CN113109566A - Fluorescence immunochromatography joint inspection method for food-borne pathogenic bacteria based on antigen colorization - Google Patents
Fluorescence immunochromatography joint inspection method for food-borne pathogenic bacteria based on antigen colorization Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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Abstract
The invention provides a fluorescence immunochromatographic joint inspection method for food-borne pathogenic bacteria based on antigen colorization, which comprises a coloring agent and a test strip, wherein a sample is subjected to coloring culture by the coloring agent and then is detected by the test strip; the staining agent is tetra [4- (4' -carboxyphenyl) phenyl ] ethylene, the test strip comprises a sample pad, a detection reaction area, a water absorption pad and a bottom plate, the sample pad, the detection reaction area and the water absorption pad are sequentially arranged on the bottom plate along the chromatography direction, the detection reaction area comprises at least three detection lines and an NC membrane, and the detection lines are arranged on the NC membrane in parallel at intervals. In the application, the fluorescence immunochromatographic combined detection method for the food-borne pathogenic bacteria based on antigen colorization is provided, shigella boydii, salmonella and escherichia coli O157: H7 are subjected to dyeing culture by using a dyeing agent, and combined detection is performed by using a test strip. The three detection lines can be used for capturing the three pathogenic bacteria dyed by the coloring agent and displaying the detection result, and can be used for detecting the three food-borne pathogenic bacteria independently and simultaneously in a combined manner, and have no cross reaction to other food-borne pathogenic bacteria and good specificity.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a fluorescence immunochromatographic joint inspection method for food-borne pathogenic bacteria based on antigen colorization.
Background
Food-borne diseases are diseases caused by ingestion of contaminated food products and are one of the most widespread health problems in the world today. The incidence of food-borne diseases is reported to be the second of the overall incidence of the various diseases. However, food-borne pathogenic bacteria are one of the main factors causing outbreak of food-borne diseases, mainly exist in beef, milk, chicken, vegetables, fruits and products thereof, and can cause death due to serious infection after being ingested by human bodies. Therefore, the method is particularly important for timely detecting the food-borne pathogenic bacteria in the food.
The immunochromatographic test strip is a cheap and portable technology suitable for on-site rapid detection, can realize accurate detection within a few minutes, and is widely applied to the fields of food supervision, agricultural production, environmental protection, clinical diagnosis and the like. The immunochromatographic test strip mostly adopts a double-antibody sandwich mode for detection, and adopts nano materials such as colloidal gold and the like to realize double functions of marking antibodies and reporting results, so that the quality of the test strip detection performance seriously depends on the stability of the nano materials, the pairing antibodies and the material marking antibodies. However, the preparation requirement of the labeling material with stable property, good dispersibility and uniform particle size is higher, the labeling material is not easy to obtain, the acquisition period of the monoclonal antibody which can be used for the double-antibody sandwich is long, the acquisition difficulty of the antibody with stable performance is higher, and the factors become the bottleneck of further development and application of the immunochromatographic test strip. In addition, most immunochromatographic test strips for detecting food-borne pathogenic bacteria are single-target detection of a single sample, that is, one test strip can only detect one pathogenic bacteria in one sample, and in the face of multiple pathogenic bacteria to be detected, respective corresponding test strips need to be developed, which undoubtedly increases the complexity of establishing a detection method.
Disclosure of Invention
The invention aims to provide a fluorescence immunochromatographic joint detection method of food-borne pathogenic bacteria based on antigen colorization, which is used for getting rid of the serious dependence of an immunochromatographic test strip on an antibody marking material and a paired antibody and establishing a rapid joint detection method for various food-borne pathogenic bacteria (Escherichia coli O157: H7, salmonella and Shigella bodyii) in food without a nano marking material and only one specific antibody.
In order to achieve the purpose, the invention provides a fluorescence immunochromatographic joint inspection method for food-borne pathogenic bacteria based on antigen colorization, which comprises a coloring agent and a test strip, wherein a sample is subjected to colorization culture by the coloring agent and then is detected by the test strip; the staining agent is tetra [4- (4' -carboxyphenyl) phenyl ] ethylene, the test strip comprises a sample pad (1), a detection reaction zone, a water absorption pad (4) and a bottom plate (7), the bottom plate (7) is sequentially provided with the sample pad (1), the detection reaction zone and the water absorption pad (4) along the chromatography direction, the detection reaction zone comprises at least three detection lines and an NC membrane (6), and the detection lines are arranged on the NC membrane (6) in parallel at intervals.
