Lateral flow strip based on loop-mediated isothermal amplification for detecting bovine brucella

Abstract

The present invention relates to a lateral flow strip based on loop-mediated isothermal amplification for detecting bovine Brucella and, more specifically, to a lateral flow assay kit for detecting Brucella abortus and a method for diagnosing bovine Brucellosis using the kit, the kit comprising: a primer set for loop-mediated isothermal amplification; a probe specifically binding to an amplification product amplified by the primer set; and a lateral flow strip.

Description

A lateral flow strip based on ring-mediated isothermal amplification for detection of bovine brucellosis

[0001] The present invention relates to a lateral flow strip based on loop-mediated isothermal amplification (LAMP) for detecting bovine Brucella, and more particularly, to a method for detecting bovine Brucella using a probe having a biotin-spacer-complementary bases to target-biotin structure that can specifically bind to an amplification product generated by LAMP from the BruAb2_0168 gene of bovine Brucella, and a lateral flow strip based on a Bio-receptor (biotin-albumin and FITC specific antibody).

[0002]

[0003] This invention was carried out with the support of the Livestock Disease Response Technology Advancement Support Project of the National Institute of Agricultural Science and Technology Planning and Evaluation and the Science and Engineering Research Infrastructure Construction Project of the National Research Foundation of Korea (Project Number: 122001022SB010 and 2022R1A6A3A01087516).

[0004] Brucellosis is a statutory Class 2 bacterial infectious disease of livestock (added Class 1 wildlife disease) that mainly occurs in mammals (cattle, pigs, goats, sheep, goats, rodents, and marine mammals). Infection with Brucella bacteria causes fever, chills, sweating, malaise, weight loss, and miscarriage. Although it is not highly pathogenic, it is difficult to treat because it is an intracellular parasite, and there are no specific symptoms that can indicate the infection externally. Brucellosis is one of the most common zoonotic diseases worldwide and one of the zoonotic diseases with serious economic damage, with the World Health Organization (WHO) estimating that more than 500,000 people are infected annually.

[0005] Brucellosis can be diagnosed by directly isolating the causative organism from tissue samples such as aborted fetuses, placentas, amniotic fluid, and lymphoid tissues from euthanasia, or by testing using specific cell-mediated or serological reactions to Brucella antigens. However, care must be taken in differential diagnosis with bacteria such as Coxiella burnetii and Chlamydia, which are morphologically similar to Brucella, and Yersinia enterocolitica, which causes immunological cross-reactivity. In Korea, the rose-bengal test (RBT) is used as a primary screening test according to the “Guidelines for the Prevention of Tuberculosis and Brucellosis,” and the standard tube agglutination test (STAT) and competitive enzyme-linked immunosorbent assay (C-ELISA) are used as secondary confirmatory tests for individuals diagnosed positive in the RBT.

[0006] Isothermal nucleic acid amplification is similar to the conventional PCR (polymerase chain reaction), but it is a technology that allows amplification reactions to be performed at a constant temperature without the temperature changes required for DNA denaturation, primer annealing, and polymerase extension during the PCR reaction. Since isothermal nucleic acid amplification technology does not require temperature control, the reaction time is relatively short, enabling amplification of target nucleic acids in a shorter period of time. In addition, it has the advantage of showing very high amplification efficiency because it can prevent DNA loss and damage due to temperature changes. Among various isothermal nucleic acid amplification technologies, loop-mediated isothermal amplification (LAMP) technology can amplify up to 10 in a short reaction time of about 1 hour. 9By implementing target nucleic acid amplification efficiency of the ship, it has been considered a technology with high potential for use as a point-of-care testing (POCT) technology.

[0007] Meanwhile, Korean Patent Publication No. 2014-0022194 discloses 'A ring-mediated isothermal amplification primer set for identification of Brucella abortus and a method for detecting Brucella abortus using the same', and Korean Patent Publication No. 2016-0121673 discloses 'A fluorescent immunodiagnostic kit for diagnosing brucellosis', but the 'lateral flow strip based on ring-mediated isothermal amplification for detection of bovine Brucella' of the present invention is not described.

