Composition for diagnosing schistosoma infection and use thereof

Abstract

The present invention relates to a composition comprising a primer set for detecting or diagnosing Schistosoma and use thereof. When the composition of the present invention is used, it is possible to provide a diagnostic kit and a method for providing information for diagnosis, which can accurately, rapidly and effectively diagnose Schistosoma haematobium and Schistosoma mansoni, which account for the majority of schistosomiasis prevalence, without separate equipment and trained manpower. In addition, the present invention can be easily adopted in resource-constrained countries where schistosomiasis is prevalent, thereby contributing to enhancing the capacity for the diagnosis and management of Schistosoma infection.

Description

Composition for diagnosing schistosome infection and use thereof

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2023-0093115, filed on July 18, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition comprising a primer set for detecting or diagnosing schistosome DNA and to a use thereof.

[0003] The present invention was completed with the support of the Ministry of Health and Welfare of the Republic of Korea under grants No. 800-20210577, No. 800-20220247, and No. 800-20230203.

[0004]

[0005] Schistosomiasis, one of the six major tropical diseases designated by the World Health Organization (WHO), is a global public health problem. More than 250 million people are infected in tropical and subtropical regions, including South America, Africa, and Asia, and approximately 800 million people are at risk of infection. Schistosomiasis causes more than 200,000 deaths annually in sub-Saharan Africa alone, making it the second leading cause of death after malaria. Schistosomiasis is a parasitic disease that carries a particularly high burden of disease, particularly among children.

[0006] There are six species of schistosomes that cause human infection, and among them, the number of patients with schistosomiasis caused by Schistosoma cystis, Schistosoma mansoni, and Schistosoma japanese is estimated to be approximately 150 million worldwide, accounting for the majority of the prevalence.

[0007] When the eggs of Schistosoma blood fluke are discharged from the body through the urine or feces of an infected person, they grow into the miradicium stage and then infect intermediate hosts, shellfish, where they develop into the cercariae stage. The free-swimming cercariae swim in the water and enter the body through the skin when the host, including humans, comes into contact with water. They parasitize in the liver or bladder veins and cause liver disease, dysentery symptoms, chronic bladder disease with hematuria, and bladder cancer.

[0008] Existing diagnostic methods for Schistosoma infection include direct microscopic observation of Schistosoma eggs after collecting stool and urine samples, immunoassays (ELISA, CCA), and PCR (Polymerase chain reaction). Microscopic examination methods require experience in ova examination and diagnosis, and there are difficulties in collecting and transporting urine and fecal samples for large-scale testing. Furthermore, fluctuations in egg shedding over time necessitate multiple tests for accurate results. ELISA and CCA are used as auxiliary methods, but these methods have the disadvantage of low sensitivity and accuracy due to variations in values ​​depending on the skill of the tester and a high false-positive rate. Molecular diagnosis using PCR has high sensitivity and specificity, but it requires expensive equipment and personnel, making it difficult to utilize in field laboratories in countries where Schistosoma is endemic due to limitations in laboratory capacity and cost.

[0009] Lysothermal amplification method (LAMP), discovered in 2000, is a genetic amplification method. Because the amplification reaction proceeds at a single temperature of 60-65°C, eliminating the need for expensive thermal cyclers, it boasts rapid amplification rates and high specificity, making it widely used in the diagnosis of various infectious diseases. However, molecular diagnostics such as PCR or LAMP typically require DNA extraction from specimens. This involves dissolving and precipitating various proteins and RNA present within parasite eggs or filtering them through a column to extract only the DNA. While this method can yield high-quality DNA, it requires equipment such as a centrifuge, takes more than two hours, and is expensive when using commercial kits, making it unsuitable for point-of-care diagnostics. Therefore, the development of a method to resolve these issues and secure DNA for use in LAMP is urgently needed.

[0010] Previously, LAMP, which utilized urine or feces from patients infected with Schistosoma haematobium, had low sensitivity, long reaction time, expensive equipment, and problems in the specimen collection stage, making it difficult to use as a simple on-site diagnostic test.

[0011] To achieve the World Health Organization (WHO) schistosome elimination target by 2030, the development of diagnostic methods that are more sensitive than existing methods and easier to implement in resource-limited low-income countries is essential. In particular, the development of simple diagnostic kits that utilize urine, which facilitates patient cooperation during specimen collection, and target gene extraction methods that are easy to handle in the field remains essential.

[0012]

[0013] [Prior Art Literature]

[0014] [Patent Document]

[0015] (Patent Document 1) KR 10-2010-0100501 (2010-10-14)

[0016]

[0017] The present inventors have made efforts to provide a method for detecting or diagnosing Schistosoma DNA that can be used in the field quickly while providing high sensitivity using easily collected specimens. As a result, they have completed the present invention by completing a method for easily obtaining Schistosoma DNA from a patient's urine specimen and an isothermal amplification primer set targeting a Schistosoma species-specific gene sequence.

[0018] Accordingly, an object of the present invention is to provide a composition comprising a primer set for detecting or diagnosing Schistosoma DNA and a use thereof.

[0019]

[0020] The present invention comprises a first primer set including a primer consisting of a base sequence of SEQ ID NO: 1, a primer consisting of a base sequence of SEQ ID NO: 2, a primer consisting of a base sequence of SEQ ID NO: 3, and a primer consisting of a base sequence of SEQ ID NO: 4;

[0021] A second primer set comprising a primer consisting of a base sequence of SEQ ID NO: 7, a primer consisting of a base sequence of SEQ ID NO: 8, a primer consisting of a base sequence of SEQ ID NO: 9, and a primer consisting of a base sequence of SEQ ID NO: 10;

[0022] A third primer set comprising a primer consisting of a base sequence of SEQ ID NO: 13, a primer consisting of a base sequence of SEQ ID NO: 14, a primer consisting of a base sequence of SEQ ID NO: 15, and a primer consisting of a base sequence of SEQ ID NO: 16;

[0023] A fourth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 19, a primer consisting of a base sequence of SEQ ID NO: 20, a primer consisting of a base sequence of SEQ ID NO: 21, and a primer consisting of a base sequence of SEQ ID NO: 22;

