CN111505278A - Staphylococcus aureus detection test strip, detection method and application - Google Patents
Staphylococcus aureus detection test strip, detection method and application Download PDFInfo
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- CN111505278A CN111505278A CN202010294483.6A CN202010294483A CN111505278A CN 111505278 A CN111505278 A CN 111505278A CN 202010294483 A CN202010294483 A CN 202010294483A CN 111505278 A CN111505278 A CN 111505278A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
A test paper strip for detecting staphylococcus aureus, a detection method and application thereof are provided, the test paper strip comprises a lining plate, a nitrocellulose membrane is pasted on the lining plate, a detection line is arranged on a detection area of the nitrocellulose membrane, and pig immunoglobulin is coated on the detection line. The invention only uses the pig immunoglobulin to scratch on the nitrocellulose membrane for direct detection, breaks through the traditional detection method which needs to adopt the antibody, greatly saves the cost, avoids the complex processes of producing the antibody and matching, improves the stability and the sensitivity, and is simpler, more convenient and more novel.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a staphylococcus aureus detection test strip, a detection method and application.
Background
Staphylococcus aureus is a widespread gram-positive pathogen that under the right conditions can pose a serious threat to human health. It is one of the major food-and iatrogenic pathogens that may cause various dangerous infectious diseases such as sepsis, osteomyelitis, pneumonia, toxic shock syndrome and endocarditis. The method can quickly, accurately, sensitively and simply detect the staphylococcus aureus, and has important significance in the aspects of medical health, food health, animal epidemic disease monitoring and the like.
In recent years, new methods have been developed that rely on various recognition molecules (e.g., aptamers, antibodies, and phage) that can specifically bind to bacterial cells, including enzyme-linked immunoassays, surface plasmon resonance, fluorescence detection, electrochemical measurements, Surface Enhanced Raman Scattering (SERS), and the like. Although the above methods are powerful and accurate, the labor intensive operations, time consuming culturing and extraction and the requirement for extensive laboratory equipment all limit the application in point of care (POCT) and rapid screening.
In order to improve the sensitivity of the test strip detection method, different labels such as fluorescent nanospheres, quantum dots, magnetic microspheres, up-conversion nanomaterials, graphene, composite materials and the like are used; although the sensitivity is improved, the synthesis method of the nano materials is complex and the synthesis conditions are high. In addition, antibodies are often used in bacterial assays, and the antibody generation and paired antibody screening process is a laborious operation, time consuming and extensive laboratory equipment, all of which limit the widespread use of antibody-based lateral flow assay strips for rapid assays.
Therefore, further efforts are urgently needed to develop a stable, low-cost signal carrier, a detection method that simplifies the analysis steps and is easy to handle, while improving stability, sensitivity and specificity.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the staphylococcus aureus detection test strip, the detection method and the application provided by the invention have the advantages that the pig immunoglobulin is scratched on the nitrocellulose membrane for direct detection, bright red strips can be obtained only by simply incubating bacteria and gold nanoparticles solidified with vancomycin, the traditional detection method needing an antibody is broken through, the cost is greatly saved, the complicated processes of antibody production and pairing are avoided, the stability, the sensitivity and the specificity are improved, and the method is simpler, more convenient and more novel.
In order to achieve the technical effects, the technical scheme adopted by the invention is as follows:
the test paper strip for detecting staphylococcus aureus comprises a lining plate, wherein a nitrocellulose membrane is attached to the lining plate, a detection line is arranged on a detection area of the nitrocellulose membrane, and the detection line is coated with porcine immunoglobulin.
Preferably, one end of the nitrocellulose membrane covers the water absorption pad, the other end of the nitrocellulose membrane covers the sample pad and the combination pad in sequence, and the sample pad and the combination pad are respectively sealed by sealing liquid; the detection zone is a non-covered surface of the nitrocellulose membrane.
Preferably, the preparation method of the detection line coated with the pig immunoglobulin comprises the steps of dissolving the pig immunoglobulin in a coating solution to prepare a coating solution of 4mg/m L, coating the coating solution on the detection line of the nitrocellulose membrane at the speed of 1 mu L/cm, and adding water into the coating solution of 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach the volume of 100m L to obtain the detection line coated with the pig immunoglobulin.
A detection method of staphylococcus aureus, a detection method of the test strip.
