRT-qPCR and are shown in Tables 4 and 5 below. Statistical analysis was performed using SPSS software (version 26.0, IBM, Armonk, NY, U.S.A.), and a P-value less than 0.05 was considered statistically significant.

Diagnosis results nRT-qPCR+ nRT-qPCR- DS+ 37 0 DS- 7 67 MS+ 40 0 MS- 4 67 NALFA+ 42 0 NALFA- 2 67

As a result, as shown in Table 4 above, none of the DS-, MS-, or NALFA-positive patients were negative for nRT-qPCR. Then, the specificity and positive predictive value of each test were both 100%. The results of nRT-qPCR were positive in 7 of 74 DS-negative patients, 4 of 71 MS-negative patients, and 2 of 69 NALFA-negative patients.

Diagnosis method Sensitivity (%)
(95% CI ^b ) Specificity (%)
(95% CI) Positive predictive value (%) Speech predictability (%)
(95% CI) accuracy (%)
(95% CI) P-value DS 84.1
(70.0 - 93.4) 100
(94.6 - 100) 100 90.5
(82.9 - 95.0) 93.7
(87.4 - 97.4) 0.016 M.S. 90.9
(78.3 - 97.5) 100
(94.6 - 100) 100 94.4
(86.8 - 97.7) 96.4
(91.0 - 99.0) 0.125 NALFA 95.5
(84.5 - 99.4) 100
(94.6 - 100) 100 97.1
(89.6 - 99.2) 98.2
(93.6 - 99.8) 0.500

Additionally, as shown in Table 5 above, the sensitivity and negative predictive value of DS were 84.1% (95% confidence interval (CI), 70.0 - 93.4) and 90.5% (95% CI, 82.9 - 95.0), respectively, while that of MS 90.9% (95% CI, 78.3 to 97.5) and 94.4% (95% CI, 86.8 to 97.7), and NALFA was 95.5% (95% CI, 84.5 to 99.4) and 97.1% (95% CI, 89.6 to 99.2). . McNemar's test comparing the results of DS with those of nRT-qPCR was statistically significant with P = 0.016 < 0.05. When the results of MS and NALFA were compared with those of nRT-qPCR, the P values were 0.125 and 0.500, respectively, which were not statistically significant.

Example 4: Sensitivity measurement of NALFA

To evaluate the detection limit of NALFA, PCR and NALFA were performed on samples of serially diluted cloned DNA from 1 ng to 10 ^-7 ng. As a result, as shown in Figure 2, it was confirmed that a transparent band was observed up to 10 ^-2 ng and a very weak band was observed at 10 ^-3 ng.

This is the first study to develop a nucleic acid-based lateral flow assay (NALFA) for scabies diagnosis and compare it with currently available scabies testing methods such as DS, MS, and nRT-qPCR. When the results of NALFA were compared with the results of nRT-qPCR, there was no significant difference with P = 0.500 > 0.05. Therefore, NALFA can be used for scabies diagnosis instead of nRT-qPCR.