[0024] A fifth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 25, a primer consisting of a base sequence of SEQ ID NO: 26, a primer consisting of a base sequence of SEQ ID NO: 27, and a primer consisting of a base sequence of SEQ ID NO: 28; and

[0025] A sixth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 31, a primer consisting of a base sequence of SEQ ID NO: 32, a primer consisting of a base sequence of SEQ ID NO: 33, and a primer consisting of a base sequence of SEQ ID NO: 34;

[0026] A composition for diagnosing schistosomiasis infection is provided, comprising at least one primer set selected from the group consisting of:

[0027] According to a preferred embodiment of the present invention, the first primer set additionally includes a primer consisting of a base sequence of SEQ ID NO: 5 and / or a primer consisting of a base sequence of SEQ ID NO: 6;

[0028] The second primer set further comprises a primer comprising a base sequence of SEQ ID NO: 11 and / or a primer comprising a base sequence of SEQ ID NO: 12;

[0029] The third primer set further comprises a primer consisting of a base sequence of SEQ ID NO: 17 and / or a primer consisting of a base sequence of SEQ ID NO: 18;

[0030] The fourth primer set further comprises a primer comprising a base sequence of SEQ ID NO: 23 and / or a primer comprising a base sequence of SEQ ID NO: 24;

[0031] The fifth primer set further comprises a primer consisting of the base sequence of SEQ ID NO: 29 and / or a primer consisting of the base sequence of SEQ ID NO: 30; or

[0032] The above 6th primer set additionally includes a primer consisting of a base sequence of SEQ ID NO: 35 and / or a primer consisting of a base sequence of SEQ ID NO: 36;

[0033] It is to do.

[0034] According to a preferred embodiment of the present invention, the schistosome is at least one selected from the group consisting of Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum.

[0035] According to a preferred embodiment of the present invention, the first primer set, the second primer set or the third primer set detects a specific gene IGS (ribosomal intergenic spacer) of Schistosoma haematobium.

[0036] According to a preferred embodiment of the present invention, the fourth primer set detects the specific gene 16SrRNA (16s mitochondrial rRNA) of Schistosoma haematobium.

[0037] According to a preferred embodiment of the present invention, the fifth primer set detects a specific gene IGS (ribosomal intergenic spacer) of Schistosoma mansoni.

[0038] According to a preferred embodiment of the present invention, the sixth primer set detects the specific gene ITS (Internal transcribed spacer 1-5.8S-ITS2) of Schistosoma.

[0039] According to a preferred embodiment of the present invention, the primer set is a primer set used in loop-mediated isothermal amplification (LAMP).

[0040] According to a preferred embodiment of the present invention, the detection limit of the composition is plasmid 1 ~10 copies.

[0041] In addition, the present invention provides a kit for diagnosing schistosomiasis comprising the composition.

[0042] In addition, the present invention comprises the steps of: i) manufacturing a dipstick;

[0043] ii) a step of adsorbing the gene in the sample separated from the object onto the dipstick manufactured in step i);

[0044] iii) a step of releasing the adsorbed gene; and

[0045] iv) a step of reacting the above-described gene with the above-described composition;

[0046] Provides a method for providing information for diagnosing schistosome infection including .

[0047] According to a preferred embodiment of the present invention, the dipstick of step i) is made of filter paper.

[0048] According to a preferred embodiment of the present invention, the sample of step ii) is at least one selected from the group consisting of urine, feces, hair, sweat, saliva, body fluid, blood, cerebrospinal fluid, skin, cells, and tissues.

[0049] According to a preferred embodiment of the present invention, the reaction of step iv) is carried out at 60 to 67°C for 20 to 50 minutes.

[0050] According to a preferred embodiment of the present invention, the reaction of step iv) is carried out by a loop-mediated isothermal amplification (LAMP) method.

[0051]

[0052] Hereinafter, the present invention will be described in more detail.

[0053]

[0054] The present invention aims to improve the limitations of existing schistosome infection diagnosis methods and isothermal amplification methods, and has completed a method for securing DNA for specimens that can be used in LAMP without separate equipment, and an isothermal amplification method that enables rapid diagnosis at a single temperature through optimization of primer combinations, reactants, and reaction conditions. The present invention effectively diagnoses schistosome infection with high sensitivity, high specificity, and high precision (high repeatability, high reproducibility), thereby saving time and cost, and thus has the effect of being useful for strengthening national schistosome diagnosis and management capabilities in countries where schistosomes is endemic.

[0055]

[0056] Accordingly, the present invention provides a first primer set comprising a primer consisting of a base sequence of SEQ ID NO: 1, a primer consisting of a base sequence of SEQ ID NO: 2, a primer consisting of a base sequence of SEQ ID NO: 3, and a primer consisting of a base sequence of SEQ ID NO: 4;

[0057] A second primer set comprising a primer consisting of a base sequence of SEQ ID NO: 7, a primer consisting of a base sequence of SEQ ID NO: 8, a primer consisting of a base sequence of SEQ ID NO: 9, and a primer consisting of a base sequence of SEQ ID NO: 10;

[0058] A third primer set comprising a primer consisting of a base sequence of SEQ ID NO: 13, a primer consisting of a base sequence of SEQ ID NO: 14, a primer consisting of a base sequence of SEQ ID NO: 15, and a primer consisting of a base sequence of SEQ ID NO: 16;

[0059] A fourth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 19, a primer consisting of a base sequence of SEQ ID NO: 20, a primer consisting of a base sequence of SEQ ID NO: 21, and a primer consisting of a base sequence of SEQ ID NO: 22;

[0060] A fifth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 25, a primer consisting of a base sequence of SEQ ID NO: 26, a primer consisting of a base sequence of SEQ ID NO: 27, and a primer consisting of a base sequence of SEQ ID NO: 28; and

[0061] A sixth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 31, a primer consisting of a base sequence of SEQ ID NO: 32, a primer consisting of a base sequence of SEQ ID NO: 33, and a primer consisting of a base sequence of SEQ ID NO: 34;

[0062] A composition for diagnosing schistosomiasis infection comprising at least one primer set selected from the group consisting of can be provided.