Furthermore, the detection lines sequentially arranged on the NC membrane (6) along the chromatography direction are a Shigella bodyii detection line (2), a salmonella detection line (3) and an Escherichia coli O157: H7 detection line (4) respectively.
Further, the Shigella boydii detection line (2) comprises a Shigella boydii monoclonal antibody, the salmonella detection line (3) comprises a Salmonella monoclonal antibody, and the Escherichia coli O157: H7 detection line (4) comprises an Escherichia coli O157: H7 monoclonal antibody.
Further, the sample pad (1) and the absorbent pad (4) are respectively overlapped and adhered above two ends of the NC film (6).
Further, the detection lines are sprayed on the NC membrane (6) by a spotting instrument at 1. mu.L/cm.
Further, the width of the test strip is 3mm, the lengths of the sample pad (1) and the absorbent pad (4) are both 2cm, the length of the bottom plate (7) is 6cm, the length of the NC membrane (6) is 2.5cm, and the distance between adjacent detection lines is 0.5 cm.
Further, the test strip was stored dry at 4 ℃ in the dark.
Further, the bottom plate (7) is a PVC (polyvinyl chloride) adhesive bottom plate, and the NC membrane (6) is a nitrocellulose membrane.
Compared with the prior art, the invention has the following beneficial effects: shigella bodyii, salmonella and escherichia coli O157: H7 are subjected to dyeing culture by using a coloring agent, and are subjected to high-sensitivity combined detection by using a test strip, a detection line is used for capturing three pathogenic bacteria dyed by the coloring agent and displaying a detection result, and three detection lines can simultaneously and jointly detect the three food-borne pathogenic bacteria without cross reaction.
Drawings
FIG. 1 is a schematic structural diagram of the test strip of the present invention;
FIG. 2 is a schematic diagram illustrating the determination of the detection result of the joint test strip in the embodiment of the present invention;
FIG. 3a is a schematic diagram showing the observation result of Escherichia coli O157: H7 stained by TCBPE in the example of the present invention under a fluorescent microscope;
FIG. 3b is a schematic representation of the results of fluorescent microscopic staining of Salmonella in an embodiment of the present invention with TCBPE;
FIG. 3c is a schematic diagram showing the observation result of Shigella boydii stained by TCBPE in the embodiment of the present invention;
FIG. 4 is a schematic diagram of the detection sensitivity measurement of the joint test strip according to the embodiment of the present invention;
FIG. 5 is a diagram showing the specific results of various food-borne pathogenic bacteria of the test strip for joint inspection in the embodiment of the present invention;
FIG. 6 is a schematic diagram showing the measurement results of the test strip for joint inspection performed on food samples according to the embodiment of the present invention simulating the presence of Escherichia coli O157: H7;
FIG. 7 is a schematic diagram showing the results of a test strip performed in a combined test on a food sample to simulate the presence of Salmonella in accordance with an embodiment of the present invention;
FIG. 8 is a schematic diagram showing the measurement result of a food sample simulated Shigella boydii by using the combined test strip in the embodiment of the invention;
FIG. 9 is a schematic diagram of the test strip for joint inspection in the embodiment of the invention, which simulates the food sample and simultaneously shows the measurement results of Escherichia coli O157: H7, Salmonella and Shigella boydii.
Detailed Description
The present invention will be described in more detail below with reference to schematic drawings, in which preferred embodiments of the present invention are shown, and it is to be understood that one skilled in the art can modify the present invention described herein while still achieving the advantageous effects of the present invention. Accordingly, the following description should be construed as broadly as possible to those skilled in the art and not as limiting the invention.
In the description of the present invention, it should be noted that, for the terms of orientation, such as "central", "lateral", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., it indicates that the orientation and positional relationship shown in the drawings are based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, but does not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated without limiting the specific scope of protection of the present invention.
In the present invention, unless otherwise specified and limited, "above" or "below" a first feature may include the first and second features being in direct contact, and may also include the first and second features not being in direct contact but being in contact with each other through another feature therebetween. Also, the first feature being "above," "below," and "above" the second feature includes the first feature being directly above and obliquely above the second feature, or simply an elevation which indicates a level of the first feature being higher than an elevation of the second feature. The first feature being "above", "below" and "beneath" the second feature includes the first feature being directly below or obliquely below the second feature, or merely means that the first feature is at a lower level than the second feature.
The invention is described in more detail in the following paragraphs by way of example with reference to the accompanying drawings. The advantages and features of the present invention will become more apparent from the following description. It is to be noted that the drawings are in a very simplified form and are not to precise scale, which is merely for the purpose of facilitating and distinctly claiming the embodiments of the present invention.
