Kit for diagnosing infection with Methicillin-resistant <i>Staphylococcus aureus </i>(MRSA) by detecting magnesium ions

Abstract

Provided is a diagnostic kit for determining infection with Methicillin-Resistant Staphylococcus aureus (MRSA) in a specimen, and a method for determining the infection with MRSA using the diagnostic kit is performed by visually observing a color change after LAMP reaction, and the color change is caused by a change in a magnesium concentration and confirmed using a specific dye compound which sensitively reacts with magnesium ions. The amplification of the MRSA DNA is performed using the loop-mediated isothermal amplification (LAMP), so that the diagnostic kit has advantages of being conveniently used anytime and anywhere and quickly diagnosing.

Claims

1. A kit for diagnosing an infection caused by Methicillin-Resistant Staphylococcus aureus (MRSA) through a change in a concentration of magnesium ions, the kit comprising: a dye compound selected from at least one of compounds represented by the following Chemical Formulas 3, 4, 7, and 9: ##STR00014## ##STR00015##

2. The kit of claim 1, wherein the MRSA is amplified by loop-mediate isothermal amplification (LAMP).

3. A method for diagnosing an infection caused by Methicillin-Resistant Staphylococcus aureus (MRSA) through loop-mediate isothermal amplification (LAMP) using at least one of compounds represented by the following Chemical Formulas 3, 4, 7, and 9, wherein each of the compounds represented by Chemical Formulas 3, 4, 7, and 9 forms a complex with magnesium in a LAMP reaction solution and color-developed wavelengths of the dye compounds represented by Chemical Formulas 3, 4, 7, and 9 are changed by a change in a magnesium concentration generated in the LAMP reaction process. ##STR00016## ##STR00017##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates results of evaluating the solubility and the sensitivity to magnesium ions of 9 types of compounds used in exemplary embodiments of the present invention.

(2) FIG. 2 illustrates results of evaluating color development according to the presence of MRSA by loop-mediated isothermal amplification (LAMP) of the compounds in accordance with exemplary embodiments of the present invention. Herein, P represents a positive control and N represents a negative control.

(3) FIG. 3 illustrates results of confirming the amplification of MRSA DNA by electrophoresis. Herein, P represents a positive control and N represents a negative control.

(4) FIG. 4 illustrates results of evaluating sensitivity to MRSA DNA of the compounds in accordance with exemplary embodiments of the present invention by loop-mediated isothermal amplification (LAMP). Herein, NC represents a negative control.

(5) FIG. 5 illustrates results of evaluating color development of a compound of Chemical Formula 7 to MRSA DNA. Herein, NC represents negative control.

(6) FIG. 6 illustrates results of confirming sensitivities in detecting MRSA according to an exemplary method of the present invention and detecting MRSA using a PCR reaction in the related art.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(7) Methicillin-resistant Staphylococcus aureus (MRSA) is bacteria mainly causing hospital-acquired infection such as wound infections, pneumonia, and sepsis. The MRSA is multiple drug resistant bacteria which are resistant to all β-lactam agents such as penicillin, cephalosporin, and carbapenem due to altered penicillin binding proteins (PBP 2a, PBP 2′) mainly generated by a mecA gene and resistant even to other types of antimicrobial agents such as macrolide, clindamycin, tetracycline, and aminoglycoside. Accurate and rapid bacterial identification and an antimicrobial susceptibility test are required for the selection of appropriate antimicrobial agents and management of hospital-acquired infection.

(8) As the accurate identification method of the MRSA, there are a method of directly detecting a mecA gene using a DNA probe or a polymerase chain reaction (PCR) as a molecular biological test method, a method of detecting a product of the mecA gene, PBP 2a using immunoblotting or immunoradiometric assay (IRMA), and the like. However, various conditions such as special equipments, expensive reagents, and complicated test counts and test time are not suitable to be used generally in general laboratories, and an antimicrobial susceptibility test, which has been traditionally used, requires an incubation time of 24 hours and has a difference in the expression of resistance depending on a type of culture medium or a culture condition, so that it is difficult to expect rapid and accurate results.