Preferably, the method comprises the steps of mixing gold nanoparticles solidified with vancomycin with a sample to be detected to obtain a liquid to be detected, and determining staphylococcus aureus in the liquid to be detected through the color displayed by the contact of a test strip coated with porcine immunoglobulin and the liquid to be detected.
Preferably, the gold nanoparticles solidified with vancomycin are prepared by mixing chloroauric acid solution and vancomycin solution, and then adding sodium hydroxide solution for reaction.
Preferably, the vancomycin solution and the chloroauric acid solution in a molar ratio of 1:2 are mixed, and the sodium hydroxide solution is added to be stirred for 24 hours under the condition of keeping out light and at room temperature.
Preferably, the mass percentage concentration of the vancomycin-immobilized gold nanoparticles in the solution to be detected is 20%.
Preferably, the amount of pig immunoglobulin is 4mg/m L.
The test strip for detecting staphylococcus aureus or the method for detecting staphylococcus aureus are applied to detecting staphylococcus aureus in drinking water.
Due to the adoption of the technical scheme, the method has the following beneficial effects:
(1) the test strip for detecting staphylococcus aureus has high sensitivity, the minimum detection limit of the test strip for detecting staphylococcus aureus is 103 cfu.m L-1, and the value of the test strip is lower than that reported in some literatures.
(2) The staphylococcus aureus detection method does not need to be marked, only needs to simply incubate the staphylococcus aureus and the gold nanoparticles solidified with the vancomycin, and obtains bright red strips by specifically combining the liquid to be detected obtained by incubation and the immunoglobulin of the pig, thereby avoiding complex marking process, breaking through the traditional sandwich detection method which needs to adopt the antibody, greatly saving cost, solving the problem of difficulty in screening the antibody, and being quicker, simpler, more convenient and novel.
(3) The staphylococcus aureus detection method disclosed by the invention is high in specificity, and the used pig immunoglobulin can only identify the protein A on the surface of the staphylococcus aureus with high specificity and has no specificity to other bacteria of non-staphylococcus aureus.
(4) The staphylococcus aureus detection method and the detection test strip can detect staphylococcus aureus in drinking water, have the same sensitivity as a standard strain, can reach 103 cfu.m L-1, and have good application prospect.
Drawings
FIG. 1 is a schematic diagram of the assembly of a lateral flow test strip for rapid detection of Staphylococcus aureus according to the present invention;
FIG. 2 is a schematic flow chart of the lateral flow test strip for rapid detection of Staphylococcus aureus according to the present invention;
FIG. 3 is a graph showing the results of example 2;
FIG. 4 is a graph showing the results of example 3;
FIG. 5 is a graph showing the results of example 4;
FIG. 6 is a graph showing the results of example 5;
the invention is described in detail below with reference to the drawings and the detailed description.
Detailed Description
The working principle of the invention is as follows: based on the antibiotic affinity principle, gold nanoparticles solidified with vancomycin are enriched on the surface of staphylococcus aureus, mixed bacterial suspension is dripped onto a test strip, incubated bacteria migrate along a nitrocellulose membrane due to capillary action, and are captured by immunoglobulin of a T line based on the principle that the specific binding of protein A on the surface of the staphylococcus aureus and the immunoglobulin of a pig causes the T line to display a strong red strip. Through the process, staphylococcus aureus can be effectively captured through the immune globulin on the T line, the traditional test strip form requiring antibodies is broken through, only the immune globulin is used, the cost is greatly reduced, and highly sensitive detection is realized. In addition, the simple sensor greatly simplifies the early preparation process of immunoassay-related assay reagents, such as the preparation process of conventional probes. The device has the advantages of high sensitivity, strong specificity, simple structure, high cost benefit, no need of equipment, quick analysis time and portability, and great application potential to meet the requirements of POC diagnosis and determination. The method is successfully applied to the detection of staphylococcus aureus in drinking water, and the practicability and the applicability of the method are verified.
The gold nanoparticles with vancomycin solidified can be prepared by the existing method: and mixing the vancomycin solution and the chloroauric acid solution to obtain the gold nanoparticles with the vancomycin solidified under the action of the sodium hydroxide solution. The porcine immunoglobulin may be a commercially available product.