As shown in figure 1, the invention provides a fluorescence immunochromatographic joint inspection method for food-borne pathogenic bacteria based on antigen colorization, which comprises a coloring agent and a test strip, wherein a sample is subjected to colorization culture by the coloring agent and then is detected by the test strip; the coloring agent is Tetra [4- (4' -carboxyphenyl) phenyl ] ethylene (1,1,2,2-Tetra (4-carboxylphenyl) ethylene), which is called TCBPE for short. The test strip comprises a sample pad 1, a detection reaction area, a water absorption pad 4 and a bottom plate 7, wherein the sample pad 1, the detection reaction area and the water absorption pad 4 are sequentially arranged on the bottom plate 7 along the chromatography direction, the detection reaction area comprises at least three detection lines and an NC membrane 6, and the detection lines are arranged on the NC membrane 6 in parallel at intervals. In order to increase the food-borne pathogenic bacteria to be detected, the detection line of a pathogenic bacteria antibody is only needed to be added on the test strip, so that the multi-target combined detection can be realized on one test strip, the detection cost is greatly reduced, and the research, development and commercial application of the immunochromatographic test strip for rapidly detecting the food-borne pathogenic bacteria are greatly developed.
Specifically, in the present embodiment, the detection lines sequentially arranged along the chromatography direction on the NC membrane 6 are a shigella bodyii detection line 2, a salmonella detection line 3, and an escherichia coli O157: H7 detection line 4, respectively. The Shigella boydii detection line 2 comprises a Shigella boydii monoclonal antibody, the Salmonella detection line 3 comprises a Salmonella monoclonal antibody, and the Escherichia coli O157: H7 detection line 4 comprises an Escherichia coli O157: H7 monoclonal antibody.
Further, in this embodiment, the test strip has a width of 3 mm.
Further, the detection lines were each sprayed on the NC membrane 6 at 1. mu.L/cm by a spotting instrument. The bottom plate 7 is a PVC sticky bottom plate, and the NC membrane 6 is a nitrocellulose membrane.
The test paper strip is prepared by respectively spraying 1mg/mL Shigella baumannii monoclonal antibody, salmonella monoclonal antibody and Escherichia coli O157H 7 monoclonal antibody on an NC membrane in parallel at 1 muL/cm by using a spotting instrument to form three detection lines, wherein the distance between every two adjacent detection lines is 0.5cm, and after spraying, placing the NC membrane at 4 ℃ in a dark condition for dry storage. The sample pad 1 and the absorbent pad 4 are both 2cm in length, the bottom plate 7 is 6cm in length, the NC film 6 is 2.5cm in length, and the sample pad 1 and the absorbent pad 4 are respectively overlapped and adhered above two ends of the NC film 6. Overlapping the treated NC film, the sample pad and the absorption pad on a test strip pasting machine, then sequentially pasting the test strip pasting machine on a PVC base plate, cutting the test strip into 3mm wide by a test strip cutting machine, and drying and storing the test strip at 4 ℃ in a dark place.
Specifically, the detection of the joint test strip and the judgment of the result are carried out, as shown in fig. 2, when a sample is dripped on a test strip sample pad, the solution is chromatographed to an NC membrane along with the capillary action, and if the sample solution does not contain dyed targets Shigella boydii, salmonella and Escherichia coli O157: H7, the detection line does not develop color; if the sample solution only contains one dyed target bacterium, the dyed bacterium can be captured and gathered by the corresponding monoclonal antibody on the detection line, so that one corresponding detection line of the test strip is developed; similarly, if the sample contains two target bacteria, the two corresponding detection lines on the test strip are colored; if the sample simultaneously contains Shigella bodyii, salmonella and Escherichia coli O157: H7, the three detection lines of the joint detection test strip are all colored.
Meanwhile, as shown in FIG. 3, bacteria stained by TCBPE can be observed by a fluorescence microscope, and the experiment comprises the following steps of sucking bacterial liquid of Shigella bodyii, salmonella and Escherichia coli O157: H7 which are freshly cultured for 5 hours, centrifuging at 5000rpm for 3min, discarding the culture liquid, resuspending with ultrapure water of the same volume, and dropwise adding the suspended liquid to a glass slide; after the bacteria are dried and fixed, the TCBPE solution is dripped, after the bacteria are kept stand for 1min, the dye solution is washed by ultrapure water, and after the bacteria are dried, the bacteria are placed under a fluorescence microscope for observation. As can be seen from FIGS. 3a to 3c, TCBPE can effectively stain Escherichia coli O157: H7, Salmonella and Shigella bodyii, so that the Escherichia coli O157: H7, Salmonella and Shigella bodyii can be stained with green fluorescent signals.
