CN111983240B

CN111983240B - Visual detection method for clostridium perfringens alpha toxin

Info

Publication number: CN111983240B
Application number: CN202010883292.3A
Authority: CN
Inventors: 黄金海; 郭艳余; 张丽琳; 曹爱萍
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2023-11-21
Anticipated expiration: 2040-08-27
Also published as: CN111983240A

Abstract

The invention discloses a visual detection method for clostridium perfringens alpha toxin, which combines a smart phone image processing technology and silica microsphere antibody coupling detection for food-borne clostridium perfringens alpha toxin (CPA). According to the double-antibody sandwich principle, the prepared CPA polyclonal antibody is coupled with the silicon dioxide microsphere, CPA fluorescence detection is carried out through FITC fluorescence marked by the antibody, and finally, the real-time detection of CPA content is realized by combining with the image processing technology of the smart phone APP. Compared with the traditional clostridium toxin detection method, the nano microsphere combined smart phone CPA detection system is a more sensitive, stable and convenient detection method, and provides a new method for detecting natural CPA in food.

Description

Visual detection method for clostridium perfringens alpha toxin

Technical Field

The invention belongs to the field of biotechnology, and relates to a detection method of food-borne pathogenic bacteria clostridium perfringens alpha toxin.

Background

Clostridium perfringens shapeBacillus (Clostridium perfringens), also known as food-borne gastroenteritis and other infectious diseases. As a conditionally pathogenic bacterium, it can contaminate many types of retail meat products and produce alpha toxins in the small intestine of livestock ^[1] Food poisoning may result when milk and dairy products contaminated with such bacteria or toxins are consumed. Clostridium perfringens causes a variety of diseases in various hosts due to the production of various toxins and extracellular enzymes ^[2] . To date, at least 20 exotoxins have been found, of which the major deadly toxins are α, β, ε and iota. Because all types of toxins produce alpha toxin and result in hemorrhagic enteritis and acute death in livestock ^[3] And alpha toxin is the most important virulence factor of clostridium perfringens type a ^[4] . It has cytotoxicity, hemolytic activity, lethality, skin necrosis, and myonecrosis ^[5] Granulocytogenesis ^[6] Erythroid differentiation inhibition ^[7] Platelet aggregation and increased vascular permeability.

The detection of clostridium perfringens is currently mainly based on molecular biological assays PCR or immunological assays ELISA, etc. Oisvik et al established a four-layer sandwich method for the detection of clostridium perfringens enterotoxins. This method can detect enterotoxins at 0.1 μg/mL, but the entire experiment takes 4 days to complete. McClane et al ^[8] Established indirect ELISA methods can detect enterotoxins of 25ng/mL and shorten the experimental time. Hale et al ^[9] An antigen capture ELISA method is established for detecting clostridium perfringens toxin, and has good specificity and sensitivity, and the minimum detection amount of alpha toxin is 19ng/mL. These methods can be used to diagnose Clostridium perfringens using PCR techniques instead of serum neutralization assays ^[10] The toxin content of goat feces and gastrointestinal tract was successfully detected. In 1997, a PCR technique was established to determine four different toxins in goat feces. However, the method established by Uzal is cumbersome and uneconomical and the sample needs to be tested four times. Yoo et al ^[11] Multiplex PCR was established to detect the four main toxins of clostridium perfringens, but this method requires extraction and purification of the bacterial genome. Baums CG and the like ^[12] Establishes a kind ofA multiplex PCR method capable of typing clostridium perfringens, which can directly detect and distinguish between cpa (alpha), cpb (beta 1), etx (epsilon), iap (iota), cpe (enterotoxin) and cpb2 (beta 2) and other toxin genes, and has good specificity and reproducibility. Furthermore, gurjar et al ^[13] Fluorescent quantitative PCR was established. The main toxin genes of clostridium perfringens can be detected directly from fecal samples and can be used for epidemiological investigation of diseases. In summary, the sensitivity of the existing technology for detecting clostridium perfringens by ELISA is high, the minimum detection limit can reach 19ng/mL, but the common problem is that the operation process is time-consuming and complicated; the PCR method can be used for simultaneously detecting various clostridium perfringens toxins, but the toxins need to be purified in the detection process, and the sensitivity is low, and the detection is time-consuming and tedious. Therefore, it is necessary to establish a highly efficient, sensitive, and convenient detection system for clostridial toxins.