(9) Accordingly, exemplary embodiments of the present invention provide a method for visually determining infection and amplification of MRSA by detecting a change in the concentration of magnesium ions by a dye compound to detect the MRSA bacteria and has an important advantage that required cost and time are remarkably low compared with the related art. Further, exemplary embodiments of the present invention use loop-mediate isothermal amplification (LAMP) without using the PCR method to amplify a gene of MRSA so that the amplification time of the gene is remarkably short.

(10) Dyes used for detecting magnesium ions in the related art have many problems in that the color is developed (discolored) by magnesium only at a predetermined pH condition, and when the temperature or pH condition is changed, the color is developed to the same color as when magnesium exists even in the absence of magnesium ions, and thus reliability is deteriorated, and there is an inconvenience that the pH needs to be controlled every detection. Accordingly, the inventors of the present invention have developed a dye compound capable of visually confirming the presence of magnesium ions while being stable to changes in temperature and pH and dye compounds which are color-developed in a different concentration range of magnesium ions, thereby completing the present invention.

(11) Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, the following Examples should not be construed as limiting the scope of the present invention and will be described to help in the understanding of the present invention.

Synthesis Example 1. Preparation of Compound of Chemical Formula 2

(12) ##STR00005##

(13) 2-amino-4-nitrophenol (0.385 g, 2.50 mmol, 1 eq) is dispersed in 5.8 ml of distilled water. After 0.75 g of hydrochloric acid (35%) is added, the temperature is lowered to 0° C. by adding ice. 0.19 g of sodium nitrite is added and stirred at 5° C. or lower for 1 hour, and then 0.019 g of sulfamic acid is added and stirred for 3 minutes.

(14) After 4-hydroxynaphthalene-1-sulfonic acid (0.58 g, 2.59 mmol, 1.04 eq) is added to 9 ml of distilled water, the mixture is adjusted to pH 10 and fully dissolved, and then added to a reaction solution and stirred for 2 hours at pH 10 and 10° C. or lower.

(15) A material obtained by filtering the reaction solution is purified by silica gel column chromatography to obtain a pure compound (128 mg, 13.2%).

(16) R.sub.f=0.4 (RP-C18, acetonitrile/water 1:2 v/v)

(17) MALDI-TOF/MS, calculated value C.sub.16H.sub.11N.sub.3O.sub.7S 389.34, measured value 388.11

Synthesis Example 2. Preparation of Compound of Chemical Formula 3

(18) ##STR00006##

(19) 2-amino-5-nitrophenol (0.385 g, 2.50 mmol, 1 eq) is dispersed in 5.8 ml of distilled water. After 0.75 g of hydrochloric acid (35%) is added, the temperature is lowered to 0° C. by adding ice. 0.19 g of sodium nitrite is added and stirred at 5° C. or lower for 1 hour, and then 0.019 g of sulfamic acid is added and stirred for 3 minutes.

(20) 7-amino-4-hydroxynaphthaloene-2-sulfonic acid (0.628 g, 2.62 mmol, 1.05 eq) is added to 9.4 ml of distilled water and fully dissolved, and then the mixture is added to a reaction solution and stirred for 2 hours at 10° C. or lower.

(21) A material obtained by filtering the reaction solution is purified by silica gel column chromatography to obtain a pure compound (13 mg, 1.3%).

(22) R.sub.f=0.3 (RP-C18, acetonitrile/water 1:2 v/v)

(23) LC/MS, calculated value C.sub.16H.sub.12N.sub.4O.sub.7S 404.35, measured value 403.0

Synthesis Example 3. Preparation of Compound of Chemical Formula 4

(24) ##STR00007##

(25) 2-amino-4,6-dinitrophenol (0.498 g, 2.50 mmol, 1 eq) is dispersed in 7.4 ml of distilled water. After 0.75 g of hydrochloric acid (35%) is added, the temperature is lowered to 0° C. by adding ice. 0.19 g of sodium nitrite is added and stirred at 5° C. or lower for 1 hour, and then 0.019 g of sulfamic acid is added thereto and stirred for 3 minutes.