Compared with the traditional method, the preparation method does not need any complex material to label the probe of the antibody; the antibody is not used, and only the pig immunoglobulin is used, so that the difficulty of antibody production and pairing is solved; the preparation process is greatly simplified, the structure is simple, the probe does not need to be loaded on the bonding pad in advance, only the detection line is needed, and no quality control line exists. The method is novel, simple, rapid, low in cost and short in manufacturing period, the whole process is completed within 20 minutes, the method can be used for detecting actual sample drinking water, and the immunoglobulin can be used as a method for detecting staphylococcus aureus and is suitable for field detection.
Example 1: preparation method of gold nanoparticles solidified with vancomycin
The preparation method of the gold nanoparticles solidified with the vancomycin comprises the following steps of preparing by direct one-step reaction between the vancomycin and the chloroauric acid, uniformly mixing 14M L vancomycin (4mM) and 14M L chloroauric acid aqueous solution (8mM) and stirring for 10 minutes, dripping 140 mu L NaOH (10M), stirring the solution for 24 hours at room temperature in the dark, and collecting the obtained deep red solution, namely the gold nanoparticle solution solidified with the vancomycin.
Example 2: optimization of each condition of test strip for rapidly detecting staphylococcus aureus
1) Preparation of nitrocellulose membranes
Coating the detection line, dissolving pig immunoglobulin in 0.01 mol/L phosphate buffer solution to prepare 4mg/m L coating solution, transversely coating the coating solution at a speed of 1 mu L/cm at a position 30mm away from the cellulose nitrate film (namely on the detection line) by a scribing mode, and then drying for 30 minutes at 37 ℃.
2) The preparation of the sample pad comprises cutting the glass fiber membrane into pieces with the length of 15mm and the width of 3mm, soaking in a sealing solution, drying at 37 deg.C for 10-16 hr to obtain the sample pad, and storing in a drier at room temperature, wherein the sealing solution is prepared by adding 2g bovine serum albumin water to constant volume of 100m L.
3) Preparation of the bonding pad: cutting the glass fiber membrane into pieces with length of 8mm and width of 3mm, soaking in sealing solution, taking out, drying at 37 deg.C for 10-16 hr, and storing at room temperature in a dryer.
4) Preparation of absorbent pad
Cutting the absorbent paper into pieces with the length of 18mm and the width of 3mm to obtain the absorbent pad.
5) The microbial culture comprises the steps of respectively inoculating activated staphylococcus aureus to L B culture mediums, standing at 37 ℃ for 24 hours, selecting single colonies, inoculating the single colonies to 250m L L B broth culture mediums, culturing at 37 ℃ for 24 hours by a 150r/min shaking table, centrifuging bacteria liquid at 4000r/min for 15 minutes, collecting thalli, respectively washing the thalli for 3 times by phosphate buffer solution with the pH of 7.4 of 0.01 mol/L, re-suspending by phosphate buffer solution with the concentration of 10m L0.01.01 mol/L, adding 0.5% formalin solution, standing at room temperature for 24 hours for inactivation, respectively washing for 3 times by phosphate buffer solution with the concentration of 0.01 mol/L after inactivation is finished, adjusting the proper concentration by PBS with the concentration of 0.01 mol/L, and adjusting the antigen concentration to 108cfu·mL-1And storing at-20 deg.C for use.
6) Assembling the test strip: firstly, attaching the nitrocellulose membrane to a lining plate, then pressing the sample pad by 1-3mm, pressing the nitrocellulose membrane by 1-3mm, and sequentially attaching the nitrocellulose membrane by 1-3mm through the water absorption pad to the lining plate, thus obtaining the immunochromatography test strip for rapidly detecting salmonella enteritidis.
7) Optimization of immunoglobulin coating amount in pig
The coating amount of immunoglobulin can affect the signal intensity on the detection line, and different concentrations of immunoglobulin are coated on the detection line, and the concentration range is 2-6 mg.m L-1。
8) Optimization of time for incubation of nanomaterials with bacterial solutions
The time of incubation may also affect the signal intensity on the detection line. The vancomycin-immobilized gold nanoparticle solution and the bacterial mixture were incubated for various times ranging from 0 to 120 seconds.
9) Optimization of volume of vancomycin-immobilized gold nanoparticle solution
The volume of the gold nanoparticle solution with vancomycin immobilized has a significant effect on the capture effect of bacteria. At a fixed bacterial concentration of 108cfu·mL-1In the following, different material volumes of 10 to 30 μ L, respectively, were investigated.