Disclosure of Invention

The invention aims to provide a visual detection method for clostridium perfringens alpha toxin, which is used for solving the problems of low detection limit, long time, and the like in the prior art and making up for the defect of single detection technology.

The invention aims at realizing the following technical scheme:

a visual detection method for rapidly detecting food-borne clostridium perfringens alpha toxin in real time based on combination of silica microspheres and a smart phone comprises the steps of preparing antibodies by injecting a prokaryotic-expressed CPA truncated protein into animals, coupling the antibodies with the silica microspheres after Fluorescein Isothiocyanate (FITC) labeling, capturing alpha toxin in food, uploading a fluorescent picture obtained by detection to a mobile phone APP, and immediately obtaining the concentration of the alpha toxin in a sample to be detected in real time.

The prokaryotic expression CPA protein truncated CPA _N Comprises two mutation sites (D56G, D130G) of CPA protein, and the preparation of the antibody _N The protein sequence of the truncated SEQ ID No. 1.

The method comprises the following main steps:

(1) CPA protein truncate CPA _N Is constructed by the following steps: due to CPA eggsWhite is toxic and lethal, and to facilitate subsequent antisera preparation, mutants of CPA were used in experiments to eliminate toxic effects on immunized animals. The complimentary 4-amino acid mutant plasmid (pET 30 a-CPA) was taught by Chen Xiaoyun _m4 ) And a C-terminal three-copy tandem plasmid (pET 30a-CPA _C3 ) ^[14,15] . In CPA _m4 Primers (CPA) were designed based on _m4 Dele TM-F, SEQ ID No.2 and CPA _m4 Delete the transmembrane domain of delete TM-R, SEQ ID No. 3) to obtain N-terminal truncated plasmid pET30a-CPA _N The plasmid contains only two amino acid mutations (D56G, D130G).

(2) Antibody preparation: the constructed pET30a-CPA _N pET30a-CPA given away by Chen professor _C3 The prokaryotic expression vector converts competent cells of escherichia coli BL21 (DE 3), IPTG induces and purifies recombinant proteins. Immunization of rabbits three times with 1mg/mL protein amount resulted in anti-CPA _C3 And CPA (CPA) _N The polyclonal antibody was purified from rabbit serum by Saturated Ammonium Sulfate (SAS) precipitation and the antibody titer was detected by Western blot and ELISA.

(3) CPA using semi-permeable membrane process _N The general principle of the coupling and fluorescence detection of the subsequent silica microspheres is shown in figure 7, and the fluorescence intensity of the microspheres detected by a fluorescence microscope is the concentration of the corresponding clostridium alpha toxin by utilizing the antigen-antibody combined double-antibody sandwich principle.

The CPA protein truncated silica microsphere coupling comprises the following steps:

(1) Quantification of clostridium perfringens alpha toxin using virulence substitution experiment-lecithin hydrolysis experiment, and final measured natural CPA concentration of 10LD ₅₀ 。

(2) Activation of silica microspheres: carboxylated silica microsphere surface coupling CPA _N Polyclonal antibody, adding coupling agent N-hydroxysuccinimide (NHS) and carbodiimide (EDC) with final concentration of 10mg/mL, and finally blocking.

(3) Condition optimization of silica microsphere detection experiment: the optimal conditions for the fluorescence detection experiment are determined according to the experimental data, namely at 2Capture antibody (CPA) at 0.1mg/ml at 5 ℃ _C3 ) After 2h interaction with activated silica microspheres, blocking overnight at 4℃with 5% BSA, alpha toxin and 0.2mg/ml detection antibody (FITC-CPA _N ) And (3) performing interaction with the silica microspheres coupled with the capture antibody for 1h, so as to perform detection by a fluorescence microscope.

Establishment of alpha toxin capture standard curve, detection line sensitivity of 0.625LD ₅₀ The detection time was about 90min.