[0063] According to a preferred embodiment of the present invention, the first primer set additionally includes a primer consisting of a base sequence of SEQ ID NO: 5 and / or a primer consisting of a base sequence of SEQ ID NO: 6;

[0064] The second primer set further comprises a primer comprising a base sequence of SEQ ID NO: 11 and / or a primer comprising a base sequence of SEQ ID NO: 12;

[0065] The third primer set further comprises a primer consisting of a base sequence of SEQ ID NO: 17 and / or a primer consisting of a base sequence of SEQ ID NO: 18;

[0066] The fourth primer set further comprises a primer comprising a base sequence of SEQ ID NO: 23 and / or a primer comprising a base sequence of SEQ ID NO: 24;

[0067] The fifth primer set further comprises a primer consisting of the base sequence of SEQ ID NO: 29 and / or a primer consisting of the base sequence of SEQ ID NO: 30; or

[0068] The above 6th primer set additionally includes a primer consisting of a base sequence of SEQ ID NO: 35 and / or a primer consisting of a base sequence of SEQ ID NO: 36;

[0069] It could be doing.

[0070] According to a preferred embodiment of the present invention, the schistosome may be at least one selected from the group consisting of Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum.

[0071] Schistosoma haematobium enters the skin of its host, including humans, and parasitizes in the vesical venous plexus, laying approximately 1,500 eggs per day. These eggs destroy the bladder mucosa, protrude into the bladder along with the blood, and are then expelled in the urine. Consequently, lesions primarily appear in the urogenital system, particularly the bladder wall. Symptoms include hematuria, but may also include frequent urination, incontinence, dysuria, and perineal pain. Eggs can damage the bladder mucosa, leading to hypertrophy and papillary growth within the bladder. Calcification of the eggs can lead to the formation of bladder stones. Urethral strictures and obstructions can also cause reflux of urine, leading to renal lesions and even renal failure. In some patients, bladder wall lesions can worsen, eventually leading to bladder cancer.

[0072] Schistosoma mansoni parasitizes the hepatic portal vein, and a single female lays about 300 eggs a day, which is relatively small. The number of eggs discharged into the intestinal cavity and the number of eggs deposited in the wall of the intestine or the tissue around blood vessels in the liver are small, so the lesions are relatively mild. On the other hand, Schistosoma japanese has the highest number of eggs ovulated per day at 3,500. Chronic stages of Schistosoma mansoni and Japanese schistosomiasis result in severe hepatomegaly and splenomegaly. The liver progresses to hepatosplenomegaly over time, and the mesentery and omentum gradually thicken, adhering to the colon and causing significant abdominal distension.

[0073] According to a preferred embodiment of the present invention, the first primer set, the second primer set or the third primer set may detect a specific gene IGS (ribosomal intergenic spacer) of Schistosoma haematobium.

[0074] According to a preferred embodiment of the present invention, the fourth primer set may detect a specific gene 16SrRNA (16s mitochondrial rRNA) of Schistosoma haematobium.

[0075] According to a preferred embodiment of the present invention, the fifth primer set may detect a specific gene IGS (ribosomal intergenic spacer) of Schistosoma mansoni.

[0076] According to a preferred embodiment of the present invention, the sixth primer set may detect a specific gene ITS (Internal transcribed spacer 1-5.8S-ITS2) of Schistosoma.

[0077] According to a preferred embodiment of the present invention, the primer set may be a primer set used in loop-mediated isothermal amplification (LAMP).

[0078] According to a preferred embodiment of the present invention, the detection limit of the composition may be 1 to 10 copies of the plasmid. More preferably, the detection limit of the composition may be 1 copy of the plasmid.

[0079]

[0080] In addition, the present invention can provide a kit for diagnosing schistosoma infection comprising the composition.

[0081] The above schistosome may be at least one selected from the group consisting of Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum.

[0082] The kit of the present invention may be comprised of one or more other component compositions, solutions, or devices suitable for commonly used expression level analysis methods. For example, a kit for measuring gene amplification levels may include a substrate, a suitable buffer solution, a secondary antibody labeled with a colorimetric enzyme or fluorescent substance, a chromogenic substrate, etc. for detecting the amplification product.

[0083] The kit of the present invention may include a sample extraction means for obtaining a sample from a subject of evaluation. The sample extraction means may include a dipstick, a disposable dropper, a Pasteur pipette, or the like. The kit may include a sample collection container for receiving the extracted sample, which may be a liquid. The kit may further include instructions for use. The sample may be any body sample in which a gene of Schistosoma haematobium is present or may be secreted. For example, the sample may be urine, feces, hair, sweat, saliva, body fluid, blood, cerebrospinal fluid, cells, or tissue. The measurement of the gene of Schistosoma haematobium in a body sample may be performed on a whole sample or a processed sample.

[0084] When the above-mentioned schistosome infection diagnostic kit is applied to an isothermal amplification method, it may include reagents necessary for isothermal amplification, such as a buffer, a DNA polymerase (e.g., a heat-stable DNA polymerase obtained from Bacillus stearothermophilus (Bst), Bacillus caldotenax (Bca), or Thermus aquaticus (Taq)), a DNA polymerase cofactor, dNTPs, and additives.

[0085] The above-mentioned schistosome infection diagnostic kit may include, for example, an isothermal amplification kit comprising a mixture of a dipstick, a wash buffer, a primer mix, a master mix, and an isothermal amplification buffer, a positive sample (schistosome plasmid DNA) diluted to a certain concentration as a control DNA, and a flat cap of a strip tube used when performing isothermal amplification.

[0086] The above-described kit for diagnosing schistosomiasis infection can be manufactured into a number of separate packages or compartments containing the above-described reagent components.

[0087]

[0088] In addition, the present invention comprises the steps of: i) manufacturing a dipstick;

[0089] ii) a step of adsorbing the gene in the sample separated from the object onto the dipstick manufactured in step i);

[0090] iii) a step of releasing the adsorbed gene; and

[0091] iv) a step of reacting the above-described gene with the above-described composition;

[0092] Provides a method for providing information for diagnosing schistosomiasis, including .

[0093] According to a preferred embodiment of the present invention, the dipstick of step i) is made of filter paper.

[0094] According to a preferred embodiment of the present invention, the sample of step ii) is at least one selected from the group consisting of urine, feces, hair, sweat, saliva, body fluid, blood, cerebrospinal fluid, skin, cells, and tissues. Preferably, the sample of step ii) may be urine.