(26) 4-hydroxy-7-(phenylamino) naphthalene-2-sulfonic acid (0.817 g, 2.62 mmol, 1.05 eq) is added to 12.4 ml of distilled water and fully dissolved, and then the mixture is added to a reaction solution and stirred for 2 hours at 10° C. or lower.

(27) A material obtained by filtering the reaction solution is purified by silica gel column chromatography to obtain a pure compound (45 mg, 3.4%).

(28) R.sub.f=0.4 (RP-C18, acetonitrile/water 1:2 v/v)

(29) LC/MS, calculated value C.sub.22H.sub.15N.sub.5O.sub.9S 525.45, measured value 523.9

Synthesis Examples 4 to 8: Preparation of Compounds of Chemical Formulas 5 to 9 was Synthesized in the Similar Method to Synthesis Examples 1 to 3

Synthesis Example 4. Preparation of Compound of Chemical Formula 5

(30) ##STR00008##

(31) (70 mg, 0.7%)

(32) R.sub.f=0.2 (RP-C18, acetonitrile/water 1:2 v/v)

(33) LC/MS, calculated value C.sub.16H.sub.15N.sub.5O.sub.7S 421.38, measured value 419.0

Synthesis Example 5. Preparation of Compound of Chemical Formula 6

(34) ##STR00009##

(35) (200 mg, 22.5%)

(36) R.sub.f=0.5 (RP-C18, acetonitrile/water 1:2 v/v)

(37) LC/MS, calculated value C.sub.12H.sub.9N.sub.3O.sub.8S 355.28, measured value 350.9

Synthesis Example 6. Preparation of Compound of Chemical Formula 7

(38) ##STR00010##

(39) (40 mg, 2.9%)

(40) R.sub.f=0.8 (RP-C18, acetonitrile/water 1:2 v/v)

(41) LC/MS, calculated value C.sub.22H.sub.16N.sub.4O.sub.10S.sub.2 560.51, measured value 561.1

Synthesis Example 7. Preparation of Compound of Chemical Formula 8

(42) ##STR00011##

(43) (35 mg, 4.0%)

(44) R.sub.f=0.2 (RP-C18, acetonitrile/water 1:2 v/v)

(45) LC/MS, calculated value C.sub.16H.sub.10N.sub.4O.sub.6 354.27, measured value 352.9

Synthesis Example 8. Preparation of Compound of Chemical Formula 9

(46) ##STR00012##

(47) (110 mg, 9.4%)

(48) R.sub.f=0.7 (RP-C18, acetonitrile/water 1:2 v/v)

(49) LC/MS, calculated value C.sub.16H.sub.10N.sub.3NaO.sub.10S.sub.2 491.38, measured value 489.8

Synthesis Example 9. Preparation of Compound DKLS of Chemical Formula 10

(50) ##STR00013##

(51) 2-amino-4-nitrophenol-6-sulfonic acid (0.5 g, 2.14 mmol, 1 eq) is dispersed in 7.4 ml of distilled water. After 0.75 g of hydrochloric acid (35%) is added, the temperature is lowered to 0° C. by adding ice. 0.19 g of sodium nitrite is added and stirred at 5° C. or lower for 1 hour, and then 0.019 g of sulfamic acid is added and stirred for 3 minutes.

(52) 4-(benzamido)-5-hydroxynaphthalene-1,7-disulfonic acid (0.949 g, 2.24 mmol, 1.05 eq) is added to 12.4 ml of distilled water and fully dissolved, and then the mixture is added to a reaction solution and stirred for 2 hours at 10° C. or lower. A material obtained by filtering the reaction solution is purified by silica gel column chromatography to obtain a pure compound (64 mg, 4.5%).

(53) R.sub.f=0.5 (RP-C18, acetonitrile/water 1:2 v/v)

(54) LC/MS, calculated value C.sub.24H.sub.16N.sub.4O.sub.14S.sub.3 668.59, measured value 667.0

(55) Hereinafter, a method for checking the presence of magnesium ions to monitor an amplification process of MRSA bacteria using the loop-based isothermal amplification method (LAMP) using the dye compounds will be described using specific Examples. It is apparent that the scope of the present invention is not limited to the following Examples.