The method comprises the following steps:

(1) Establishment of a standard curve: a double gradient dilution of clostridium perfringens alpha toxin (CPA) was performed for a total of 5 dilutions, each dilution being 3 biological replicates, after which a set of fluorescence images was obtained by detecting the fluorescence intensity of silica microspheres coupled to FITC-labeled antibodies.

(2) The fluorescence intensity in the picture is subjected to gray value analysis (conventional image analysis software such as Photoshop, imageJ and the like is used), the research uses ImageJ software to perform gray value analysis, firstly, the picture is converted into an 8-bit gray image and background interference is eliminated, then brightness values in all image areas are selected for average analysis, finally, the average gray value of the corresponding image is obtained, and data are arranged to obtain a regression equation Y=0.01915X+0.002875 (R ² = 0.9972). Wherein X represents LD ₅₀ Y represents an average gray value.

(3) The image processing technology is programmed into the intelligent mobile phone APP, the general principle is that the picture is firstly subjected to RGB value analysis, then the RGB color value is converted into hexadecimal color code (gray), so that the gray value of the image is obtained, and finally, different gray value ranges are corresponding to CPA concentration by using an IF algorithm. After APP is prepared, the corresponding CPA concentration can be directly obtained by uploading the fluorescence image.

Based on the method, the invention claims a method for detecting clostridium perfringens alpha toxin in real time by combining the silicon dioxide microspheres with a smart phone.

The invention has the beneficial effects that: the invention prepares the alpha toxin antibody with FITC label and the alpha toxin antibody with FITC label according to the double-antibody sandwich methodA method for detecting food-borne clostridium perfringens alpha toxin by coupling silica microspheres. And analyzing and processing the fluorescence micrograph through APP software which is universal to the android smart phone, so that the alpha toxin content can be determined. The minimum detection limit of the method is 0.625LD ₅₀ The whole detection time is less than or equal to 90min. The overall data processing time was < 5 seconds. The method has the advantages of good stability, high sensitivity, convenient and quick operation and the like, and provides a new thought and direction for detecting the food-borne clostridium perfringens alpha toxin.

Drawings

Fig. 1: CPA recombinant protein CPA _C3 And CPA (CPA) _N SDS-PAGE results of expression and purification; (a) CPA (CPA) _C3 Expression electrophoretogram of recombinant protein. Lanes 1-2 are purified CPA _C3 2 samples of recombinant protein; (b) CPA (CPA) _N Electrophoresis results M of recombinant proteins: protein molecular Mass Standard (Blue Plus) lanes 3-4 are purified CPA _N Two parallel samples of recombinant protein;

fig. 2: clostridium perfringens alpha toxin (CPA) _C3 And CPA (CPA) _N ) Is determined by the concentration and titer of polyclonal antibodies;

(a) An antibody concentration standard curve measured by Bradford method;

(b) Recombinant protein CPA _C3 Western blot results of (M: protein marker (GenStar M221) lanes 1-2: CPA _C3 Recombinant proteins);

(c) Recombinant protein CPA _C3 And CPA (CPA) _N Western blot of (M: protein marker (GenStar M221) lanes 3-4: CPA _N Recombinant proteins);

(d) Recombinant protein CPA _C3 Is an indirect ELISA result of (2): detected CPA _C3 The titer of the antibody was 1:3200;

(e) Recombinant protein CPA _N Is an indirect ELISA result of (2): detected CPA _N The titer of the antibody was 1:6400;

fig. 3: degree of hydrolysis of CPA by lecithin hydrolysis;

fig. 4: optimal conditions for silica microsphere fluorescence detection experiments;

(a) Optimal antibody coating conditions were determined (fixed other conditions, selected toxin concentration 10LD50, antibody dilution concentration 0.02, 0.1, 0.5mg/ml, assay temperature and time 37℃for 2h,25℃for 2h,4℃for 12h; 3 biological replicates per group);

(b) Optimal blocking conditions were determined (fixing other conditions, selecting toxin concentration as 10LD50, selecting blocking agent as 5% milk and 5% BSA, selecting experimental temperature and time as 37℃for 2h,25℃for 2h,4℃for 12h; 3 biological replicates per group);