[0095] The gene of step iii) above may be DNA.

[0096] According to a preferred embodiment of the present invention, the reaction of step iv) is carried out at 60 to 67°C for 20 to 50 minutes. More preferably, the reaction of step iv) is carried out at 61 to 65°C for 30 to 40 minutes.

[0097] According to a preferred embodiment of the present invention, the reaction of step iv) is carried out by a loop-mediated isothermal amplification (LAMP) method.

[0098] Detection of the amplified gene product according to the above reaction may be performed through colorimetric analysis, fluorescence analysis, or lateral flow immunoassay.

[0099] Colorimetric analysis can mean testing or quantifying the concentration of a chemical compound or solution by measuring the absorbance of a specific wavelength of light using a color reagent, etc. Fluorescence analysis is a chemical analysis method that utilizes the fluorescence of a substance. It can mean changing a non-fluorescent sample into a fluorescent substance through a chemical reaction and analyzing the fluorescence. Lateral flow immunoassay is an analysis method that utilizes an antigen-antibody reaction. It performs amplification by attaching a fluorescent substance to a primer and flows the amplified product onto an absorbent paper coated with a secondary antibody that binds to the fluorescent substance. In case of a positive reaction, two red lines can be observed, and in case of a negative reaction, only the control line can be visible.

[0100] Since the isothermal amplification method (LAMP) performs amplification using six types of primers, interference may occur between the primers, so it is important to establish optimal conditions by modifying various conditions when performing isothermal amplification.

[0101] In the above method for providing information for diagnosing schistosome infection, the isothermal amplification reaction may use various DNA polymerases for the amplification reaction, and preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which may include Bacillus stearothermophilus (Bst), Bacillus caldotenax (Bca), and Thermus aquaticus (Taq).

[0102] In the above method for providing information for diagnosing schistosome infection, the amplification reaction solution may include a dNTP mixture (dATP, dCTP, dGTP, dTTP), an isothermal amplification buffer, a DNA polymerase cofactor, and an additive.

[0103] In the above method for providing information for diagnosing schistosome infection, the amplification process can be performed in a single reactant without changing conditions such as addition of a reactant.

[0104] It may be at least one selected from the group consisting of Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum.

[0105]

[0106] The composition of the present invention can provide a diagnostic kit and a method for providing information for diagnosis that can accurately, quickly, and effectively diagnose Schistosoma haematobium and Schistosoma mansoni, which account for the majority of schistosoma prevalence, without separate equipment or trained personnel. Furthermore, the present invention can be readily introduced to resource-poor, schistosoma-endemic countries, thereby contributing to strengthening their capacity for diagnosing and managing schistosoma infections.

[0107]

[0108] Figure 1 illustrates a method for diagnosing schistosomiasis using the dipstick of the present invention.

[0109] Figure 2 shows a flow chart of a method for diagnosing schistosomiasis using the dipstick of the present invention.

[0110] Figure 3a shows the results of an isothermal amplification diagnostic experiment for Schistosoma haematobium infection using a primer combination of sequence numbers 1 to 6 of the present invention.

[0111] Figure 3b shows the results of an isothermal amplification diagnostic experiment for Schistosoma haematobium infection using a primer combination of sequence numbers 7 to 12 of the present invention.

[0112] Figure 3c shows the results of an isothermal amplification diagnostic experiment for Schistosoma haematobium infection using a primer combination of sequence numbers 13 to 18 of the present invention.

[0113] Figure 3d shows the results of an isothermal amplification diagnostic experiment for Schistosoma haematobium infection using a primer combination of sequence numbers 19 to 24 of the present invention.

[0114] Figure 3e shows the results of an isothermal amplification diagnostic experiment for Schistosoma mansoni infection using a primer combination of sequence numbers 25 to 30 of the present invention.

[0115] Figure 3f shows the results of an isothermal amplification diagnostic experiment for Schistosoma haematobium infection using a primer combination of sequence numbers 31 to 36 of the present invention.

[0116] Figure 4 shows the results of a cross-reaction confirmation experiment with Schistosoma haematobium, S. mansoni, and S. japonicum using the primer set of the present invention. SYBR Green I was used, and positive results were shown in fluorescent green, and negative results were shown in orange.

[0117] Figure 5 shows the results of an experiment comparing the detection limit of Schistosoma DNA in urine using the dipstick of the present invention and when using a micropipette. SYBR Green I was used, and positive results were shown in fluorescent green, and negative results in orange.

[0118] Figure 6 shows the results of an isothermal amplification test for diagnosing cystic haematobium infection using the dipstick of the present invention on a urine specimen of an actual schistosoma infected patient.

[0119] Figure 7 shows the isothermal amplification results when the dipstick of the present invention is used after drying at room temperature for one week after target gene adsorption.

[0120] Figure 8 shows the results of visualizing the product obtained by attaching a fluorescent material to the primers of sequence numbers 13 to 18 of the present invention and performing an isothermal amplification test using a lateral flow assay.

[0121]

[0122] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0123]

[0124] [Example 1]

[0125] Primer production

[0126] The present inventors obtained the IGS (ribosomal intergenic spacer), 16S (16S mitochondrial rRNA), and ITS (Internal transcribed spacer 1-5.8S-ITS2) gene sequences of schistosomiasis, particularly Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum, from the National Center for Biotechnology Information (NCBI), and received genomic DNA of each schistosome through BEI resources. Using the donated genomic DNA as a template, each gene sequence was amplified by PCR, and then TA cloning was performed. Colony PCR and sequencing were performed to confirm the IGS, 16S rRNA, and ITS genes of each schistosome, which were then used as the standard sequences of the present invention.

[0127] Based on the above-obtained base sequence, primers capable of diagnosing Schistosoma were developed using the NEB LAMP Primer Design Tool. The developed primers are shown in [Table 1] below.

[0128]

[0129]

[0130] [Example 2]

[0131] Dipstick manufacturing

[0132] The present inventors have manufactured a dipstick to easily isolate the genes of schistosomes from the urine of a subject.