Specifically, the detection sensitivity of the joint test strip is measured, as shown in fig. 4, shigella boydii, salmonella and escherichia coli O157: H7 are respectively cultured in a colored manner to be more than 109CFU/mL, 3 bacteria are respectively mixed according to the same final concentration, the mixture is diluted by using ultrapure water in a 10-fold gradient manner, 100 μ L of each gradient bacteria liquid is respectively dripped onto a sample pad of the joint test immunochromatographic test strip, and the stained culture medium is used as a negative control. Wherein 9-4 represents the bacterial concentration of 109-104 CFU/mL, and N represents a negative control.
As can be seen from the part a in the observation and detection result in FIG. 4, when the concentration of Shigella boydii is 105-109 CFU/mL, a green fluorescent strip is arranged at the line of the joint test strip T1, and when the concentration is 104CFU/mL, no strip appears in T1, which indicates that the sensitivity of the joint test strip to Shigella boydii detection is 105CFU/mL when Shigella boydii exists alone; as shown in part b of FIG. 4, when the salmonella is 105-109 CFU/mL, the test strip T2 is developed, and when the concentration is 104CFU/mL, the test strip T2 is not developed, which indicates that the detection sensitivity of the joint test strip for the salmonella is 105CFU/mL when the salmonella exists alone; as shown in part c of FIG. 4, when only Escherichia coli O157: H7 exists, the detection sensitivity of the joint test strip is still 105 CFU/mL; as shown in part d in FIG. 4, when 3 bacteria mixed liquor with the concentration of 105-109 CFU/mL is detected, green fluorescent strips appear on T1, T2 and T3 of the joint detection test strip, and when the concentration is lower than 105CFU/mL, three detection lines of the joint detection test strip do not develop color, which indicates that the joint detection test strip can simultaneously detect Shigella boydii, Salmonella and Escherichia coli O157: H7 with the detection sensitivity of 105CFU/mL, and is consistent with the sensitivity of detecting each pathogenic bacterium individually, and also indicates that the 3 bacteria of Shigella boydii, Salmonella and Escherichia coli O157: H7 dyed by BPTCE do not influence the detection result when being detected by the joint detection test strip.
In addition, the cross reaction of 26 strains of food-borne pathogenic bacteria is determined by adopting the joint test strip, TCBPE colored culture is carried out on all strains in the following table 1 to be more than 108CFU/mL, and the test strip is used for detecting each bacterial liquid to evaluate the detection specificity of the invention. As shown in FIG. 5, the joint test strip can specifically detect positive results only for Escherichia coli No. 3-5O 157: H7, Salmonella No. 15-21 and Shigella boydii No. 25, and the negative control is not false positive. Wherein, 1-30 are the corresponding strain numbers in the table 1, 31-37 are positive controls of simultaneous existence, respective existence of every two and independent existence of 3 strains, and N is a negative control; the combined detection immunochromatographic test strip has the advantages that the combined detection immunochromatographic test strip only has specific detection on Shigella bodyii, salmonella and Escherichia coli O157: H7 stained by TCBPE, has no cross reaction on other food-borne pathogenic bacteria, and has good detection specificity.
TABLE 1 strains used in the Cross-reactivity assay of the invention
Further, a joint test strip is used for testing the food sample with simulated bacteria, the joint test immunochromatography test strip is used for testing the food with simulated bacteria, and the feasibility of the joint test strip in the food sample test based on the TCBPE dyeing method is verified. Beef, milk, Chinese cabbage and chicken samples are purchased from local markets, 25g or 25mL of the beef, milk, Chinese cabbage and chicken samples are respectively weighed and added into 225mL of nutrient broth liquid culture medium, the mixture is homogenized for 2min and then transferred into a conical flask to be sterilized at 121 ℃ for 15min, after the temperature is reduced to room temperature, about 250CFU Shigella baumannii, 250CFU Salmonella and 250CFU Escherichia coli O157: H7 are respectively added into the sterile environment, 3 kinds of bacteria are mixed according to the same inoculation amount and then added into the culture medium of a food sample, the mixture is placed into a 37 ℃ incubator to be cultured for 12H, and meanwhile, the culture solution collected every hour is subjected to joint detection immunochromatography test paper strip detection. As shown in FIGS. 6 to 9, the test strip for joint inspection of the invention shows the results of the determination of the single existence and the total existence of Shigella bodyii, salmonella and Escherichia coli O157: H7 in sequence, wherein a to d are samples of Chinese cabbage, chicken, milk and beef in sequence, the enrichment time H is 1 to 10, N is a negative control, and P is a positive control.
