CN115060899A - Colloidal gold detection card for fish-derived Shewanella putrefaciens and preparation method and application thereof - Google Patents

Colloidal gold detection card for fish-derived Shewanella putrefaciens and preparation method and application thereof Download PDF

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CN115060899A
CN115060899A CN202210501353.4A CN202210501353A CN115060899A CN 115060899 A CN115060899 A CN 115060899A CN 202210501353 A CN202210501353 A CN 202210501353A CN 115060899 A CN115060899 A CN 115060899A
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putrefaciens
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蒋昕彧
孙艳辉
刘国威
王啸宇
孔祥会
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Henan Normal University
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Abstract

The invention discloses a colloidal gold detection card for fish-derived Shewanella putrefaciens and a preparation method and application thereof, wherein the bottom layer of the detection card is a support plate, a sample pad, a gold-labeled binding pad, a nitrocellulose membrane and a siphon pad are sequentially arranged on the support plate, inactivated Shewanella putrefaciens is arranged on the nitrocellulose membrane and is used as a detection line, a rabbit anti-mouse IgM antibody is used as a quality control line, the detection line is positioned at one side close to the sample pad, and the gold-labeled binding pad is a mouse anti-mouse IgM antibody adsorbed with a colloidal gold labelS.putrefaciensNitrocellulose membrane of polyclonal antibody. The invention also specifically discloses a preparation method of the detection card and application of the detection card in rapid detection of fish-derived Shewanella putrefaciens. The detection card prepared by the invention is simple to operate, high in detection speed, free of special instruments and equipment and low in detection cost, so that the problem of the existing detection method for the fish-derived Shewanella putrefaciens is solved.

Description

Colloidal gold detection card for fish-derived Shewanella putrefaciens and preparation method and application thereof
Technical Field
The invention belongs to the technical field of immunological detection, and particularly relates to a colloidal gold detection card for fish-derived Shewanella putrefaciens, and a preparation method and application thereof.
Background
Shewanella putrefaciens (Shewanella putrefaciens) (II)Shewanella putrefaciens) Is a gram-negative facultative anaerobe. Shewanella putrefaciens is widely distributed in nature, mainly exists in dead and decayed organisms, is commonly used in the preservation process of chilled aquatic products, has strong low-temperature adaptability, can generate a large amount of trimethylamine and putrefactive volatile substances, and has strong corrosivity. The reports of the Shewanella putrefaciens infecting the living organisms are relatively few, and researches show that the Shewanella putrefaciens can generate hemolysin and has pathogenicity and lethality for aquatic animals such as Chinese mitten crabs, carassius auratus gibelio, silver carps, grass carps and the like.
Currently, detection of Shewanella putrefaciens mainly comprises pathogenic morphology identification, Polymerase Chain Reaction (PCR), loop-mediated isothermal amplification technology and the like. The Chinese invention patent 'separation and detection method of Shewanella putrefaciens in aquatic products' (CN 103820544B) discloses that the Shewanella putrefaciens in aquatic products can be separated and identified by designing a primer pair by using the genomic DNA of hydrogen sulfide producing bacteria and carrying out PCR amplification. However, the method has the defects of complex operation, long detection time, expensive required instruments and equipment, inconvenience in carrying and the like, so that the rapid detection on the diseased fish pond is difficult, and the rapid and accurate detection on the Shewanella putrefaciens is difficult due to the reasons of long culture period, poor specificity and the like of facultative anaerobic bacteria in the pathogenic morphology identification and loop-mediated isothermal amplification technology. Therefore, the development of a reagent capable of rapidly and accurately detecting Shewanella putrefaciens on site at a pond mouth is a problem to be solved in aquaculture profession at present.