(56) <Selection of Dye Compounds>

(57) 1) Synthesis of Candidate Substances

(58) Compounds developed by magnesium ions through a compound library were screened, 70 types of candidate compounds were synthesized based on the screened compounds, and the candidate substances were selected through solubility and spectroscopic analysis and further modeled based on the structure of the selected compounds to finally obtain 9 types of compounds such as compounds of Chemical Formulas 2 to 9 and DKLS.

(59) 2) Solubility Test

(60) The solubility in water was measured to confirm the solubility of the synthesized candidate compounds. Specifically, 10 mg of hydroxynaphthalene blue (HNB), which is a compound known to be developed by magnesium ions as a control, and 10 mg of each of the candidate compounds were dissolved in 1 ml of deionized water, and then the solubility was confirmed. It was confirmed that the 9 types of compounds among 70 types of the candidate compounds and hydroxynaphthalene blue were dissolved in water. As a result, the 9 types of compounds showed excellent solubility in water.

(61) <Evaluation of Color Development Characteristics>

(62) 1) Preparation of Solution

(63) 200 mM of a magnesium chloride solution was prepared by dissolving 0.9521 g of magnesium chloride in 50 ml of deionized water.

(64) 59.99 mg of sodium phosphate monobasic and 70.98 mg of sodium phosphate dibasic were dissolved in 50 ml of distilled water to prepare 10 mM of a sodium phosphate buffer solution, and then 28.85 ml of a sodium phosphate monobasic solution and 21.15 ml of a sodium phosphate dibasic solution were mixed to prepare a sodium phosphate buffer solution at pH 7. In a similar manner, the two solutions were mixed to prepare sodium phosphate buffer solutions having acidity of pH 4, pH 7, pH 9, and pH 11, respectively.

(65) For determination of color development, the HNB and the candidate substance were prepared at concentrations of 240 μM and used for the test.

(66) 2) Preparation of Specimen

(67) Hydroxynaphthalene blue (HNB) and 9 types of water-soluble candidate substances selected by the solubility test were diluted with distilled water to prepare specimens at concentrations of 240 μM, respectively.

(68) 3) Evaluation of Magnesium Ion Detection Characteristics and pH Stability

(69) It was confirmed whether the candidate substances according to the present invention reacted with magnesium ions to be color-developed (discolored). In addition, pH stability was confirmed according to whether the candidate substances were specifically color-developed by reacting only with magnesium ions regardless of a pH change.

(70) The color development of the control (HNB) and 35 types of candidate substances was confirmed under conditions of blank, a MgCl.sub.2 solution, pH 4, pH 7, pH 9, and pH 11 using a 96 well plate (12×6 holes). Specifically, 100 μl of deionized water (blank) was added in a first row of the plate, 100 μl of the MgCl.sub.2 solution was added in a second row thereof, and 100 μl of each of solutions at pH 4, pH 7, pH 9, and pH 11 was added in third to sixth rows thereof using 10 mM of the sodium phosphate buffer solution prepared in step a) above. In columns of the plate, 100 μl of specimens prepared at a concentration of 240 μM were added to 6 rows, respectively, and a total of 36 specimens were screened. The final concentration of the MgCl.sub.2 solution contained in each well was 100 mM and the final concentration of the specimen was 120 μM.

(71) Each specimen showed a unique color in the deionized water (blank), and 9 types of compounds color-developed (discolored) in the presence of 200 mM MgCl.sub.2 were confirmed.

(72) In particular, the compounds of Chemical Formulas 3 and 4 were developed to a complementary color to be clearly distinguished from the blank in the presence of magnesium ions, and were developed to other colors completely different from the developed color in the presence of magnesium ion even in a pH change, and as a result, it was confirmed that the pH stability was excellent.