[0008] The present invention was derived from the above-mentioned needs, and the inventors of the present invention developed a lateral flow strip equipped with a probe capable of specifically binding to a loop-mediated amplification product for field testing of bovine Brucella based on isothermal amplification, and a bio-receptor capable of effectively detecting the amplification product to which the probe is bound, and completed the present invention by confirming that the lateral flow assay (LFA) kit according to the present invention has a superior false positive correction effect compared to a commercially available Brucella diagnostic LFA kit.

[0009] To solve the above problem, the present invention provides a lateral flow assay kit for detecting Brucella abortus, comprising: a primer set for loop-mediated isothermal amplification; a probe that specifically binds to an amplification product amplified by the primer set; and a lateral flow strip.

[0010] In addition, the present invention provides a method for diagnosing bovine brucellosis, comprising: a step of isolating nucleic acid from a biological sample of a bovine suspected of brucellosis; a step of amplifying a target sequence through a loop-mediated isothermal amplification reaction using the isolated nucleic acid as a template and a primer set for loop-mediated isothermal amplification consisting of the base sequences of SEQ ID NOs: 10 to 15 and a probe consisting of the base sequence of SEQ ID NO: 16; and a step of applying a product of the amplification step to a lateral flow strip to detect a signal.

[0011] The method for detecting bovine Brucella using a lateral flow strip based on a ring-mediated isothermal amplification method of the present invention is characterized by high field usability, the ability to provide rapid diagnostic results, and an excellent false positive correction effect compared to a commercially available Brucella diagnostic LFA (lateral flow assay) kit.

[0012] Figure 1 shows the principle (left) of the loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA) for rapid diagnosis of bovine Brucella according to the present invention and the conditions established for manufacturing a lateral flow strip (right).

[0013] Figure 2 shows the locations of the LAMP primer set and probe (dotted box) for the BruAb2_0168 gene.

[0014] Figure 3 shows the results of DNA probe optimization for the rapid diagnostic LAMP-LFA for bovine Brucella. These results include the LAMP reaction using a probe with the biotin-spacer-target gene-biotin structure and the LFA strip application results. The upper part of the figure shows the electrophoresis results for the LAMP amplification product, and the lower part shows the strip results. P: positive; Brucella abortus DNA, N: negative; Escherichia coli DNA.

[0015] Figure 4 shows the results of an experiment to optimize the concentration ratio of the internal and external primer sets of LAMP primers. The upper left shows the electrophoresis results for the LAMP amplification product, the lower left shows the strip results, the upper right shows the test line (TL) on the strip, and the lower right shows the quantification results for the TL.

[0016] Figure 5 shows the results of confirming the detection limit under optimized LAMP internal and external primer set concentration ratio conditions.

[0017] Figure 6 shows the results of an optimal concentration experiment of a DNA probe.

[0018] Figure 7 shows the results of an experiment to optimize the concentration of anti-FITC antibody to be treated on the test line (TL) of a nitrocellulose membrane.

[0019] Figure 8 shows the results of an experiment to optimize the concentration of albumin-biotin to be treated on the control line (CL) of a nitrocellulose membrane.

[0020] Figure 9 shows the results of selecting a developing solvent for LFA.

[0021] Figure 10 shows the results of confirming the detection limit using the LAMP-LFA method optimized through Example 2.

[0022] Figure 11 shows the results of cross-reactivity analysis using the LAMP-LFA method optimized through Example 2.

[0023] Figure 12 shows the results confirming the applicability of the LAMP-LFA method to LAMP amplification products in which a precipitate (magnesium pyrophosphate; Mg2P2O7) is generated.

[0024] Figure 13 shows the results of analysis of a Brucella abortus positive plasma sample using the LAMP-LFA method according to the present invention.

[0025] Figure 14 shows the results of analysis of a Brucella abortus negative plasma sample using the LAMP-LFA method according to the present invention.

[0026] Figure 15 shows the results of analyzing a false positive plasma sample for Brucella abortus using the LAMP-LFA method according to the present invention. (a) is the analysis result using a commercially available PCR kit, (b) is the result using the LAMP-LFA method according to the present invention, and (c) is the analysis result using a commercially available LFA kit.

[0027] In order to achieve the object of the present invention, the present invention provides a lateral flow assay kit for detecting Brucella abortus, comprising: a primer set for loop-mediated isothermal amplification; a probe that specifically binds to an amplification product amplified by the primer set; and a lateral flow strip.