The principle of colloidal gold chromatography is that an antigen/antibody is fixed on a solid phase carrier (nitrocellulose membrane), the corresponding antibody/antigen is labeled with colloidal gold, and when the antibody/antigen labeled with colloidal gold is specifically combined with the corresponding antigen/antibody on the solid phase carrier by using the siphon principle, the colloidal gold label can display a specific color, thereby judging whether a sample contains a target detection object. The commonly used colloidal gold chromatography includes a competitive method, a double antibody sandwich method, an indirect method and the like. Compared with other detection technologies, the colloidal gold chromatography does not need to carry out special treatment on a sample, does not need other instruments and equipment, has small dosage and strong stability, and ensures the specificity due to the use of the antibody. In recent years, the technology has been widely applied to the detection of new coronary pneumonia and veterinary clinical detection. However, the colloidal gold immunodiagnosis reagent for fish-derived Shewanella putrefaciens is only rarely reported at home and abroad.
Disclosure of Invention
Aiming at the defects of the existing fish-derived Shewanella putrefaction detection technology, the invention provides the colloidal gold detection card for the fish-derived Shewanella putrefaction, which is simple in operation, rapid in detection, good in stability and strong in specificity, and the preparation method and the application thereof, and is used for effectively solving the defects of the existing fish-derived Shewanella putrefaction detection method.
In order to solve the problems of timeliness and specificity of detection of fish-derived Shewanella putrefaciens, the invention adopts the following technical scheme: shewanella putrefaciens of fish origin (Shewanella putrefaciens) The colloidal gold detection card is characterized in that: the bottom layer of the detection card is a support plate, a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and a siphon pad are sequentially arranged on the support plate, inactivated Shewanella putrefaciens is arranged on the nitrocellulose membrane to serve as a detection line, a rabbit anti-mouse IgM antibody serves as a quality control line, the detection line is located on one side close to the sample pad, and the gold-labeled combination pad is formed by adsorbing a colloidal gold-labeled mouse anti-mouse IgM antibodyS. putrefaciensNitrocellulose membranes of polyclonal antibodies.
The preparation method of the colloidal gold detection card for fish-derived Shewanella putrefaciens is characterized by comprising the following specific steps of:
(1) mouse antibodyS. putrefaciensPreparation of polyclonal antibodies
Will be separatedS. putrefaciensExpanding the culture in LB liquid medium to OD 600 Centrifuge at 4000rpm/min at 4 deg.C for 3min, resuspend and wash 3 times with sterile PBS, adjustS. putrefaciensThe concentration is 1.5X10 5 CFU/mL, mixing with Freund's complete adjuvant, shaking to completely emulsify, intraperitoneal injecting into mice, immunizing 3 times at intervals of 7d, collecting blood 7d after last immunization, separating serum, and mixing withThe serum is primarily purified by an octanoic acid-saturated ammonium sulfate method, and affinity chromatography is carried out by a Protein A column, so as to obtain the mouse antibodyS. putrefaciensPolyclonal antibody, storing at-20 deg.C;
(2) preparation of colloidal gold-labeled antibody
Mixing 1mL of colloidal gold particle stock solution with diameter of 60nm, concentration of 40 μ g/mL, and 20 μ L of 0.1mg/mL prepared mouse antibodyS. putrefaciensShaking and incubating the polyclonal antibody in a refrigerator at 4 deg.C for 12h, centrifuging and ultrafiltering to remove free antibody and small molecular impurities, and adding 1wt% BSA as stabilizer to obtain colloidal gold-labeled antibodyS. putrefaciensA polyclonal antibody;
（3）S. putrefaciensinactivation of (2)
Labelled with colloidal gold prepared by inactivating a volume fraction of 0.7% formalin reagent at 28 ℃S. putrefaciensCollecting thallus by centrifuging at 8000rpm/min for 10min for 48h, washing thallus with PBS for 5 times to reduce formalin solution residue, re-suspending thallus with PBS reagent, and adjusting bacteria solution concentration to 1.0X10 5 CFU/mL to obtain inactivated colloidal gold labeledS. putrefaciensPolyclonal antibody, storing at-20 deg.C;
(4) preparation of nitrocellulose membranes
To be inactivated separatelyS. putrefaciensAnd 1mg/mL of a three-dimensional spraying point platform system for the commercial rabbit anti-mouse IgM antibody, spraying the point on a nitrocellulose membrane, and respectively and correspondingly serving as a detection line, namely a T line, and a quality control line, namely a C line, wherein the widths of the detection line and the quality control line are both 1mm, the using amount is 5 muL/cm, the distance between the detection line and the quality control line is 5mm, the detection line is positioned at the central position of the nitrocellulose membrane, the sprayed nitrocellulose membrane is placed in a drying box, and the nitrocellulose membrane is packaged for later use after being dried;
(5) preparation of test cards
Adsorbing the colloidal gold-labeled polyclonal antibody on the binding pad to form a gold-labeled binding pad, fixing the nitrocellulose membrane on the support plate, sequentially fixing the gold-labeled binding pad and the sample pad at the T-line end, respectively keeping the overlapping width of the nitrocellulose membrane and the gold-labeled binding pad as well as the overlapping width of the gold-labeled binding pad and the sample pad at 2mm, fixing the siphon pad at the C-line end, and keeping the overlapping width of the siphon pad and the nitrocellulose membrane at 2 mm.