(73) The compound of Chemical Formula 3 was developed to orange or violet in the absence of magnesium ions, but was developed to dark blue only in the presence of magnesium. The compound of Chemical Formula 4 was developed to red in the absence of magnesium ions, but was developed to dark violet only in the presence of magnesium. As a result, there was a clear difference therebetween. On the other hand, it was evaluated that the compound of Chemical Formula 2 was developed to a color clearly different from that in the presence of magnesium ions under neutral to basic conditions, and had characteristics similar to those of the control HNB.

(74) On the other hand, hydroxynaphthalene blue (HNB), a representative color developing agent that was developed in the presence of magnesium ions, was discolored (developed) to a color similar to that in the presence of magnesium ions even in an acidity range of pH 7 to 11 in the absence of magnesium ions. Therefore, it was confirmed that the hydroxynaphthalene blue had a problem that it was inconvenient to control the specimen to be acidic, specifically around pH 4, in order to detect magnesium ions, and the reliability was lowered.

(75) Through the test, it was confirmed that the compounds of Chemical Formula 3 and 4 were dye compounds which were particularly effective for the detection of magnesium ions by being developed at a specific wavelength only in the presence of magnesium ions without being influenced by the pH change.

(76) 4) Evaluation of Temperature Stability

(77) It was confirmed whether the candidate substances according to the present invention had color changes depending on the temperature. 100 μl of the specimens (HNB and 35 types of candidate substances) prepared in step 2) and 100 μl of the deionized water were added into a 1.5 ml tube (final volume of 200 μl, final specimen concentration of 120 μM). After diluting well at room temperature, the color developed state was photographed, the mixture was incubated at 80° C. for 12 hours, and then the color change was observed.

(78) As a result of the test, the specimens according to exemplary embodiments of the present invention were not influenced by the temperature and the colors were preserved.

(79) 5) Evaluation of Molar Absorption Coefficient

(80) HNB and 9 types of candidate substances were dissolved in deionized water to prepare 100 μg/ml of a stock and then diluted with deionized water to prepare samples of 50, 25 and 12.5 μg/ml. Next, an absorption spectrum was obtained using an analyzer UV-vis spectrometer (Agilent 8453), and then absorption coefficient values at a concentration of 10 mg/ml were measured and shown in Table 1 below.

(81) TABLE-US-00001 No. Compound Molar absorption coefficient 1 HNB 13,000 2 Chemical Formula 2 12,000 3 Chemical Formula 3 44,000 4 Chemical Formula 4 31,000 5 Chemical Formula 5 33,579 6 Chemical Formula 6 18,000 7 Chemical Formula 7 25,000 8 Chemical Formula 8 5,117 9 Chemical Formula 9 23,000

(82) As shown in Table 1, it was confirmed that the compound of Chemical Formula 3 had the highest molar absorption coefficient of 44,000 and an absorption coefficient 3.38 times higher than that of HNB, and the compound of Chemical Formula 4 had an absorption coefficient of 31,000, which was 2.38 times higher than that of HNB, and thus it was evaluated that the compounds were industrially useful.

(83) <Evaluation of Magnesium Ion Sensitivity>

(84) 1) Evaluation of Magnesium Ion Sensitivity at Concentration of Less than 10 mM

(85) Through the evaluation, the compounds of Chemical Formulas 2, 3, and 4 were selected as excellent dye compounds for detecting magnesium ions. The sensitivities of the selected 4 types of compounds and HNB for detecting magnesium ions were evaluated.

(86) Magnesium chloride was dissolved in deionized water and diluted to 8, 10, 12, 14 and 16 mM, respectively, to prepare specimens. Next, as the dye compounds, the HNB and the compound of Chemical Formula 2 were prepared at a concentration of 240 μM, but the compounds of Chemical Formulas 3 and 4 were prepared to be diluted at a concentration of 120 μM by 50% because the molar absorption coefficient of the compounds was two times higher than that of the control (HNB).

(87) In each row of a 96-well plate, 1 ml of the dye compound was filled, and in each column, 1 ml of a diluted magnesium chloride solution was filled, and then the color changes were observed and absorbance was measured. The final concentrations of magnesium chloride in the 96-well plate were 0, 4, 5, 6, 7, and 8 mM, and the final concentrations of HNB and Chemical Formula 2 were 120 μM, respectively, and the final concentrations of Chemical Formulas 3 and 4 were 60 μM, respectively.