(c) Optimal incubation time and dilution (fixation) of enzyme-labeled antibody, unchanged conditions, toxin concentration of 10LD50, dilution concentration of fluorescent-labeled antibody of 0.05, 0.1, 0.2mg/mL, experiment temperature and time of 37 ℃ for 0.5h,37 ℃ for 1h, and 37 ℃ for 2h, respectively; 3 biological replicates per group experiment);

fig. 5: a fluorescence diagram of CPA detection and an established standard regression equation;

(a) Fluorescence pictures at different toxin concentrations: from left to right, the concentrations are respectively 10LD ₅₀ ，5LD ₅₀ ，2.5LD ₅₀ ， 1.25LD ₅₀ ，0.625LD ₅₀ Three biological replicates per concentration gradient (i.e., three columns in the corresponding graph);

(b) Y=0.01915x+0.002875 (R ² ＝0.9972)。

Fig. 6: a smart phone APP detection interface;

fig. 7: silica microsphere coupled fluorescent antibody is combined with a smart phone APP detection system principle.

Detailed Description

The invention is further illustrated in the following, in conjunction with the accompanying drawings and specific embodiments.

Example 1 basic preparation of the detection method

Step one, standard strain type A (C57-1) was purchased from a veterinary drug test. Alpha toxin 4 amino acid mutant plasmid (CPA) _m4 ) And in alpha toxin (CPA) _C3 ) The C-terminal three-copy tandem prokaryotic expression vector (pET 30 a) was constructed by the teachings of Chen Xiaoyun of the China veterinary drug institute ^[14,15] 。

Because site-directed mutagenesis toxins can greatly reduce the toxicity and lethality of CPA, subsequent preparation of non-lethal rabbit serum antibodies is facilitated.

We then used the existing alpha toxin 4 amino acid mutant plasmid (CPA _m4 ) N-terminal truncation construction was performed, so that the constructed expression vector contained two mutation sites (D56G, D130G). Construction of the N-terminal expression vector of CPA, PET30a-CPA, using two primers _N The clostridium perfringens alpha toxin gene (Genbank accession number: DQ 202275) was searched for according to Genbank, and the N-terminal truncated primer (CPA) was designed by removing the transmembrane domain _m4 Dele TM-F, SEQ ID No.2 and CPA _m4 Del TM-R, SEQ ID No. 3), upstream and downstream contain restriction sites for BamHI and Xho I, respectively.

Step two, the constructed prokaryotic expression vector (CPA _C3 And CPA (CPA) _N ) Expressing and purifying prokaryotic recombinant protein, and immunizing rabbits three times with 1mg/ml protein to obtain the anti-CPA _C3 And CPA (CPA) _N The polyclonal antibody was purified from rabbit serum by Saturated Ammonium Sulfate (SAS) precipitation and the antibody titer was detected by Western blot and ELISA. The results are shown in FIG. 1, and CPA recombinant protein CPA _C3 And CPA (CPA) _N Can be expressed normally. As can be seen from FIG. 2, CPA of Clostridium perfringens alpha toxin was measured by the Bradford method _C3 And CPA (CPA) _N The polyclonal antibody concentration of (C) was 7.02mg/mL and 7.47mg/mL, respectively. FIG. 2-b, c shows the Western blot results of recombinant protein CPA _C3 And CPA (CPA) _N The specificity is good; FIG. 2-d, e indirect ELISA results showing recombinant protein CPA _C3 And CPA (CPA) _N Titers of 1:3200 and 1:6400, respectively.