[0028] In the lateral flow test kit for detecting Brucella abotus according to the present invention, the primer set for ring-mediated isothermal amplification may be composed of the base sequences of SEQ ID NOs: 10 to 15, but is not limited thereto.

[0029] In the present invention, the term "Loop-mediated isothermal amplification (LAMP)" refers to a method that can perform an amplification reaction under isothermal conditions, unlike the conventional PCR (polymerase chain reaction) method. For the LAMP reaction, four types of primers (F3, B3, FIP, BIP) are basically required, and two types of primers (LF, LB) are added to improve the reaction speed, so that a final six types of oligonucleotide primers consisting of different base sequences are required for the reaction. The forward outer primer (F3) and the reverse outer primer (B3) play a role in unwinding the DNA double strand during the non-cyclic step of the reaction. In addition, the forward inner primer (FIP) and the reverse inner primer (BIP) are composed of nucleotides corresponding to the forward and reverse base sequences so that they can form a loop essential for the loop-mediated isothermal amplification reaction. The forward loop primer (LF) and reverse loop primer (LB) are attached to the base sequence to which the internal primers (FIP and BIP) do not bind, thereby accelerating the loop-mediated isothermal amplification reaction.

[0030] In the present invention, the base sequences of sequence numbers 10 to 15 are sequentially F3, B3, LF, LB, FIP, and BIP, and FIP consisting of the base sequence of sequence number 14 has FITC (fluorescein isothiocyanate) bound to the 5' end.

[0031] In the present invention, the term "primer" refers to a single-stranded oligonucleotide sequence complementary to the nucleic acid strand to be copied, and can serve as an initiation point for the synthesis of a primer extension product. The length and sequence of the primer must allow for the initiation of the synthesis of the extension product. The specific length and sequence of the primer will depend on the complexity of the desired DNA or RNA target, as well as the conditions under which the primer is used, such as temperature and ionic strength.

[0032] In the present specification, the oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or an intercalating agent. Furthermore, the primer may incorporate additional features that do not alter the primer's fundamental properties as an initiation point for DNA synthesis. The appropriate primer length is determined by the characteristics of the primer to be used, but is typically 15 to 30 bp in length.

[0033] The primer does not need to be exactly complementary to the template sequence, but it must be complementary enough to form a hybrid complex with the template.

[0034] In addition, in the lateral flow test kit for detecting Brucella abotus according to the present invention, the probe is designed to specifically bind to a product amplified by a primer set for loop-mediated isothermal amplification (LAMP), and the probe may have a structure in which biotin, a spacer, a base sequence specific to the LAMP amplification product, and biotin are sequentially connected, and the spacer may be composed of 4 to 6 adenine bases, but is not limited thereto.

[0035] In one embodiment of the present invention, the base sequence specific to the LAMP amplification product may be composed of the base sequence of SEQ ID NO: 16, but is not limited thereto.

[0036] In the present invention, the term "probe" means a nucleic acid fragment such as RNA or DNA, which is short, ranging from a few bases to several hundred bases, and capable of forming a specific binding with a gene or mRNA.

[0037] In addition, in the lateral flow test kit for detecting Brucella abortus according to the present invention, the lateral flow strip may be a nitrocellulose membrane in the form of a strip having a conjugate pad treated with a gold nanoparticle-streptavidin conjugate; a test line on which an anti-FITC (fluorescein isothiocyanate) antibody is fixed; and a control line on which an albumin-biotin conjugate is fixed; but is not limited thereto, and the nitrocellulose membrane may additionally have a sample injection portion and an absence pad at both ends.

[0038] In one embodiment of the present invention, the anti-FITC antibody may be treated to the test line at a concentration of preferably 1.0 to 2.0 mg / ml, more preferably 1.2 to 1.6 mg / ml, and even more preferably 1.4 mg / ml, but is not limited thereto. In addition, the albumin-biotin conjugate may be treated to the control line at a concentration of preferably 0.2 to 0.4 mg / ml, more preferably 0.2 to 0.3 mg / ml, and even more preferably 0.25 mg / ml, but is not limited thereto.

[0039] The lateral flow assay kit provided in the present invention may be an immunostrip or FICT (fluorescent immunochromatographic test kit) having a sample injection portion for injecting a sample (specimen) by combining a pad made of glass fiber, cotton or cellulose material to a nitrocellulose membrane in the form of a strip, and having a condensation pad, a test line, a control line and an absorption pad sequentially positioned at a certain interval from the sample injection portion.