(88) The compound of Chemical Formula 3 exhibited a complementary color by detecting magnesium ions contained in a magnesium chloride solution diluted to a very low concentration of 4 mM, and thus the sensitivity to magnesium ions was very excellent. Therefore, detection of a small amount of magnesium ions is easy, and the industrial availability is very high.

(89) It was observed that in the dye compound represented by Chemical Formula 3 according to exemplary embodiments of the present invention, the absorption spectrum was shifted from 500 nm to 640 nm by magnesium ions. In addition, since the absorbance change is remarkable with respect to the magnesium ion concentration, magnesium contained in the measurement specimen may be quantitatively calculated through the absorbance evaluation.

(90) 2) Evaluation of Magnesium Ion Sensitivity at Concentration of 10 mM or More

(91) In order to evaluate the magnesium ion sensitivity for the HNB, and Chemical Formulas 2 and 4 which were not discolored at the magnesium ion concentration of less than 10 mM, an additional test was conducted in the same manner at a concentration of 10 mM or more. The magnesium concentration in the 96 well plate was confirmed from 10 mM to 100 mM, and the final concentration of the dye compound was set to 120 μM.

(92) The HNB started to be color-developed at a concentration of 10 mM or more, developed to violet at 20 to 30 mM, and developed to dark blue at 40 mM or more, the compound of Chemical Formula 2 was remarkably developed to cherry red instead of violet at 100 mM or more, and the compound of Chemical Formula 4 started to be developed from red purple to purple at a concentration of 10 mM and developed to blue violet at a concentration of about 20 mM.

(93) As in the test, the compound according to exemplary embodiments of the present invention reacts with magnesium ions to be discolored (developed) by shifting the absorption spectrum, and the lowest detection concentration of magnesium ions varies depending on the structure of the compound. Accordingly, the compounds having different lowest detection concentrations of magnesium ions according to exemplary embodiments of the present invention are treated to samples to be measured, and the concentration of magnesium ions contained in the samples may be visually confirmed by confirming the color development and the developed colors of the samples.

Example 1: Selection of Compounds for Configuring Methicillin-Resistant Staphylococcus aureus (MRSA) Detection Kit

(94) In Example 1, a mecA gene was selected as a target gene for detecting MRSA, and six primers of the mecA gene shown in Table 2 below were prepared.

(95) TABLE-US-00002 TABLE 2 <MRSA primer sequence> Oligomer name Base sequence F3 ATG ATT ATG GCT CAG GTA CTG B3 AAC CCA ATC ATT GCT GTT AAT ATT FIP TAC ATA AAT GGA TAG ACG TCA TCT ATC  CAC CCT CAA ACA GGT G BIP GGC ATG AGT AAC GAA GAA TAT AAT CCT  GGT GAA GTT GTA ATC TGG AAC LF ATG AAG GTG TGC TTA CAA GTG C LB CCG AAG ATA AAA AAG AAC CTC TGC T

(96) 1) Selection of Compound According to Solubility Test and Magnesium Sensitivity

(97) Various color developing dyes represented by Chemical Formula 1 above were dissolved in distilled water (D.W) and the solubility was tested to select a compound having high solubility. The selected compound was diluted at each concentration (100 uM, 50 uM, 25 uM, 12.5 uM, 6.25 uM, and 3.13 uM) and the reaction sensitivity with magnesium (magnesium concentration of 100 mM, 10 mM) by concentration was tested.

(98) As a result of the test, 9 types of compounds which had high solubility in D.W and sensitively reacted with magnesium were selected. FIG. 1 illustrates the selected 9 types of compounds (Chemical Formulas 2 to 9 and DKLS).

(99) 2) Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Loop-Mediated Isothermal Amplification (LAMP).