[0133] Specifically, 95 g of Paraplast Plus wax chips and 0.67 g of candle dye were placed in a glass flask and melted using an alcohol burner or hot plate. Once the wax was melted, it was poured into a 120x120 mm square petri dish, and a 100x75 mm Whatman grade 1 filter paper was coated by immersing it about two-thirds in the melted wax. The top 20 mm was kept free of wax. The waxed filter paper was placed on aluminum foil and dried for about 1 minute. The uncoated portion of the dried filter paper was cut with clean scissors, leaving 6 mm. Copy paper was cut to a size of 105x50 mm, folded in half to make a size of 105x25 mm, and the dried filter paper was inserted into the folded area. The blade of the pasta machine was adjusted to a width of 2 mm, and the filter paper wrapped in copy paper was cut. After removing the copy paper from the completed dipstick, store it in an airtight container.

[0134]

[0135] [Example 3]

[0136] Diagnosis of Schistosoma infection

[0137] <3-1> Production of related samples

[0138] Wash buffer was prepared by dissolving 1.21 g of Tris base in 900 ml of water to make a 10 mM Tris (pH 8) solution, adjusting the pH to 8 with HCl, and making 1 L with water.

[0139] The 10X primer mix was prepared by mixing all six primers of [Table 1], F3, B3, FIP, BIP, LF, and LB, to produce a primer mixture with a final concentration of 2, 2, 16, 16, 4, and 4 uM, respectively.

[0140] The LAMP master mix was prepared by adding 3.5 μl of 10X primer mixture, 3.5 μl of 10X Isothermal amplification buffer (New Enland Biolabs, NEB), 2.1 μl of 100 mM Magnesium Sulfate (NEB), 4.9 μl of 10 mM dNTP mix (NEB), 1.4 μl of Bst2.0 WarmStart DNA polymerase (NEB), 7 μl of 5M Betaine (Sigma), and 9.1 μl of distilled water to a microtube and mixing well to prepare a reaction mixture.

[0141]

[0142] <3-2> Diagnosis of Schistosoma infection using LAMP

[0143] The LAMP master mix manufactured in the above Example <3-1> was transferred to 3 microtubes (9 ㎕ each) and prepared for the experimental group, positive control group, and negative control group without amplification template, respectively. The dipstick manufactured in the above <Example 2> was immersed in the urine specimen of a patient infected with Schistosoma for 3 seconds to adsorb Schistosoma DNA. The dipstick with the adsorbed target DNA was washed by immersing in 800 ㎕ of the wash buffer (10 mM Tris (pH 8)) manufactured in the above Example <3-1> for 3 seconds. The washed dipstick was immersed in the reaction medium for the experimental group for 10 seconds to release the template DNA. The reaction medium for the positive control group contained 1 ㎕ of Schistosoma genomic DNA at a concentration of 0.1 ng / ㎕ or 10 IGS plasmid instead of a template. 5 Add 1 ㎕ of copy, and add 1 ㎕ of distilled water to the negative control reaction.

[0144] The above mixtures were placed in a heat block or water bath and reacted at 63°C for 30 minutes. After removing the reacted mixture from the incubation device, 2 μl of 10,000X SYBR Green I (Invitrogen) reagent diluted 1:10 was added each time, and the color change was confirmed. If no amplification occurred, the orange color of the SYBR Green I reagent was maintained, and if amplification occurred, the color changed to fluorescent light green (Fig. 1, Fig. 2).

[0145]

[0146] [Example 4]

[0147] Diagnostic performance verification test by primer combination

[0148] To determine the difference in diagnostic efficiency according to the combination of primers for diagnosing Schistosoma infection and to determine the optimal combination and reaction time, isothermal amplification tests were performed using four combinations for each primer pair.

[0149] The primer combinations were as follows: 4 primers (F3, B3, FIP, BIP); 4 primers + LF; 4 primers + LB; 6 primers (4 primers + LF + LB). LAMP assays were performed at 63°C using each primer combination and WarmStart Colorimetric LAMP 2X Master mix (NEB) according to the conventional method. Yellow color indicated positive, and pink color indicated negative.

[0150] As a result, when LF and LB were combined with any one of the four primers (F3, B3, FIP, BIP), a clear positive reaction was observed from 30 minutes, indicating that the amplification reaction was improved. When the reaction was performed for more than 50 minutes, false positive reactions occurred in some negative control groups due to interference between primers, so it was determined that it was appropriate to determine the result between 30 and 40 minutes after the start of the reaction (incubation) (Fig. 3a to Fig. 3e). In addition, the primer targeting the Schistosoma common SchITS gene showed a complete positive result in just 20 minutes after the start of incubation when using a combination of 6 primers, and showed positive and false positive results in all combinations after 30 minutes (Fig. 3f), so it was determined that it was appropriate to determine the result within 20 minutes when using the primers.

[0151]

[0152] [Example 5]

[0153] Isothermal amplification results for real-time diagnosis of Schistosoma haematobium infection using fluorescent materials

[0154] <5-1> Production of related samples

[0155] The LAMP master mix was prepared by adding 25 μl of the 10X primer mixture prepared in the same manner as in Example <3-1>, 125 μl of WarmStart Multi-Purpose LAMP / RT-LAMP 2X Master MIX with UDG (NEB), 5 μl of fluorescence dye, 25 μl of 5% w / v Pullulan, and 50 μl of distilled water to a microtube and mixing well to prepare a reaction product.

[0156]

[0157] <5-2> Diagnosis of Schistosoma haematobium infection using real-time LAMP

[0158] The LAMP master mix prepared in the above Example <5-1> was transferred to 8 microtubes at 23 ㎕ each to prepare a positive control and a negative control without amplification template, respectively. The positive control reaction was prepared by gradually reducing the copy number of Schistosoma haematobium IGS, 16S rRNA, or ITS plasmid by 10-fold (5*10 5 copies / ㎕ ~ 0.5 copies / ㎕) and add 2 ㎕ of distilled water to the negative control reaction.

[0159] The above mixtures were placed in a CFX Opus 96 Real-Time PCR System (Biorad) and reacted at 63°C for 60 minutes, and the amplified fluorescence signal was measured every minute. The above reaction was repeated twice, and the primers targeting ShIGS and Sh16S showed positive results with an average Ct value of 30 or less even at a low copy number of 1 copy / Rx, indicating excellent sensitivity and precision (reproducibility) (Table 2).