[0040] Additionally, the lateral flow assay kit of the present invention may include one or more other components, solutions, or devices suitable for the detection method. For example, it may include a test tube or other suitable container, a reaction buffer, and the like.

[0041] Additionally, the kit of the present invention may further include a user guide describing optimal reaction conditions. The guide is a printed document explaining how to use the kit, such as how to prepare a buffer solution, the proposed reaction conditions, etc. The guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and descriptions on the surface of the package containing the kit. Furthermore, the guide includes information disclosed or provided through electronic media, such as the Internet.

[0042] The lateral flow test kit for detecting Brucella abortus of the present invention amplifies a target sequence by a primer set for loop-mediated isothermal amplification (LAMP) including a FIP primer conjugated with FITC when Brucella abortus nucleic acid is present in a sample (specimen), and a probe having biotin linked to both ends specifically binds to the target sequence of the LAMP amplification product, so that the final amplicon contains both FITC and biotin. Therefore, when the LAMP product of a positive sample is applied to a lateral flow strip, FITC in the amplicon is detected by an anti-FITC antibody located at the test line, thereby confirming a positive reaction.

[0043] The present invention also provides a method for diagnosing bovine brucellosis, comprising: isolating nucleic acid from a biological sample of a bovine suspected of having brucellosis; amplifying a target sequence through a loop-mediated isothermal amplification reaction using the isolated nucleic acid as a template and a primer set for loop-mediated isothermal amplification consisting of the base sequences of SEQ ID NOs: 10 to 15 and a probe consisting of the base sequence of SEQ ID NO: 16; and applying a product of the amplification step to a lateral flow strip to detect a signal.

[0044] In the diagnostic method according to the present invention, the biological sample may be selected from the group consisting of blood, nasal fluid, saliva, nasal mucus, sputum, urine, and combinations thereof, but is not limited thereto. A method for obtaining body fluids and tissues from a mammal may utilize a conventional method known in the art. In addition, a method for isolating the nucleic acid may utilize a method known in the art, and for example, the CTAB (Cetyltrimethylammonium Bromide) method may be utilized, or the Wizard prep kit (Promega) may be utilized, but is not limited thereto.

[0045] In the diagnostic method according to the present invention, the primers, probes, and lateral flow strips for ring-mediated isothermal amplification are as described above.

[0046] In the diagnostic method according to one embodiment of the present invention, the ring-mediated isothermal amplification reaction may be performed at 57 to 58°C for 48 to 52 minutes, but is not limited thereto. In addition, when applying the product of the amplification step to the lateral flow strip, it may be preferable to dilute the amplification product 1 / 3 times in a developing solvent [3X PBS (Phosphate-buffered saline) containing 0.1% SDS, 0.1% PVP, and 0.2% Tween 20]) and apply it to the sample injection port, but is not limited thereto.

[0047]

[0048] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0049]

[0050] Example 1. Development of a loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA) for rapid diagnosis of bovine Brucella.

[0051] To develop a highly sensitive and rapid diagnostic technology for bovine Brucella, a lateral flow strip based on loop-mediated isothermal amplification (LAMP) was designed as shown in Fig. 1. Through a literature review, the BruAb2_0168 gene (SEQ ID NO: 1), which was most frequently used in the LAMP method for detecting bovine Brucella, was selected as the target gene. After confirming the reactivity using three LAMP primer sets developed and reported for the BruAb2_0168 gene, primer set 3 was finally selected. Considering the possibility of application to LFA during oligonucleotide synthesis, FITC (fluorescein isothiocyanate) was tagged at the 5' end of the FIP primer.

[0052] LAMP primer set for detection of bovine brucellosis Base sequence (5'-3') Sequence number LAMPprimer 1F3GGG TGG AAC GAC CTT TGC2B3ATT GCC ACC AAT CTC TCC G3FIPCAA AAC CTT GGC TGT CAC CGC TTG GCA GTC TGG TGC AGA A4BIPCGG TAC GAC CAC GGT GTC GAC ACC GCT ATT CAC CGT CAC5LAMPprimer 2F3GCG GAA GCA AGA ATG ACC6B3TCT TAC CTT GTG ACG CCT G7FIPGGA CTG CGT TGA GTA CGC ATC CGC AAG CAC AGA CCA CAG C8BIPCGC TCT TAC AAG TCT TGC GCC CTT CTG AGA TGT GCC CAC TG9LAMPprimer 3F3GGG CTT ACG CTT GGA TCG10B3CGT AGC TGG GGC TAT TCG T11LFTCG TTG ACC TGC TGG TTG AT12LBGGC AAT GGC ACC TGG ACA13FIPAGT GGC GCC AGT AGT ATT GAC GTT TTT GAG CGT GGA CAA GGT GT14BIPCGC AGG GCG ATG GTC TGT TGC TTC AGT TGA AGT TGC CCG TAG15