Step three, CPA is carried out by utilizing semi-permeable membrane method _N Labeling of antibody Fluorescein Isothiocyanate (FITC) the protein concentration of the antibody solution was corrected to 10mg/mL using 0.025M, pH 9.0 carbonate buffer and placed in a dialysis bag. Simultaneously, the amount of antibody protein 1/20FITC, namely, the amount (mg) required by FITC (Beijing Sotel technologies Co., ltd.) =protein content (mg/mL) ×antibody solution amount (mL) ×1/20 was weighed; FITC was dissolved in 10 times 0.025M antibody solution, pH 9.0 carbon in salt buffer, gently stirred to avoid air bubbles; in dialysis bagsThe antibody was immersed in the FITC solution and allowed to stand at 4℃for 16-18 hours, during which time it was stirred periodically 1-2 times per hour with a magnetic stirrer for a total stirring time of about 6 hours. Taking out the dialysis bag, and putting into 0.01M PH7.2 PBS for dialysis for 4 hours for standby.

And step four, quantifying clostridium perfringens alpha toxin by using a virulence substitution experiment, namely a lecithin hydrolysis experiment. Firstly, mashing and mixing fresh cooked yolk, and adding 1% CaCl ₂ Normal saline 40 mg/g and 6% Al (OH) ₃ Gel 3ml/g, mix and place in a refrigerator at 4℃overnight. The next day was taken out, and after centrifugation at 720g for 20 minutes, the supernatant was taken as lecithin. With physiological saline (2) ⁰ 、2 ^-1 、2 ^-2 、 2 ^-3 、2 ^-4 、2 ^-5 ) The alpha toxin was diluted, 1mL lecithin was added to each dilution, and then left to stand at 37℃1 time. Hours. Negative medium and 1mL lecithin were used as control, at OD ₅₅₀ The absorbance (turbidity) was measured, and the dilution of each hydrolysis reaction was 1/6.4LD ₅₀ Toxicity, i.e. toxicity 2x/6.4 LD after 2-fold dilution ₅₀ . The final measured natural CPA concentration was 10LD ₅₀ . As a result, as shown in FIG. 3, the hydrolysis degree of CPA was measured by lecithin hydrolysis, three biological replicates were performed for each concentration gradient, and a series of absorbance values were obtained, and since toxicity and hydrolytic turbidity had a stable correlation, the correspondence between the hydrolysis degree and concentration of CPA was calibrated by a curve, and finally CPA at the lowest detectable dilution was compared with formula 2x/6.4 LD ₅₀ The clostridium toxin (undiluted) extracted in the experiment was calculated to be 10LD ₅₀ 。

Coupling carboxyl modified silica microspheres with fluorescent antibodies: a certain amount of silica microspheres (particle size 10 μm, tianjin all good tech Co., ltd.) was weighed out to be 0.5: EDC was added in a ratio of 1 to 1:1, adding NHS in proportion, fully mixing and shaking at room temperature. For 20 minutes, then washed with PBS and centrifuged to remove the wash (repeated three times). The optimal experimental conditions for the coupling of the antibody and silica microspheres were then determined.

Example 2: condition optimization of detection methods

Step one, determining optimal coating conditions: the variable is coated antibody (bound silica microsphere antibody CPA) _C3 ) Other conditions were fixed, the toxin concentration was chosen to be 10LD ₅₀ (3 replicates per condition) then the fluorescence image was observed by fluorescence microscopy and finally the ratio was obtained by grey value analysis. Finally, optimal coating conditions were determined to be 0.1mg/mL capture antibody and silica microspheres were mixed at 25 ℃ for 2 hours. The results are shown in FIG. 4: optimal conditions for microsphere fluorescence detection experiments; (a) determining optimal antibody coating conditions.

Step two, determining the optimal closed condition: the variables were selected blocking agent (5% nonfat milk powder or 5% BSA), the other conditions were fixed, and the selected toxin concentration was 10LD ₅₀ (3 replicates per condition) and grey values for fluorescent image analysis were obtained. Finally, the optimal sealing conditions were determined to be 5% BSA at 4℃for 12 hours. The results are shown in FIG. 4: optimal conditions for microsphere fluorescence detection experiments; (b) determining optimal closed conditions.