The invention relates to an application of a colloidal gold detection card for fish-derived Shewanella putrefaciens in rapid detection of fish-derived Shewanella putrefaciens, which is characterized by comprising the following specific steps: dripping the mucus or ascites on the surface of the diseased fish onto the sample pad, horizontally standing the detection card for 3-5min, and judging whether the diseased fish is infected by the color development conditions of the detection line and the quality control lineS. putrefaciensIf the detection line and the quality control line are both colored, the result is negative, if the detection line is not colored, the quality control line is colored, the result is positive, and if the quality control line is not colored, the detection card is invalid and needs to be re-detected.
The detection principle of the colloidal gold rapid detection card for fish source Shewanella putrefaciens is as follows: after the sample pad is dripped with a sample to be detected, the sample solution moves to the gold-labeled combining pad along the sample pad under the siphoning action of the siphoning pad, and when the sample solution moves to the gold-labeled combining pad, the sample solution dissolves the colloidal gold-labeled antibody. When the sample contains Shewanella putrefaciens to be tested, the colloidal gold is labeled on the combination padS. putrefaciensPolyclonal antibody is combined and moves to a siphon pad at the same time to reach spraying inactivationS. putrefaciensThe detection line of (3) is such that the colloidal gold-labeled antibody is already bound to the Shewanella putrefaciens antigen, and therefore the detection line does not develop color. When the sample solution does not contain the substance to be detected, the antibody in the gold-labeled binding pad moves to the detection line and is bound with the Shewanella putrefaciens antigen, so that part of the colloidal gold is retained on the detection line, and the detection line shows red. Whether the sample contains the substance to be detected or not, a part of the gold-labeled antibody can be combined with the rabbit anti-mouse antibody of the quality control line and stays on the quality control line, so that the quality control line is developed. Therefore, if the quality control line is not colored, the detection card is invalid, the detection card needs to be replaced for re-detection, namely if the detection line and the quality control line are both red, the sample is negative; if the detection line does not develop color and the quality control line develops red, the sample is positive.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the Shewanella putrefaciens is inactivated and is sprayed on the nitrocellulose membrane to be used as a competitive antigen; preparing a murine Shewanella putrefaciens polyclonal antibody, and incubating and combining the murine Shewanella putrefaciens polyclonal antibody with colloidal gold to be used as a color developing source; the rabbit anti-mouse IgM monoclonal antibody is sprayed on a nitrocellulose membrane as a quality control antibody, and the fish-derived Shewanella putrefaciens colloidal gold rapid detection card is prepared. The detection card prepared by the invention is simple to operate, high in detection speed, free of special instruments and equipment and low in detection cost, so that the problem of the existing detection method for the fish-derived Shewanella putrefaciens is solved.
2. The fish-derived Shewanella putrefaciens rapid detection card prepared by the invention can obtain a detection result 3-5min after sample addition, has strong specificity, no cross reaction and sensitivity up to 10 3 CFU/mL can meet the requirements of low cost and on-site rapid detection, and has potential popularization and application values.