[0053] LAMP reaction composition ComponentStock conc.Volume (μl)Nuclease-free water-2110 template8.85×10 3 CFU / ㎖6Total volume (㎕)50

[0054]

[0055] Example 2. Optimization of conditions for the development of a rapid diagnostic LAMP-LFA assay for bovine brucellosis.

[0056] 2-1. Probe design

[0057] The BruAb2_0168 gene-specific DNA probe used in the experiment was designed to target the BIP site so that it can bind to the loop structure of the molecular beacon. Considering the possibility of application to LFA and the possibility of non-specific reaction, biotin was tagged at the 5' and 3' ends, and 5 adenine bases were placed in the spacer. LAMP reaction was performed using the designed DNA probe (biotin-AAA AAG ACA TAG ATA TCG TTG ACC TGC TGG TTG AT-biotin; SEQ ID NO: 16), and the reaction product was applied to LFA strip [Absorbent pad (AP22, Grade 222, Boreda Biotech), sample pad (Glass Fiber, Grade 8964), nitrocellulose membrane (LFT Membrane, LFNC-C-HS03-15μ, Nupore Filtration System Pvt. Ltd.), backing card (PJ EGO Co., Ltd.)]. As a result, no nonspecific reaction was observed, and 57.5℃, which had the highest nucleic acid amplification efficiency and probe hybridization efficiency, was selected as the optimal reaction temperature (Fig. 3).

[0058]

[0059] 2-2. Primer and probe concentration

[0060] ① Primer concentration

[0061] The concentration ratio of the internal primer set (F3+B3) and the external primer set (FIP+BIP) of the LAMP primer was adjusted as shown in Table 3, and the LAMP reaction was performed using primer set 3 in Table 1. As a result, condition 3, which produced the most ideal reaction in the electrophoresis and strip results, was selected as the optimized condition (Fig. 4).

[0062] Experimental conditions for determining the optimal concentration of primers Concentration of primers (Inner: outer primers, μM) Condition 1 Condition 2 Condition 3 Condition 4 40:10 30:7.5 20:5 10:2.5

[0063] In addition, the detection limit was confirmed based on the changed conditions, and the primer concentration of the above-established condition 3 was the detection limit (8.85×10 0 It was confirmed that it did not affect the CFU / ㎖ (Fig. 5).

[0064]

[0065] ② Concentration of the probe

[0066] As a result of performing the LAMP reaction by varying the concentration of the DNA probe whose structure was optimized in Example 2-1, condition 2, which showed the highest intensity in the test line (TL) of the LFA strip, was selected as the optimized condition (Fig. 6).

[0067] Experimental conditions for determining the optimal concentration of the probe Concentration of the probe (μM) Condition 1 Condition 2 Condition 3 Condition 4 20 10 5 2.5

[0068]

[0069] 2-3. Components of the lateral flow strip

[0070] In the present invention, gold nanoparticles (GNP) were synthesized according to the literature data reported by Frens, G (Nature Physical Science (1973) 241;2022), and the wavelength of the synthesized colloidal gold solution was scanned (400-800 nm), and the peak SPR wavelength (PSW; nm) of the solution was observed to confirm the size of the gold nanoparticles. In the present invention, it was decided to use 30 nm gold nanoparticles, and it was confirmed that the stability of the GNP-streptavidin (SA) conjugate was maintained under the condition that 40 ㎕ or more of streptavidin (streptavidin unlabeled, EDM Millipore Corp., cat. no. SA101) was added to 500 ㎕ of colloidal gold solution.

[0071] In addition, in the present invention, a nitrocellulose (NC) membrane having a capillary flow time of 90±23-180±45 sec / 4cm was manufactured to a width of 3 mm and used as a strip for LFA based on preliminary experimental results.