10) Optimization of the ratio of the reinforcing fluids
Another important parameter affecting the strength of the test line after reinforcement is the ratio of the reinforcement liquid at a fixed hydroxylamine hydrochloride concentration of 0.1 M. L-1And the volume is 100 mu L, the concentration of chloroauric acid is 25mmol L-1The different volumes below are 1, 3, 5 μ L, respectively.
As a result, referring to FIG. 3a, the color intensity on the T line gradually deepens as the concentration of immunoglobulin in swine increases until it reaches 4 mg.m L-1 and then is maintained at an almost constant level, and thus, the optimal coating amount of immunoglobulin is 4 mg.m L-1.
As shown in fig. 3b, the color intensity on the T-line did not change substantially with increasing incubation time, and therefore, no extra incubation was required to enhance the intensity of the detection line.
Referring to fig. 3c, when the addition amount of the vancomycin-cured gold nanoparticle solution was 20 μ L, the strength of the T-line was maintained at an almost constant level, and thus the material addition amount of 20 μ L was taken as an optimum condition.
Referring to FIG. 3d, when the amount of chloroauric acid solution added was 3 μ L, i.e., the ratio was 1: 3, the difference between the positive and negative values on the T-line was the greatest, and thus the optimal ratio of hydroxylamine hydrochloride to chloroauric acid was 1: 3.
Example 3: sensitivity determination of test strip for rapidly detecting staphylococcus aureus
The test strip preparation and bacterial culture process steps are the same as 1) -6) in example 2.
7) And (3) detection process: diluting the bacterial liquid to 10% with 0.01M phosphate buffer solution1-108cfu·mL-1Taking 100 mu L solution as detection solution at each concentration respectively, mixing and incubating with 20 mu L immobilized vancomycin gold nanoparticles, adding a sample pad of a test strip dropwise, taking 100 mu L0.01.01M phosphate buffer solution as negative control solution, adding the sample pad of another test strip dropwise in the same way as the above, taking out the test strip after 10 minutes, washing off redundant materials by using 100 mu L0.05.05% tween phosphate buffer solution, putting the test strip into 100 mu L freshly prepared enhancement solution, reacting for 5 minutes, and reading the result.
And (3) detection results: (1) Positive: when the detection line of the detection test strip shows a colored line, the result is positive, which indicates that the concentration of the staphylococcus aureus in the sample to be detected is higher than or equal to 103cfu·mL-1. (2) Negative: when the detection line of the detection test strip does not display color, judging as a negative result, and indicating that the staphylococcus aureus in the sample to be detected is less than 103cfu·mL-1。
As shown in FIG. 4, as the concentration of Staphylococcus aureus decreases, the red-black band of the T line of the test strip becomes lighter and lighter, and the concentration of the T line is 103cfu·mL-1Therefore, the invention can detect the staphylococcus aureus with the minimum concentration of 103cfu·mL-1. The method can detect the staphylococcus aureus with high sensitivity, can detect without an antibody, and is rapid and convenient.
Example 4: specificity determination of test strip for rapidly detecting staphylococcus aureus
The test strip preparation and bacterial culture process steps are the same as 1) -6) in example 2.
7) And (3) detection process: respectively diluting the bacterial liquid of Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, Vibrio parahaemolyticus, Shigella dysenteriae, Campylobacter and Enterobacter sakazakii to 10% with 0.01M phosphate buffer solution8cfu·mL-1The concentration of (A) is 10, which is obtained by diluting Staphylococcus aureus with 0.01M hydrochloride buffer solution7cfu·mL-1Taking 100 mu L solution as detection solution at each concentration respectively, mixing and incubating with 100 mu L solution of gold nanoparticles solidified with vancomycin, dropwise adding the solution into a sample pad of a test strip, taking 100 mu L0.01.01M phosphate buffer solution as negative control solution, operating the same way as the above, dropwise adding the solution into a sample pad of another test strip, and reading the result after 10 minutes.
And (3) detection results: (1) positive: and when the detection line of the detection test strip shows a line, judging the test strip to be positive. (2) Negative: and when the detection line of the detection test strip does not display the color, judging as a negative result.