Drawings
FIG. 1 is a schematic view of a test card;
FIG. 2 is a gram stain of Shewanella putrefaciens from fish;
FIG. 3 is a schematic diagram of the fast detection of negative, positive and ineffective card by colloidal gold of Shewanella putrefaciens from fish;
FIG. 4 shows the bacterial concentration 10 4 、10 3 And 10 2 Color results of the CFU/mL test card are shown schematically.
In the figure: 1-sample pad, 2-gold label combined pad, 3-detection line, 4-support plate, 5-nitrocellulose membrane, 6-quality control line, and 7-siphon pad.
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
As shown in figure 1, the colloidal gold detection card for fish source Shewanella putrefaciens, the bottom layer of which is a support plate 4, a sample pad 1, a gold label combination pad 2, a nitrocellulose membrane 5 and a siphon pad 7 are sequentially arranged on the support plate 4, inactivated Shewanella putrefaciens is arranged on the nitrocellulose membrane 5 as a detection line 3, namely a T line, a rabbit anti-mouse IgM antibody as a quality control line 6, namely a C line, wherein the detection line 3 is positioned onNear one side of the sample pad 1, the gold-labeled binding pad 2 is made of a mouse antibody adsorbed with colloidal gold labelS. putrefaciensNitrocellulose membranes of polyclonal antibodies.
The preparation method of the colloidal gold detection card for the fish-derived Shewanella putrefaciens specifically comprises the following steps:
(1) mouse antibodyS. putrefaciensPreparation of polyclonal antibodies
Will be separatedS. putrefaciensExpanding culture in LB liquid culture medium to OD 600 Centrifuge at 4000rpm/min at 4 deg.C for 3min, resuspend and wash 3 times with sterile PBS, adjustS. putrefaciensThe concentration is 1.5X10 5 CFU/mL, mixing with Freund's complete adjuvant, shaking to completely emulsify, intraperitoneal injecting into mice, immunizing for 3 times, each time at an interval of 7d, taking blood 7d after the last immunization, separating serum, primarily purifying the serum by octanoic acid-saturated ammonium sulfate method, and performing affinity chromatography with Protein A column to obtain the final productS. putrefaciensPolyclonal antibody, storing at-20 deg.C;
(2) preparation of colloidal gold-labeled antibody
Mixing 1mL of colloidal gold particle stock solution with diameter of 60nm, concentration of 40 μ g/mL, and 20 μ L of 0.1mg/mL prepared mouse antibodyS. putrefaciensShaking and incubating the polyclonal antibody in a refrigerator at 4 deg.C for 12h, centrifuging and ultrafiltering to remove free antibody and small molecular impurities, and adding 1wt% BSA as stabilizer to obtain colloidal gold-labeled antibodyS. putrefaciensA polyclonal antibody;
（3）S. putrefaciensinactivation of (2)
Labelled with colloidal gold prepared by inactivating a volume fraction of 0.7% formalin reagent at 28 ℃S. putrefaciensCollecting thallus by centrifuging at 8000rpm/min for 10min for 48h, washing thallus with PBS for 5 times to reduce formalin solution residue, re-suspending thallus with PBS reagent, and adjusting bacteria solution concentration to 1.0X10 5 CFU/mL to obtain inactivated colloidal gold labeledS. putrefaciensPolyclonal antibody, storing at-20 deg.C;
(4) preparation of nitrocellulose membranes
To be inactivated separatelyS. putrefaciensAnd 1mgThe method comprises the following steps that a/mL commercial rabbit anti-mouse IgM antibody is spotted on a nitrocellulose membrane through a three-dimensional spot spraying platform system, the spot spraying is correspondingly used as a detection line, namely a T line, and a quality control line, namely a C line, respectively, the widths of the detection line and the quality control line are both 1mm, the using amount of the detection line and the quality control line is 5 muL/cm, the detection line and the quality control line are 5mm apart and are located at the center of the nitrocellulose membrane, the sprayed nitrocellulose membrane is placed in a drying box, and the nitrocellulose membrane is packaged for later use after being dried;
(5) preparation of test cards
Adsorbing the colloidal gold-labeled polyclonal antibody on the binding pad to form a gold-labeled binding pad, fixing the nitrocellulose membrane on the support plate, sequentially fixing the gold-labeled binding pad and the sample pad at the T-line end, respectively keeping the overlapping width of the nitrocellulose membrane and the gold-labeled binding pad as well as the overlapping width of the gold-labeled binding pad and the sample pad at 2mm, fixing the siphon pad at the C-line end, and keeping the overlapping width of the siphon pad and the nitrocellulose membrane at 2 mm.