[0072]

[0073] ① Concentration of anti-FITC antibody treated on the test line (TL) of the nitrocellulose (NC) membrane

[0074] In order to establish the optimal concentration of anti-FITC antibody (HyTest, cat. no. 5F3cc) to be treated in TL, reaction results were analyzed in the range of 0.35-2.8 mg / ml, and the concentration of 1.4 mg / ml, which showed the highest intensity in TL without nonspecific reaction, was finally selected (Fig. 7).

[0075]

[0076] ② Albumin-biotin concentration treated in the control line (CL) of the nitrocellulose (NC) membrane

[0077] To establish the optimal concentration of albumin-biotin (product name: albumin, biotin labeled bovine, Sigma-Aldrich, cat. no. A8549-10MG) to be treated in CL, the reaction results were analyzed in the range of 0.0625-0.5 mg / mL. Since clear dots could be observed in CL when albumin-biotin was used at a concentration of 0.5 mg / mL through preliminary experiments, a solution serially diluted by two times from that concentration up to 0.0625 mg / mL was tested. As a result of the analysis, the concentration of 0.25 mg / mL, which showed the highest intensity with the minimum amount, was finally selected (Fig. 8).

[0078]

[0079] ③ Running buffer for LFA

[0080] Experiments were conducted on the three buffers below to determine the developing solvent to be applied to the lateral flow strip. As a result, Condition 1, which used a buffer containing surfactants such as PVP (Polyvinylpyrrolidone), SDS (sodium dodecyl sulfate), and Tween 20 as the developing solvent, yielded clear experimental results without nonspecific reactions, and thus the buffer condition was selected as the final developing solvent (Fig. 9).

[0081] Buffers used to select a developing solvent for LFA Type of running buffer Condition 1 3X PBS (0.1% SDS, 0.1% PVP and 0.2% Tween 20 added) Condition 2 1X PBS (0.05M, pH 7.4) Condition 3 TE buffer (pH 8.0)

[0082] In addition, as a result of applying the LAMP amplification product to the strip by diluting it by various multiples using the developing solvent of the above-mentioned condition 1, it was determined that it would be good to dilute the LAMP amplification product by 1 / 3.

[0083] In the present invention, an experiment was performed by fixing the nitrocellulose membrane of the present invention to a commercially available test strip cassette (Kang-jia, cat. no. KJ7015).

[0084]

[0085] Example 3. Evaluation of the LAMP-LFA method under optimized conditions.

[0086] Based on the conditions optimized through Example 2, 0-8.85×10 9 Genomic DNA extracted from B. abortus at CFU / ㎖ was added as a template to prepare a LAMP mixture, and the LAMP reaction was performed at 57.5°C for 40, 50, or 60 minutes, respectively. The analysis results showed that 8.85×10 6 It was confirmed that it was detected from a sample of CFU / ㎖, and 8.85×10 for a reaction time of 50 minutes or more. 0 It was confirmed that detection was possible from a sample of CFU / ㎖, and the optimal reaction time was determined to be 50 minutes (Fig. 10).

[0087] In addition, the cross-reactivity of the LAMP-LFA method according to the present invention was confirmed for various standard strains, including the main causative bacteria that immunologically cross-react with bovine Brucella. The strains used in the reaction were Brucella abortus, Yersinia enterocolitica, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes, and Vibrio parahaemolyticus, and each was administered at a concentration of 1×10 8 Genomic DNA extracted from a bacterial count of CFU / ㎖ was used as a template. As a result of the analysis, as shown in Figure 11, the LAMP-LFA method according to the present invention showed a specific positive reaction only for Brucella abortus, a bovine Brucella, without cross-reactivity.

[0088] To verify the applicability of the LAMP-LFA method to LAMP amplification products that produced a precipitate (magnesium pyrophosphate; Mg2P2O7), the developed strip was tested with positive control samples with precipitate, positive control samples without precipitate, and negative control samples without precipitate. As a result, all positive samples showed positive results in the TL of the strip developed regardless of the presence or absence of precipitate (Fig. 12).