In the above example, the cut-off value of nRT-qPCR was defined as Ct ≤ 30 to minimize false positives. Therefore, the difference between MS and PCR results was relatively small compared to previous studies. In a previous study, when the sensitivity of traditional nested PCR was 100%, the sensitivity of microscopy was 75.68%, a difference of 24.32%. When the sensitivity of nested real-time PCR was 100%, the sensitivity of microscopy was 85.90%, a difference of 14.10%, but in the present invention, when the sensitivity of nested real-time PCR was 100%, the sensitivity of microscopy was 90.91%. This showed a difference of 9.09%.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as single may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION HALLYM UNIVERSITY <120> Method of providing information for Sarcoptes scabiei diagnosis using nucleic acid-based lateral flow analysis <130>P214470 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 1 tttttcggac acccagaagt 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 2 ccaattgccc aatataga agga 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 gctatgattt ctattgcaac ct 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 actcctgtag gaacagcaat a 21 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 5 actgtaggtt tagatgttga tacccgagc 29 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 6 gctatgattt ctattgcaac ct 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 7 actcctgtag gaacagcaat a 21 <210> 8 <211> 233 <212> DNA <213> Sarcoptes Scabiei <400> 8 ataatttctc acattattac ctattcaagt aataaaagag aaccttttgg atctttaggt 60 ataatttatg ctatgatttc tattgcaacc ttaggtttta ttgtatgagc tcatcatata 120 tttactgtag gtttagatgt tgatacccga gcttatttta cttcagctac tataattatt 180 gctgttccta caggagtaaa aatttttagt tgattatcta caatattagg ggg 233 <210> 9 <211> 125 <212> DNA <213> Sarcoptes Scabiei <400> 9 atgatttcta ttgcaacctt aggttttatt gtatgagctc atcatatatt tactgtaggt 60 ttagatgttg atacccgagc ttattttact tcagctacta taattattgc tgttcctaca 120 ggagt 125 <210> 10 <211> 122 <212> DNA <213> Sarcoptes Scabiei <400> 10 atttctattg caaccttagg ttttattgta tgagctcatc atatatttac tgtaggttta 60 gatgttgata cccgagctta ttttacttca gctactataa ttattgctgt tcctacagga 120 gt 122 <210> 11 <211> 128 <212> DNA <213> Sarcoptes Scabiei <400> 11 gctatgattt ctattgcaac cttaggtttt attgtatgag ctcatcatat atttactgta 60 ggtttagatg ttgatacccg agcttatttt acttcagcta ctataattat tgctgttcct 120 acaggagt 128 <210> 12 <211> 131 <212> DNA <213> Sarcoptes Scabiei <400> 12 tatgctatga tttctattgc aaccttaggt tttattgtat gagctcatca tatatttact 60 gtaggtttag atgttgatac ccgagcttat tttacttcag ctactataat tattgctgtt 120 cctacaggag t 131 <210> 13 <211> 119 <212> DNA <213> Sarcoptes Scabiei <400> 13 tctattgcaa ccttaggttt tattgtatga gctcatcata tatttactgt aggtttagat 60 gttgataccc gagcttattt tacttcagct actataatta ttgctgttcc tacaggagt 119 <210> 14 <211> 196 <212> DNA <213> Sarcoptes Scabiei <400> 14 tttggatctt taggtataat ttatgctatg atttctattg caaccttagg ttttatgta 60 tgagctcatc atatatttac tgtaggttta gatgttgata cccgagctta ttttacttca 120 gctactataa ttattgctgt tcctacagga gtaaaaattt ttagttgatt atctacaata 180 ttaggggggaa aattag 196

Claims (17)

As a method of providing information for diagnosing scabies mites,
Obtaining a labeled PCR product from a biological sample isolated from an individual;
Obtaining a conjugate solution by adding detection particles to the PCR product; and
It includes the step of treating the conjugate solution on a support,
The PCR product includes a first label and a second label,
The detection particle includes a third label and is capable of specifically binding to the first label,
The support includes a first detection unit capable of specifically binding to the second label; And a second detection unit capable of specifically binding to the third label,
How to provide information for diagnosing scabies mites.
According to paragraph 1,
A method of providing information, wherein the first label is fluorescein isothiocyanate (FITC).
According to paragraph 1,
A method of providing information, wherein the second label is biotin or a derivative thereof.
According to paragraph 3,
Wherein the first detection unit includes at least one selected from the group consisting of avidin, streptavidin, neutravidin, and captavidin.
According to paragraph 1,
A method of providing information, wherein the PCR product is obtained by performing 32 to 34 PCR cycles.
According to clause 5,
The PCR cycle is,
A denaturation process performed at 92°C to 97°C for 30 to 90 seconds;
Annealing process performed at 63°C to 66°C for 30 to 90 seconds; and
A method of providing information, comprising: an extension process performed at 70°C to 74°C for 30 to 90 seconds.
According to paragraph 1,
The method of providing information, wherein the PCR product is for amplifying the cox1 gene.
In clause 7,
The method of providing information, wherein the PCR product is obtained by the primer of SEQ ID NO: 6 and the primer of SEQ ID NO: 7.
In clause 7,
If the first detection unit does not react and the second detection unit reacts, it is determined that the scabies mite is infected,
When both the first detection unit and the second detection unit react, it is determined that the person is infected with scabies mites.
In clause 7,
A method of providing information, wherein the cox1 gene is derived from the human scabies mite (Sarcoptes scabiei var hominis).
A first primer labeled with a first label;
a second primer labeled with a second label;
A detection particle comprising a third label and capable of specifically binding to the first label; and
It includes; a support including a first detection unit and a second detection unit,
The support includes a first detection unit capable of specifically binding to the second label; A composition for diagnosing scabies mites comprising a second detection unit capable of specifically binding to the third label.
According to clause 11,
A composition wherein the first labeling moiety is fluorescein isothiocyanate (FITC).
According to clause 11,
A composition wherein the second label is biotin or a derivative thereof.
According to clause 11,
The composition wherein the first primer and the second primer are for amplifying the cox1 gene.
According to clause 14,
The first primer includes the base sequence of SEQ ID NO: 6,
The composition wherein the second primer includes the base sequence of SEQ ID NO: 7.
A kit for diagnosing scabies mites comprising the composition of any one of claims 11 to 15.
A diagnostic device for diagnosing scabies mites comprising the composition of any one of claims 11 to 15.