Specifically, as shown in fig. 6, the joint test strip of the invention can detect a positive result after 6-7 hours of enrichment before the test strip is used for testing beef, milk, Chinese cabbage and chicken samples polluted by Escherichia coli O157: H7; as shown in FIG. 7, the test strip for joint inspection of the invention can detect a positive result after increasing bacteria 5-6 hours before a food sample polluted by salmonella; as shown in FIG. 8, a positive result can be detected by adding bacteria to a shigella boydii-contaminated food sample for 5-6 hours; as shown in FIG. 9, the test strip of the invention can simultaneously detect the positive results of 3 pathogenic bacteria after increasing bacteria for 5-6 hours before shigella bodyii, salmonella and escherichia coli O157: H7 simultaneously pollute beef, milk, Chinese cabbage and chicken samples. In conclusion, the combined detection immunochromatographic test strip based on TCBPE antigen colorization can be used for combined detection of Shigella bodyii, salmonella and Escherichia coli O157: H7 in Chinese cabbage, chicken, milk and beef.
In summary, in the fluorescence immunochromatographic combined detection method for food-borne pathogenic bacteria based on antigen colorization provided in this embodiment, shigella boydii, salmonella and escherichia coli O157: H7 are subjected to dyeing culture by using a staining agent, and combined detection is performed by using a test strip, and the detection sensitivity of the test strip to the three pathogenic bacteria is 105 CFU/mL. The three detection lines can be used for capturing the three pathogenic bacteria dyed by the coloring agent and displaying the detection result, and can be used for detecting the three food-borne pathogenic bacteria independently and simultaneously in a combined manner, and have no cross reaction to other food-borne pathogenic bacteria and good specificity.
The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any way. It will be understood by those skilled in the art that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The fluorescence immunochromatographic combined detection method for the food-borne pathogenic bacteria based on antigen colorization is characterized by comprising a coloring agent and a test strip, wherein a sample is subjected to colorization culture by the coloring agent and then is detected by the test strip; the staining agent is tetra [4- (4' -carboxyphenyl) phenyl ] ethylene, the test strip comprises a sample pad (1), a detection reaction zone, a water absorption pad (4) and a bottom plate (7), the bottom plate (7) is sequentially provided with the sample pad (1), the detection reaction zone and the water absorption pad (4) along the chromatography direction, the detection reaction zone comprises at least three detection lines and an NC membrane (6), and the detection lines are arranged on the NC membrane (6) in parallel at intervals.
2. The fluorescence immunochromatographic joint detection method for food-borne pathogenic bacteria based on antigen colorization of claim 1, wherein the detection lines sequentially arranged along the chromatography direction on the NC membrane (6) are a Shigella bodyii detection line (2), a salmonella detection line (3) and an Escherichia coli O157: H7 detection line (4), respectively.
3. The fluorescence immunochromatographic assay kit for food-borne pathogenic bacteria based on antigen colorization of claim 2, wherein the Shigella boydii detection line (2) comprises Shigella boydii monoclonal antibodies, the Salmonella detection line (3) comprises Salmonella monoclonal antibodies, and the Escherichia coli O157: H7 detection line (4) comprises Escherichia coli O157: H7 monoclonal antibodies.
4. The fluorescence immunochromatographic joint inspection method for food-borne pathogenic bacteria based on antigen colorization according to claim 1, wherein the sample pad (1) and the absorbent pad (4) are respectively overlapped and adhered above the two ends of the NC membrane (6).
5. The fluorescence immunochromatographic co-detection method for food-borne pathogenic bacteria based on antigen colorization according to claim 1, characterized in that the detection lines are each sprayed on the NC membrane (6) at 1 μ L/cm by a spotting instrument.
6. The fluorescence immunochromatographic combined detection method for food-borne pathogenic bacteria based on antigen colorization according to claim 1, characterized in that the width of the test strip is 3mm, the lengths of the sample pad (1) and the water absorption pad (4) are both 2cm, the length of the bottom plate (7) is 6cm, the length of the NC membrane (6) is 2.5cm, and the distance between the adjacent detection lines is 0.5 cm.
7. The fluorescence immunochromatographic assay kit for food-borne pathogenic bacteria based on antigen colorization according to claim 1, wherein the test strip is dried and stored at 4 ℃ in the dark.
8. The fluorescence immunochromatographic co-detection method for food-borne pathogenic bacteria based on antigen colorization according to claim 1, characterized in that the base plate (7) is a PVC adhesive base plate, and the NC membrane (6) is a nitrocellulose membrane.
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Application publication date: 20210713 |