[0089]

[0090] Example 4. Evaluation of the LAMP-LFA method using plasma samples

[0091] Ten Brucella abortus-positive plasma samples and two B. abortus-negative plasma samples (foster cat plasma samples and native cat plasma samples) were provided by the College of Veterinary Medicine, Gyeongsang National University, and the LAMP-LFA method of the present invention was evaluated. As a result of the analysis, all ten B. abortus-positive plasma samples were confirmed to be positive (Fig. 13), and the negative samples were also confirmed to be negative (Fig. 14).

[0092] Although the LFA diagnostic kit has the advantage of being a diagnostic technology that can be easily and conveniently used by non-experts within a short period of time, it has the characteristic of showing cross-reactivity with pathogenic bacteria such as viruses, Y. enterocolitica O9, Escherichia coli O157, and Salmonella typhimurium morphologically and immunologically, and in particular, frequently producing false positive results.

[0093] Here, the inventors analyzed the performance of the LAMP-LFA method according to the present invention using 50 false-positive Brucella abortus plasma samples provided by the Animal and Plant Quarantine Agency. A commercially available PCR kit (Bioneer, AccuPower Brucella PCR kit, cat. no. K-2906) and Abbexa's Brucella Antibody Rapid Test kit (cat. no. Abx092069) were used for comparison. As a result of the analysis, as disclosed in Fig. 15, the commercial PCR kit showed positive reactions for 5 false-positive samples (Fig. 15a), while the LAMP-LFA method according to the present invention showed positive reactions for 6 false-positive samples (Fig. 15b). In addition, the commercial Rapid Test kit was observed to show positive reactions for 20 false-positive plasma samples (Fig. 15c). Through the above results, it was found that the LAMP-LFA method according to the present invention is superior in false-positive correction effect compared to existing rapid diagnostic kits.

Claims

1. A lateral flow assay kit for detecting Brucella abortus, comprising: a primer set for loop-mediated isothermal amplification; a probe that specifically binds to an amplification product amplified by the primer set; and a lateral flow strip.

2. A kit according to claim 1, wherein the primer set for ring-mediated isothermal amplification comprises a base sequence of sequence numbers 10 to 15.

3. A kit characterized in that, in the first paragraph, the probe has a structure in which biotin, a spacer, a base sequence specific to a ring-mediated isothermal amplification product, and biotin are sequentially connected.

4. A kit characterized in that, in the third paragraph, the base sequence specific to the ring-mediated isothermal amplification product consists of the base sequence of sequence number 16.

5. A kit according to claim 3, wherein the spacer is composed of 4 to 6 adenine bases.

6. A kit according to claim 1, wherein the lateral flow strip is a nitrocellulose membrane in the form of a strip, comprising: a conjugate pad treated with a gold nanoparticle-streptavidin conjugate; a test line on which an anti-FITC (fluorescein isothiocyanate) antibody is fixed; and a control line on which an albumin-biotin conjugate is fixed.

7. A step of isolating nucleic acid from a biological sample of a cow suspected of having brucellosis; A step of amplifying a target sequence by a loop-mediated isothermal amplification reaction using the above-mentioned separated nucleic acid as a template, a primer set for loop-mediated isothermal amplification consisting of the base sequences of SEQ ID NOs: 10 to 15, and a probe consisting of the base sequence of SEQ ID NO: 16; and A method for diagnosing brucellosis in cattle, comprising: a step of applying the product of the above amplification step to a lateral flow strip to detect a signal; 8. A diagnostic method according to claim 7, wherein the biological sample is selected from the group consisting of blood, nasal fluid, saliva, nasal mucus, sputum, urine, and combinations thereof.

9. A diagnostic method according to claim 7, wherein the primer for ring-mediated isothermal amplification consisting of the base sequence of sequence number 14 has FITC (fluorescein isothiocyanate) bound to the 5' end.

10. A diagnostic method according to claim 7, wherein the probe has a structure in which biotin, a spacer, a base sequence specific to a ring-mediated isothermal amplification product, and biotin are sequentially connected.

11. A diagnostic method according to claim 7, characterized in that the ring-mediated isothermal amplification reaction is performed at 57 to 58°C for 48 to 52 minutes.

12. A diagnostic method according to claim 7, wherein the lateral flow strip is a nitrocellulose membrane in the form of a strip, comprising: a conjugate pad treated with a gold nanoparticle-streptavidin conjugate; a test line on which an anti-FITC (fluorescein isothiocyanate) antibody is fixed; and a control line on which an albumin-biotin conjugate is fixed.

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