(100) Detection of MRSA was performed with respect to 9 types of compounds using loop-mediated isothermal amplification (LAMP). The LAMP has a cycle of enzymatic inactivation at 80° C. for 5 minutes after reaction at 63° C. for 30 minutes and is performed by reaction for total 35 minutes. After the LAMP, as illustrated in FIG. 2, in 4 types of compounds (Chemical Formulas 3, 4, 6, and 8), the changes were not observed, and in 5 types of compounds (Chemical Formulas 2, 5, 7, and 9, and DKLS), changes in color development were observed. It was confirmed that in the 5 types of compounds (Chemical Formulas 2, 5, 7, and 9, and DKLS), the MRSA detection is enabled using the LAMP.

(101) After confirming the color changes of the compounds, as a result of confirming the amplification of MRSA using a DNA electrophoresis method to confirm accurate MRSA amplification, as illustrated in FIG. 3, it was confirmed that in a positive control, the MRSA amplification was performed and in a negative control, the MRSA amplification was not performed. It was confirmed that in the compounds without color change, the MRSA amplification was performed and there was only no color change.

(102) 3) Detection Sensitivity Test of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Loop-Mediated Isothermal Amplification (LAMP).

(103) The sensitivity test of the selected 5 types of compounds was performed using LAMP. MRSA DNA was diluted 10-fold to be made at concentrations of 10 pg, 1 μg, 100 fg, and 10 fg, and then the sensitivity test was performed using the LAMP, and at this time, the concentrations of 5 types of compounds were the same as each other. As illustrated in FIG. 4, detection of up to 1 μg of DNA was enabled by all of the 5 types of compounds and detection of up to 100 fg of DNA was enabled by the compound of Chemical Formula 5.

(104) 4) Selection of Color Developing Dyes Constituting Kit

(105) Based on the results of the test of Example 1, compounds for constituting a MRSA detection kit were finally selected. The detection of up to 100 fg of MRSA is enabled by the compound of Chemical Formula 5, but it was confirmed that there was a problem that errors in detection during visual observation were caused due to color change. Therefore, a compound of Chemical Formula 7 which was easy to be visually detected due to a clear color change and can detect 1 μg of MRSA was selected.

Example 2: Comparative Verification of Detection Sensitivity

(106) 1) Comparative Verification of Detection Sensitivity of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Loop-Mediated Isothermal Amplification (LAMP)

(107) The excellence of a kit-constituting primer was verified by comparing a kit-constituting MRSA primer according to exemplary embodiments of the present invention and a primer reported in the paper (Yoshiki Misawa et al. 2007. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus in blood cultures. J infect Chemother. 13:134-14). The selected compound of Chemical Formula 7 was used and the MRSA DNA was diluted 10-fold to be made at concentrations of 1 ng, 100 μg, 10 μg, 1 μg, 100 fg, and 10 fg, respectively, and the comparative verification was performed by the LAMP reaction. As illustrated in FIG. 5, the kit-constituting MRSA primer was able to detect 1 μg of MRSA DNA. On the other hand, in the case of two types of MRSA primers reported in the paper, MRSA DNA was detected up to 1 ng by Primer 1 and 100 μg by Primer 2.

(108) In addition, comparative verification with a PCR method in the related art was performed. The excellence of exemplary embodiments of the present invention was verified by comparing a primer disclosed in the paper (Duarte C. Oliveira and Herminia de Lencastre. 2002. Multiplex PCR Strategy for Rapid Identification of Structural Types and Variants of the mec Element in Methicillin-Resistant Staphylococcus aureus. Antimicrob Agents Chemother. 46(7):2155-61) with a PCR method. MRSA DNA was diluted 10-fold to be made at concentrations of 10 ng, 1 ng, 100 μg, 10 μg, 1 μg, 100 fg, and 10 fg, and the same amount was added and reacted in the PCR method and the LAMP.

(109) As the test result, in the method of detecting MRSA using exemplary embodiments of the present invention, a total of required time was about 35 minutes and MRSA was able to be detected up to 1 μg. On the contrary, when using the PCR method in the related art, a total of required time was about 2 hours and MRSA was detected up to 100 μg. As a result, it was confirmed that the kit of exemplary embodiments of the present invention was able to confirm the infection with MRSA more efficiently than the related art, and the result was illustrated in FIG. 6.