Referring to FIG. 5, numerals 1-7 respectively represent different strains, such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, Campylobacter jejuni, Vibrio parahaemolyticus, Shigella dysenteriae; numeral 8 indicates a blank control. Except that the test strip T line for detecting the staphylococcus aureus has a bright band which can be seen by naked eyes, the test strip T line for detecting other bacteria has no color, which shows that the invention can identify the staphylococcus aureus with high specificity and has particularly high specificity.
Example 5: application of test strip for rapidly detecting staphylococcus aureus
The test strip preparation and bacterial culture process steps are the same as 1) -6) in example 2.
7) And (3) detection process: dispersing the gold nanoparticles solidified with the vancomycin in bacterial liquid with known concentration, and adding the gold nanoparticles into drinking water to form 101-108cfu·mL-1Taking 100 mu L solution as detection solution and mixing with 100 mu L solution of gold nanoparticles solidified with vancomycin, dropwise adding the solution to a sample pad of a test strip, taking 100 mu L drinking water as negative control solution, dropwise adding the solution to a sample pad of another test strip in the same operation as the above, and reading the result after 20 minutes.
And (3) detection results: (1) positive: when the detection line of the detection test strip shows a colored line, the result is positive, which indicates that the concentration of the staphylococcus aureus in the sample to be detected is higher than or equal to 103cfu·mL-1. (2) Negative: when the detection line of the detection test strip basically does not display the color, the result is judged to be negative, and the result shows that the salmonella enteritidis in the sample to be detected is less than 103cfu·mL-1。
As shown in FIG. 6, as the concentration of bacteria decreases, the red-black strip on the test strip becomes lighter and lighter, and the concentration of the red-black strip visible to the naked eye can reach 103cfu·mL-1Therefore, the invention can detect the staphylococcus aureus with the minimum concentration of 10 in the drinking water3cfu·mL-1Reflecting the good practical application value.
Claims (10)
1. The detection test strip for staphylococcus aureus is characterized in that the test strip comprises a lining plate, a nitrocellulose membrane is attached to the lining plate, a detection line is arranged on a detection area of the nitrocellulose membrane, and pig immunoglobulin is coated on the detection line.
2. The test strip for detecting staphylococcus aureus of claim 1, wherein one end of the nitrocellulose membrane covers the absorbent pad, the other end of the nitrocellulose membrane covers the sample pad and the combination pad in sequence, and the sample pad and the combination pad are respectively sealed by sealing liquid;
the detection area is a non-covered surface of the nitrocellulose membrane.
3. The test strip for detecting staphylococcus aureus in claim 1 or 2, wherein the detection line is coated with porcine immunoglobulin by a method comprising the steps of dissolving the porcine immunoglobulin in a coating solution to prepare a coating solution of 4mg/m L, and coating the coating solution on the detection line away from a nitrocellulose membrane at a speed of 1 μ L/cm;
the coating solution is prepared by adding 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate into water to reach the volume of 100m L.
4. A method for detecting Staphylococcus aureus, which is the detection method of the test strip of any one of claims 1 to 3.
5. The method for detecting staphylococcus aureus according to claim 4, wherein the method comprises the steps of mixing gold nanoparticles solidified with vancomycin with a sample to be detected to obtain a liquid to be detected, and determining the staphylococcus aureus in the liquid to be detected through the color displayed by the contact of a test strip coated with porcine immunoglobulin and the liquid to be detected.
6. The method for detecting staphylococcus aureus according to claim 5, wherein the vancomycin-immobilized gold nanoparticles are prepared by mixing a chloroauric acid solution and a vancomycin solution, and adding a sodium hydroxide solution for reaction.
7. The method for detecting Staphylococcus aureus according to claim 6, wherein the vancomycin solution and the chloroauric acid solution are mixed at a molar ratio of 1:2, and the mixture is stirred for 24 hours while adding the sodium hydroxide solution in the dark at room temperature.
8. The method for detecting staphylococcus aureus according to any one of claims 5-7, wherein the mass percentage concentration of the vancomycin-immobilized gold nanoparticles in the solution to be detected is 20%.
9. The method for detecting Staphylococcus aureus according to any one of claims 5-7, wherein the porcine immunoglobulin is present in an amount of 4mg/m L.
10. Use of the test strip for the detection of staphylococcus aureus according to claims 1-4 or the method for the detection of staphylococcus aureus according to claims 5-9 for the detection of staphylococcus aureus in drinking water.
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