Step three, determining the optimal incubation time and optimal enzyme-labeled antibody (CPA) _N ) Is a dilution of (2): the variables are dilution factor (1:50, 1:100, 1:200) of enzyme-labeled antibody and time and temperature fixed, other conditions fixed, selected toxin concentration 10LD ₅₀ (3 biological replicates per condition) and the grey scale value of the fluorescence image was analyzed. Finally, the optimal dilution concentration of the enzyme-labeled antibody was 1:50, and the silica microspheres were allowed to act at 37℃for 1 hour. The results are shown in FIG. 4, and the optimal conditions for microsphere fluorescence detection experiments, namely, at 25℃and 0.1mg/ml capture antibody (CPA _C3 ) After 2h interaction with activated silica microspheres, blocking overnight at 4℃with 5% BSA, alpha toxin and 0.2mg/ml detection antibody (FITC-CPA _N ) And (3) performing interaction with the silica microspheres coupled with the capture antibody for 1h, so as to perform detection by a fluorescence microscope.

Example 3: establishment of detection method

Step one, performing concentration gradient dilution of toxin CPA, and determining the undiluted state quantitatively in the prior artIs 10LD ₅₀ Then we dilute it in 2-fold series to obtain 10LD ₅₀ ，5LD ₅₀ ， 2.5LD ₅₀ ，1.25LD ₅₀ ，0.625LD ₅₀ Each concentration gradient was subjected to 3 biological replicates and the corresponding fluorescence image was obtained by fluorescence microscopy. As a result, see FIG. 5, CPA was diluted in a gradient to 10LD ₅₀ ，5LD ₅₀ ，2.5LD ₅₀ ，1.25LD ₅₀ ，0.625LD ₅₀ Fluorescence intensity was then measured and three biological replicates were set for each concentration gradient. The stability of the equation was demonstrated to be good.

Step two, gray value analysis is carried out by using imageJ software, and finally, the obtained gray value is drawn into a regression curve, and a regression equation is formed: y=0.01915x+0.002875 (R ² = 0.9972). The method is used for determining the mouse LD with the minimum detection limit of 0.625 times of clostridium perfringens alpha toxin ₅₀ Has higher sensitivity. The specific result is shown in figure 5 of the specification.

And thirdly, dividing the five dilutions of the detected toxin into five groups, performing three biological repetitions of each dilution, and analyzing the variation coefficient of the gray value result obtained by detection. Analysis shows that the results are lower than 15%, which indicates that the variation degree of the same sample in the same batch is low, and the established fluorescent silica microsphere toxin detection method has good stability. Specific results are shown in Table 1 attached to the specification.

Table 1. Repeatability test of silica microsphere fluorescence detection (n=3)

Example 4: detection of CPA concentration in food using silica microspheres

The effect of the detection technique established in this experiment was evaluated by performing an actual test on milk determined according to GB 4789.13-2012 to be free of clostridium welfare, i.e. the suitability and tamper resistance of the detection system for actual food products was tested. For milk, we separately took clostridium perfringens toxins low, medium, high concentrations (0.625 LD ₅₀ ， 2.5LD ₅₀ ，10LD ₅₀ ) And mixing them with 1mL of milk, respectively, and then mixing the mixture with antibody CPA _C3 With fluorescent FITC antibody CPA _N After the interaction of silica microspheres for one hour, milk is removed by washing, then the fluorescence detection experiment of the silica microspheres described above is carried out, the obtained fluorescence image is uploaded to APP, the fluorescence intensity is analyzed, the corresponding toxin concentration is obtained through conversion according to a standard regression equation, and the recovery rate is satisfactory, and is in the range of 80-108.8%. Specific results are shown in Table 2 attached to the specification.

TABLE 2 recovery of different alpha toxin concentrations in milk samples

Example 5: combination of detection method and smart phone

And the combination of the image processing technology and the smart phone is utilized, and the mobile phone APP application software is further manufactured through programming, so that the software program is applicable to all android smart phones. The method comprises the following steps of setting basic parameters: image name, average gray value and LD ₅₀ Values. The specific operation is that the fluorescence picture obtained by detection is uploaded to the mobile phone APP to obtain the corresponding clostridial toxin concentration in real time. The specific result is shown in figure 6 of the specification.

In summary, in the developed system, the whole process is less than 90 minutes, and the detection limit is 0.625LD ₅₀ CPA. The result shows that the silica microsphere combined smart phone CPA detection system is a sensitive, stable and convenient detection method, and provides a new method for detecting natural CPA in food.