The application of the colloidal gold rapid detection card for the fish-derived Shewanella putrefaciens comprises the following specific processes: dripping the surface mucus or abdominal ascites of the diseased fish onto the sample pad, horizontally standing the detection card for 3-5min, and judging whether the diseased fish is infected by the color development conditions of the detection line and the quality control lineS. putrefaciensIf the detection line and the quality control line are both colored, the result is negative, if the detection line is not colored, the quality control line is colored, the result is positive, and if the quality control line is not colored, the detection card is invalid, and the detection needs to be carried out again.
Example 1
And (3) detecting the gill mucus of the micropterus salmoides after the Shewanella putrefaction infection and the gill mucus of the micropterus salmoides after the Aeromonas veronii infection.
1. Application method of detection card
By intraperitoneal injection, 200 μ L of the extract is used at a concentration of 10 5 The Shewanella putrefaciens and Aeromonas veronii in CFU/mL infect micropterus salmoides, and the mucus at gill part is taken 5 days after infection, diluted by normal saline in equal proportion, about 100 mu L of the mucus is dripped on a sample pad of a detection card, and horizontally kept for 3 min.
2. Analyzing the results of the detection
The detection of the sample after the challenge by the aeromonas veronii is negative (reference numeral 1 in figure 3), the detection of the sample after the challenge by the Shewanella putrefaciens is positive (reference numeral 2 in figure 3), and the detection of the C line which is not developed is an invalid detection card (reference numeral 3 in figure 3), which indicates that the colloidal gold detection card for the fish-derived Shewanella putrefaciens can specifically identify Shewanella putrefaciens and has no cross reaction with the common aeromonas veronii in aquaculture.
Example 2
Sensitivity test of detection card to Shewanella putrefaciens at different concentrations
1. Sample preparation
According to the standard curve of Shewanella putrefaciens bacterial liquid concentration and absorbance value, adjusting 3 bacterial liquid concentrations to 10 respectively by using normal saline 4 CFU/mL、10 3 CFU/mL and 10 2 CFU/mL. And respectively taking 100 mu L of each of the 3 kinds of concentration bacterial liquid, dropwise adding the bacterial liquid to a sample pad of the detection card, and horizontally standing for 3 min.
2. Analyzing the results of the detection
At a concentration of 10 4 CFU/mL and 10 3 CFU/mL sample test cards showed positivity (reference numeral 1 in FIG. 4 and reference numeral 2 in FIG. 4), 10 2 The sample detection card at CFU/mL showed negative. The colloidal gold detection card for Shewanella putrefaciens in fish source of the invention can detect the Shewanella putrefaciens with the concentration of 10 3 Samples above CFU/mL have higher detection sensitivity.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.

Claims (3)

1. Colloidal gold detection card of fish source Shewanella putrefaciens, its characterized in that: the bottom layer of the detection card is a support plate, a sample pad, a gold-labeled combination pad, a nitrocellulose membrane and a siphon pad are sequentially arranged on the support plate, inactivated Shewanella putrefaciens is arranged on the nitrocellulose membrane to serve as a detection line, a rabbit anti-mouse IgM antibody serves as a quality control line, and the detection line is located close to a sampleOne side of the pad, the gold-labeled pad is made of mouse antigen adsorbed with colloidal gold labelS. putrefaciensNitrocellulose membranes of polyclonal antibodies.