[0160] Target gene template plasmid Serial Dilution Mean Ct value (Incubation time) by Primer setShIGS-Set3Sh16SSchITS10 6 copies / Rx13.2112.0617.1110 5 copies / Rx14.4313.8019.6710 4 copies / Rx25.4916.2126.0410 3 copies / Rx22.6421.7530.8010 2 copies / Rx23.1733.2850.7110 1 copies / Rx18.5132.5546.691 copies / Rx18.4421.5041.99

[0161]

[0162] [Example 6]

[0163] Specificity of the isothermal amplification test for the diagnosis of Schistosoma haematobium infection

[0164] <6-1> Confirmation of false positive reaction

[0165] To determine the accuracy of a diagnostic test, sensitivity and specificity tests must be performed using specimens that have already been confirmed positive or negative. Sensitivity refers to the 'accuracy of positive specimens diagnosed by the test method', and specificity refers to the 'accuracy of negative specimens diagnosed by the test method'. 96 negative samples were prepared by adding distilled water instead of a template to the LAMP master mix manufactured with the same composition and ratio as in Example <5-1> above, and then placed in a CFX Opus 96 Real-Time PCR System (Biorad) and reacted at 63℃ for 60 minutes to check for false positive reactions. As a result, primers targeting the ShIGS, Sh16S, and SmIGS genes showed a specificity of over 90% up to 50 minutes, which is the maximum reaction time used in the present invention, and it was confirmed that all four primers targeting the four genes used in the present invention showed excellent specificities of 90-100% up to 39 minutes of reaction. (Table 3).

[0166] Specificity by primer set (%) Reaction time ShIGS Set3Sh16SSmIGSSchITS20-2499%100%100%99%25-2998%100%100%97%30-3497%100%100%95%35-3996%100%100%90%40-4495%100%96%79%45-4995%99%94%69%50-5494%98%90%52%55-6092%97%85%39%

[0167]

[0168] <6-2> Cross-reactivity confirmation

[0169] In order to verify the specificity of the primers for diagnosing schistosome infection, 0.1 ng / ㎕ of gDNA of three species of schistosomes (Schistosome urea, Schistosoma mansoni, and Schistosoma japanese) was added as template to the LAMP master mix prepared with the same composition and ratio as in Example <5-1> above, and an isothermal amplification reaction was performed at 63℃ for 30 minutes to perform a specificity (cross-reaction) test. At this time, the results of the specificity test were repeated twice. After taking the reacted mixture out of the incubation device, 2 ㎕ of 10,000X SYBR Green I (Invitrogen) reagent diluted 1:10 was added each time, and the color change was confirmed. Orange indicates negative, and fluorescent light green indicates positive.

[0170] As a result, it was confirmed that the primers targeting the ShIGS and Sh16S genes accurately diagnosed only Schistosoma haematobium as positive and showed negative results for Schistosoma mansoni and Schistosoma nippon, indicating that they had specificity for Schistosoma haematobium. The primers targeting the SmIGS gene showed negative results for Schistosoma haematobium and positive results for Schistosoma mansoni, but some cross-reactions with Schistosoma haematobium were confirmed. The primers targeting the SchITS gene common to schistosomes diagnosed all three types of Schistosoma gDNA as positive (Fig. 4).

[0171]

[0172] [Example 7]

[0173] Sensitivity (repeatability) of the isothermal amplification test for the diagnosis of Schistosoma haematobium infection using a dipstick

[0174] To determine the sensitivity (repeatability) of the dipstick-based isothermal amplification test for Schistosoma haematobium infection, a sensitivity test was conducted using the LAMP method. This repeatability test was designed to demonstrate that the results were consistent across the first, second, and third runs. This test demonstrates the ability to obtain consistent results multiple times on the same day, and confirms the accuracy and reliability of the kit and operator.

[0175] Specifically, Schistosoma haematobium gDNA was serially diluted 10-fold (1 ng / ㎕ to 10 pg / ㎕) in ultra pure water or Schistosoma-negative urine specimens, and then sensitivity (repeatability) tests were performed by performing LAMP using a conventional method and LAMP using a dipstick at 63℃ for 30 minutes. At this time, for the LAMP test using a conventional method, 1 ㎕ of the template was dispensed into the reaction medium using a micropipette, and for LAMP using a dipstick, the dipstick was immersed in the sample 5 times (3 seconds), then immersed in the wash buffer 5 times (3 seconds), then immersed in the reaction medium 15 times (10 seconds), and then withdrawn to release the template. The dipstick method was repeated 3 times to confirm repeatability.

[0176] As a result, both when using a dipstick and when dispensing the sample with a micropipette in the usual way, a positive reaction was observed up to a concentration of 0.1 ng / ul, and at a concentration of 10 pg / ㎕, a positive reaction was observed only when using a dipstick, indicating that the sensitivity of the dipstick-based method for diagnosing Schistosoma haematobium infection is excellent (Fig. 5).

[0177] When a dipstick was used, the results of all three runs were identical up to a concentration of 0.1 ng / ul, and at 10 pg / ul, two out of three runs had identical results, so it could be judged that the repeatability was relatively stable.

[0178]

[0179] [Example 8]

[0180] Results of repeatability and reproducibility tests for the isothermal amplification test for the diagnosis of Schistosoma haematobium infection using a dipstick.

[0181] To verify the repeatability and reproducibility of the isothermal amplification test for Schistosoma haematobium infection in urine specimens from actual patients infected with Schistosoma haematobium, the target gene was isolated from each of the urine specimens pretreated in three ways (whole urine, supernatant, pellet) using a dipstick method and repeatability and reproducibility tests were performed. The reproducibility is intended to show that the results are the same whether the experiment is performed the first time or the second time. The difference with the repeatability is that the experiment is repeated on different days. It can be confirmed whether the same results can be obtained when performed multiple times on different days and whether the kit and operator are accurate and stable.

[0182] Specifically, after obtaining Schistosoma haematobium DNA by dipping a dipstick in three types of urine specimens (whole urine, supernatant, pellet), the Schistosoma haematobium infection diagnostic isothermal amplification test was performed three times in the same manner as in <Example 7> above. At this time, since the target Schistosoma haematobium DNA in the urine specimen of a patient infected with Schistosoma haematobium is mixed with the patient DNA, it is difficult to quantify it alone, so 1 ng / ㎕ of Schistosoma haematobium gDNA was used as a positive control.