The method for detecting clostridium perfringens alpha toxin based on the combination of the silica microspheres and the smart phone in real time can be realized by a person skilled in the art by referring to the content of the specification, properly changing the condition route and other links, although the method and the preparation technology of the invention have been described by the preferred embodiment examples, the related person can obviously change or recombine the method and the technical route described herein to realize the final preparation technology without departing from the content, the spirit and the scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included within the spirit, scope and content of the invention.

Reference to the literature

1.Jang,Y.S.,et al.,Prevalence,toxin-typing,and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul,Korea.Anaerobe,2020:p.102235.

2.Lacey,J.A.,et al.,In silico Identification of Novel Toxin Homologs and Associated Mobile Genetic Elements in Clostridium perfringens.Pathogens,2019.8(1).

3.Kumar,N.P.,N.V.Kumar,and A.Karthik,Molecular detection and characterization of Clostridium perfringens toxin genes causing necrotic enteritis in broiler chickens.Tropical Animal Health and Production,2019.51(9–10).

4.Oda,M.,et al.,Effect of erythromycin on biological activities induced by clostridium perfringens alpha-toxin.J Pharmacol Exp Ther,2008.327(3):p.934-40.

5.Takehara,M.,et al.,Granulocyte Colony-Stimulating Factor Does Not Influence Clostridium Perfringens alpha-Toxin-Induced Myonecrosis in Mice.Toxins(Basel),2019.11(9).

6.Takehara,M.,et al.,Clostridium perfringens alpha-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte production while triggering septic shock.Commun Biol,2019.2:p.45.

7.Takagishi,T.,et al.,Clostridium perfringens alpha-toxin impairs erythropoiesis by inhibition of erythroid differentiation.Sci Rep,2017.7(1):p.5217.

8.<Rapid Detection of Clostridium perfringens Type A Enterotoxin by Enzyme-Linked Immunosorbent Assay.pdf>.

9.<Detection of Clostridium perfringens alpha toxin using a capture antibody ELISA.pdf>.

10.<ENTEROTOXAEMIA IN GOATS.pdf>.

11.<Molecular Typing and Epidemiological Survey of Prevalence of Clostridium perfringens Types by Multiplex PCR.pdf>.

12.Baums,C.G.,et al.,Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.Veterinary Microbiology,2004.100(1-2):p.11-16.

13.Gurjar,A.A.,et al.,Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle.Mol Cell Probes,2008.22(2):p.90-5.

14. Du Jige, et al, expression of clostridium perfringens alpha toxin mutants and immunoprotection assessment chinese veterinary journal 2019.039 (002): p.265-270,280.

15.Jige,D.,et al.,Three Copies Recombinant Expression and Immunogenicity Analysis of C-terminal of Clostridium perfringensαToxin.china animal husbandry&veterinary medicine, 2018。

Sequence listing

<110> university of Tianjin

<120> a method for visual detection of clostridium perfringens alpha toxin

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Claims

1. A visual detection method for clostridium perfringens alpha toxin is characterized in that a prokaryotic expression CPA truncated protein is used for injecting animals to prepare antibodies to CPA _N After the antibody is labeled by fluorescein isothiocyanate, the capture antibody CPA is obtained _C3 The coupling silica microspheres are used for capturing alpha toxin in food, and the concentration of the alpha toxin of the sample to be detected can be obtained in real time after the fluorescence picture obtained by detection is uploaded to the mobile phone APP;

the prokaryotic expression CPA protein truncated CPA _N Comprises two mutation sites D56G and D130G of CPA protein, wherein the CPA comprises a polypeptide sequence of the CPA protein _N The truncated body is a protein sequence of SEQ ID No. 1;

said prokaryotic expression CPA protein truncated CPA _N The construction of (a) and the preparation of antibodies comprising the steps of:

(1) CPA protein truncate CPA _N Is constructed by the following steps: because CPA protein has toxicity and lethality, in order to facilitate the preparation of subsequent antisera, CPA mutants are used in experiments to eliminate the toxic effect on immunized animals; plasmid pET30a-CPA using 4-amino acid mutant _m4 And a C-terminal three-copy tandem plasmid pET30a-CPA _C3 The method comprises the steps of carrying out a first treatment on the surface of the In CPA _m4 Primer CPA is designed on the basis _m4 Dele TM-F, SEQ ID No.2 and CPA _m4 Deletion of transmembrane Domain by delete TM-R, SEQ ID No.3 to obtain N-terminal truncated plasmid pET30a-CPA _N The plasmid contains only the two amino acid mutations D56G, D130G;

(2) Antibody preparation: the constructed pET30a-CPA _N pET30a-CPA _C3 The prokaryotic expression vector converts competent cells of escherichia coli BL21 (DE 3), IPTG induces and purifies recombinant proteins; immunization of rabbits three times with 1mg/mL protein amount resulted in anti-CPA _C3 And CPA (CPA) _N Purifying polyclonal antibody by rabbit serum and Saturated Ammonium Sulfate (SAS) precipitation, and detecting antibody titer by Western-blot and ELISA;

(3) CPA using semi-permeable membrane process _N The fluorescence intensity of the microsphere detected by a fluorescence microscope is the concentration of the corresponding clostridium alpha toxin by utilizing the double antibody sandwich principle of antigen-antibody combination.

2. The method for visual detection of clostridium perfringens alpha toxin according to claim 1 wherein said CPA protein truncate silica microsphere is coupled comprising the steps of:

(1) Quantification of clostridium perfringens alpha toxin using virulence substitution experiment-lecithin hydrolysis experiment, and final measured natural CPA concentration of 10LD ₅₀ ；

(2) Activation of silica microspheres: carboxylated silica microsphere surface coupling CPA _N Polyclonal antibody, adding coupling agent N-hydroxysuccinimide (NHS) and carbodiimide (EDC) with final concentration of 10mg/mL, and finally sealing;

(3) Condition optimization of silica microsphere detection experiment: comprising optimally coupled CPA _C3 Conditions of antibody, optimal blocking conditions, optimal coupling conditions of detection antibody; finally, the optimal conditions for the fluorescence detection test, namely, the CPA of the capture antibody at 25 ℃ and 0.1mg/ml, are determined according to the experimental data _C3 After 2h interaction with activated silica microspheres, blocking with 5% BSA overnight at 4℃and finally detecting alpha toxin and 0.2mg/ml FITC-CPA at 37℃the next day _N And (3) performing interaction with the silica microspheres coupled with the capture antibody for 1h, so as to perform detection by a fluorescence microscope.

3. The method for visual detection of clostridium perfringens alpha toxin according to claim 1 wherein said alpha toxin capture standard curve is established with a detection line sensitivity of 0.625LD ₅₀ The detection time was 90min.

4. A method of visual detection of clostridium perfringens alpha toxin according to claim 3 wherein said establishment of alpha toxin capture standard curve comprises the steps of:

(1) Establishment of a standard curve: performing double gradient dilution on the natural clostridium perfringens alpha toxin, wherein the total dilution is 5, each dilution is 3 biological repetitions, and then obtaining a group of fluorescence pictures by detecting the fluorescence intensity of the silicon dioxide microspheres coupled to the FITC labeled antibodies;

(2) The fluorescent intensity in the picture is subjected to gray value analysis, the picture is firstly converted into an 8-bit gray image, background interference is eliminated, brightness values in all image areas are selected for carrying out average analysis, the average gray value of the corresponding image is finally obtained, the data are arranged to obtain a regression equation Y=0.01915X+0.002875, R ² = 0.9972; wherein X represents LD ₅₀ Y represents an average gray value;

(3) Programming an image processing technology into an APP (application) of a smart phone, firstly analyzing an RGB value of a picture, then converting the RGB color value into hexadecimal color code gray to obtain a gray value of the image, and finally utilizing an IF (IF-based) algorithm to correspond different gray value ranges to CPA (complex programmable logic) concentration; after APP is prepared, the corresponding CPA concentration can be directly obtained by uploading the fluorescence image.