2. A method for preparing the colloidal gold assay card for Shewanella putrefaciens according to claim 1, comprising the following steps:
(1) mouse antibodyS. putrefaciensPreparation of polyclonal antibodies
Will be separatedS. putrefaciensExpanding culture in LB liquid culture medium to OD 600 Centrifuge at 4000rpm/min at 4 deg.C for 3min, resuspend and wash 3 times with sterile PBS, adjustS. putrefaciensThe concentration is 1.5X10 5 CFU/mL, mixing with Freund's complete adjuvant, shaking to completely emulsify, intraperitoneal injecting into mice, immunizing for 3 times, each time at an interval of 7d, taking blood 7d after the last immunization, separating serum, primarily purifying the serum by octanoic acid-saturated ammonium sulfate method, and performing affinity chromatography with Protein A column to obtain the final productS. putrefaciensPolyclonal antibody, storing at-20 deg.C;
(2) preparation of colloidal gold-labeled antibody
Mixing 1mL of colloidal gold particle stock solution with diameter of 60nm, concentration of 40 μ g/mL, and 20 μ L of mouse antigen prepared at 0.1mg/mLS. putrefaciensShaking and incubating the polyclonal antibody in a refrigerator at 4 deg.C for 12h, centrifuging and ultrafiltering to remove free antibody and small molecular impurities, and adding 1wt% BSA as stabilizer to obtain colloidal gold-labeled antibodyS. putrefaciensA polyclonal antibody;
（3）S. putrefaciensinactivation of (2)
Labelled with colloidal gold prepared by inactivating a volume fraction of 0.7% formalin reagent at 28 ℃S. putrefaciensCollecting thallus by centrifuging at 8000rpm/min for 10min for 48h, washing thallus with PBS for 5 times to reduce formalin solution residue, re-suspending thallus with PBS reagent, and adjusting bacteria solution concentration to 1.0X10 5 CFU/mL, i.e. the inactivated colloidal gold labeledS. putrefaciensPolyclonal antibody, storing at-20 deg.C;
(4) preparation of nitrocellulose membranes
To be inactivated separatelyS. putrefaciensAnd 1mg/mL of a three-dimensional spraying point platform system for the commercial rabbit anti-mouse IgM antibody, spraying the point on a nitrocellulose membrane, and respectively and correspondingly serving as a detection line, namely a T line, and a quality control line, namely a C line, wherein the widths of the detection line and the quality control line are both 1mm, the using amount is 5 muL/cm, the distance between the detection line and the quality control line is 5mm, the detection line is positioned at the central position of the nitrocellulose membrane, the sprayed nitrocellulose membrane is placed in a drying box, and the nitrocellulose membrane is packaged for later use after being dried;
(5) preparation of test cards
Adsorbing the colloidal gold-labeled polyclonal antibody on the binding pad to form a gold-labeled binding pad, fixing the nitrocellulose membrane on the support plate, sequentially fixing the gold-labeled binding pad and the sample pad at the T-line end, respectively keeping the overlapping width of the nitrocellulose membrane and the gold-labeled binding pad as well as the overlapping width of the gold-labeled binding pad and the sample pad at 2mm, fixing the siphon pad at the C-line end, and keeping the overlapping width of the siphon pad and the nitrocellulose membrane at 2 mm.
3. The application of the colloidal gold detection card for Shewanella putrefaciens in fish source according to claim 1 in rapid detection of Shewanella putrefaciens in fish source is characterized by comprising the following steps: dripping the mucus or ascites on the surface of the diseased fish onto the sample pad, horizontally standing the detection card for 3-5min, and judging whether the diseased fish is infected by the color development conditions of the detection line and the quality control lineS. putrefaciensIf the detection line and the quality control line are both colored, the result is negative, if the detection line is not colored, the quality control line is colored, the result is positive, and if the quality control line is not colored, the detection card is invalid, and the detection needs to be carried out again.
CN202210501353.4A 2022-05-10 2022-05-10 Colloidal gold detection card for fish-derived Shewanella putrefaciens and preparation method and application thereof Pending CN115060899A (en)

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