[0183] To confirm reproducibility, a dipstick was immersed in the same infected person's urine sample to adsorb Schistosoma haematobium DNA, dried at room temperature, washed in wash buffer after 1 week, and then the DNA was released by immersing and removing the dipstick 15 times (10 seconds) in the reaction mixture, and isothermal amplification was performed at 63°C for 30 minutes.

[0184] As a result, when the isothermal amplification diagnostic method for Schistosoma haematobium infection using a dipstick was used, the same positive results were observed the first, second, and third times regardless of the specimen processing method, indicating that the repeatability of the Schistosoma haematobium infection diagnostic method using a dipstick was excellent (Fig. 6).

[0185] In addition, when the dipstick was dried for one week and the isothermal amplification diagnostic method for Schistosoma haematobium infection was performed, all three test results showed positive results, indicating that the reproducibility of the Schistosoma haematobium infection diagnostic method using the dipstick was also excellent (Fig. 7).

[0186]

[0187] [Example 9]

[0188] Visualization of isothermal amplification results for the diagnosis of Schistosoma haematobium infection using lateral flow immunoassay.

[0189] <9-1> Production of related samples

[0190] Biotin and 6-FAM were attached to the 5' end of the FIP and BIP primers of [Table 1] above, respectively, and synthesized, and mixed with all four primers of [Table 1] F3, B3, LF, and LB to prepare a 10X primer mixture having the same concentration as in Example <3-1>.

[0191] The LAMP master mix was prepared by adding 11 μl of 10X primer mixture, 55 μl of WarmStart Multi-Purpose LAMP / RT-LAMP 2X Master MIX with UDG (NEB), and 35.2 μl of distilled water to a microtube and mixing well.

[0192]

[0193] <9-2> Diagnosis of Schistosoma infection using LAMP

[0194] The LAMP master mix prepared in the above Example <9-1> was transferred to 4 microtubes (23 ㎕ each) to prepare 3 positive control groups and 1 negative control group without amplification template. The positive control group reaction mixture contained 5*10 IGS plasmids. 6 2 μl of copy was added, and 2 μl of distilled water was added to the negative control reaction.

[0195] The above mixtures were placed in a heat block or water bath and reacted at 63°C for 30 minutes. The reacted mixture was removed from the incubator and a HybriDetect strip (Milenia) was immersed and absorbed onto an adsorbent pad. This was then immersed in HybriDetect Assay buffer (Milenia) to induce an antigen-antibody reaction. Two red lines were observed in the positive sample, while only the control line was red in the negative control group (Fig. 8).

[0196]

[0197] The composition of the present invention can provide a diagnostic kit and a method for providing information for diagnosis that can accurately, quickly, and effectively diagnose Schistosoma haematobium and Schistosoma mansoni, which account for the majority of schistosoma prevalence, without separate equipment or trained personnel. Furthermore, the present invention can be easily introduced to resource-poor, schistosoma-endemic countries, thereby contributing to strengthening schistosoma infection diagnosis and management capabilities, thus demonstrating potential for industrial application.

[0198]

[0199] Sequence number 1 represents the ShIGS-Set1-F3 primer sequence for detection of ShIGS of Schistosoma haematobium.

[0200] Sequence number 2 represents the ShIGS-Set1-B3 primer sequence for detection of ShIGS of Schistosoma haematobium.

[0201] Sequence number 3 represents the ShIGS-Set1-FIP primer sequence for detection of ShIGS of Schistosoma haematobium.

[0202] Sequence number 4 represents the ShIGS-Set1-BIP primer sequence for detection of ShIGS of Schistosoma haematobium.

[0203] Sequence number 5 represents the ShIGS-Set1-LF primer sequence for detection of ShIGS of Schistosoma haematobium.

[0204] Sequence number 6 represents the ShIGS-Set1-LB primer sequence for detection of ShIGS of Schistosoma haematobium.

[0205] Sequence number 7 represents the ShIGS-Set2-F3 primer sequence for detection of ShIGS of Schistosoma haematobium.

[0206] Sequence number 8 represents the ShIGS-Set2-B3 primer sequence for detection of ShIGS of Schistosoma haematobium.

[0207] Sequence number 9 represents the ShIGS-Set2-FIP primer sequence for detection of ShIGS of Schistosoma haematobium.

[0208] Sequence number 10 represents the ShIGS-Set2-BIP primer sequence for detection of ShIGS of Schistosoma haematobium.

[0209] Sequence number 11 represents the ShIGS-Set2-LF primer sequence for detection of ShIGS of Schistosoma haematobium.

[0210] Sequence number 12 represents the ShIGS-Set2-LB primer sequence for detection of ShIGS of Schistosoma haematobium.

[0211] Sequence number 13 represents the ShIGS-Set3-F3 primer sequence for detection of ShIGS of Schistosoma haematobium.

[0212] Sequence number 14 represents the ShIGS-Set3-B3 primer sequence for detection of ShIGS of Schistosoma haematobium.

[0213] Sequence number 15 represents the ShIGS-Set3-FIP primer sequence for detection of ShIGS of Schistosoma haematobium.

[0214] Sequence number 16 represents the ShIGS-Set3-BIP primer sequence for detection of ShIGS of Schistosoma haematobium.

[0215] Sequence number 17 represents the ShIGS-Set3-LF primer sequence for detection of ShIGS of Schistosoma haematobium.

[0216] Sequence number 18 represents the ShIGS-Set3-LB primer sequence for detection of ShIGS of Schistosoma haematobium.

[0217] Sequence number 19 represents the Sh16S-F3 primer sequence for detecting Sh16S of Schistosoma haematobium.

[0218] Sequence number 20 represents the Sh16S-B3 primer sequence for detecting Sh16S of Schistosoma haematobium.

[0219] Sequence number 21 represents the Sh16S-FIP primer sequence for detecting Sh16S of Schistosoma haematobium.

[0220] Sequence number 22 represents the Sh16S-BIP primer sequence for detecting Sh16S of Schistosoma haematobium.

[0221] Sequence number 23 represents the Sh16S-LF primer sequence for detecting Sh16S of Schistosoma haematobium.

[0222] Sequence number 24 represents the Sh16S-LB primer sequence for detecting Sh16S of Schistosoma haematobium.

[0223] Sequence number 25 represents the SmIGS-F3 primer sequence for detection of SmIGS of Schistosoma mansoni.

[0224] Sequence number 26 represents the SmIGS-B3 primer sequence for detection of SmIGS of Schistosoma mansoni.

[0225] Sequence number 27 represents the SmIGS-FIP primer sequence for detecting SmIGS of Schistosoma mansoni.

[0226] Sequence number 28 represents the SmIGS-BIP primer sequence for detecting SmIGS of Schistosoma mansoni.

[0227] Sequence number 29 represents the SmIGS-LF primer sequence for detection of SmIGS of Schistosoma mansoni.

[0228] Sequence number 30 represents the SmIGS-LB primer sequence for detection of SmIGS of Schistosoma mansoni.

[0229] Sequence number 31 represents the SchITS-F3 primer sequence for Schitosoma haematobium, Schistosoma mansoni, or Schistosoma japonica.

[0230] Sequence number 32 represents the SchITS-B3 primer sequence for Schitosoma haematobium, Schistosoma mansoni, or Schistosoma japonica.

[0231] Sequence number 33 represents the SchITS-FIP primer sequence for Schitosoma haematobium, Schistosoma mansoni, or Schistosoma japonica.

[0232] Sequence number 34 represents the SchITS-BIP primer sequence for Schitosoma haematobium, Schistosoma mansoni, or Schistosoma japonica.

[0233] Sequence number 35 represents the SchITS-LF primer sequence for Schistosoma haematobium, Schistosoma mansoni, or Schistosoma japonica.

[0234] Sequence number 36 represents the SchITS-LB primer sequence for Schistosoma haematobium, Schistosoma mansoni, or Schistosoma japonica.

Claims

1. A first primer set including a primer consisting of a base sequence of sequence number 1, a primer consisting of a base sequence of sequence number 2, a primer consisting of a base sequence of sequence number 3, and a primer consisting of a base sequence of sequence number 4; A second primer set comprising a primer consisting of a base sequence of SEQ ID NO: 7, a primer consisting of a base sequence of SEQ ID NO: 8, a primer consisting of a base sequence of SEQ ID NO: 9, and a primer consisting of a base sequence of SEQ ID NO: 10; A third primer set comprising a primer consisting of a base sequence of SEQ ID NO: 13, a primer consisting of a base sequence of SEQ ID NO: 14, a primer consisting of a base sequence of SEQ ID NO: 15, and a primer consisting of a base sequence of SEQ ID NO: 16; A fourth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 19, a primer consisting of a base sequence of SEQ ID NO: 20, a primer consisting of a base sequence of SEQ ID NO: 21, and a primer consisting of a base sequence of SEQ ID NO: 22; A fifth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 25, a primer consisting of a base sequence of SEQ ID NO: 26, a primer consisting of a base sequence of SEQ ID NO: 27, and a primer consisting of a base sequence of SEQ ID NO: 28; and A sixth primer set comprising a primer consisting of a base sequence of SEQ ID NO: 31, a primer consisting of a base sequence of SEQ ID NO: 32, a primer consisting of a base sequence of SEQ ID NO: 33, and a primer consisting of a base sequence of SEQ ID NO: 34; A composition for diagnosing schistosomiasis infection, comprising at least one primer set selected from the group consisting of:

2. In the first paragraph, the first primer set additionally includes a primer consisting of a base sequence of SEQ ID NO: 5 and / or a primer consisting of a base sequence of SEQ ID NO: 6; The second primer set further comprises a primer comprising a base sequence of SEQ ID NO: 11 and / or a primer comprising a base sequence of SEQ ID NO: 12; The third primer set further comprises a primer consisting of a base sequence of SEQ ID NO: 17 and / or a primer consisting of a base sequence of SEQ ID NO: 18; The fourth primer set further comprises a primer comprising a base sequence of SEQ ID NO: 23 and / or a primer comprising a base sequence of SEQ ID NO: 24; The fifth primer set further comprises a primer consisting of a base sequence of SEQ ID NO: 29 and / or a primer consisting of a base sequence of SEQ ID NO: 30; or The above 6th primer set additionally includes a primer consisting of a base sequence of SEQ ID NO: 35 and / or a primer consisting of a base sequence of SEQ ID NO: 36; A composition characterized by:

3. A composition according to claim 1, characterized in that the schistosome is at least one selected from the group consisting of Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum.

4. In the first paragraph, the first primer set, the second primer set, or the third primer set detects a specific gene IGS (ribosomal intergenic spacer) of Schistosoma haematobium; The fourth primer set above detects the specific gene 16SrRNA (16s mitochondrial rRNA) of Schistosoma haematobium; The fifth primer set above detects the specific gene IGS (ribosomal intergenic spacer) of Schistosoma mansoni; and The above sixth primer set is characterized by detecting the specific gene ITS (Internal transcribed spacer 1-5.8S-ITS2) of Schistosoma; A composition comprising:

5. A composition according to claim 1, characterized in that the primer set is a primer set used in loop-mediated isothermal amplification (LAMP).

6. A composition according to claim 1, characterized in that the detection limit of the composition is 1 to 10 copies of the plasmid.

7. A kit for diagnosing schistosomiasis infection comprising the composition of paragraph 1. 8.i) Step of manufacturing a dipstick; ii) a step of adsorbing the gene in the sample separated from the object onto the dipstick manufactured in step i); iii) a step of releasing the adsorbed gene; and iv) a step of reacting the above-mentioned glass gene with the composition of the first clause; A method for providing information for diagnosing schistosomiasis infection, including:

9. An information providing method according to claim 8, wherein the dipstick of step i) is made of filter paper.

10. An information providing method characterized in that, in paragraph 8, the sample of step ii) is at least one selected from the group consisting of urine, feces, hair, sweat, saliva, body fluid, blood, cerebrospinal fluid, skin, cells, and tissues.

11. An information providing method characterized in that the reaction of step iv) in paragraph 8 is carried out at 60 to 67°C for 20 to 50 minutes.

12. An information providing method characterized in that the reaction of step iv) in paragraph 8 is carried out by a loop-mediated isothermal amplification (LAMP) method.

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