CN111983240A

CN111983240A - Visual detection method for clostridium perfringens alpha toxin

Info

Publication number: CN111983240A
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: 2020-11-24
Anticipated expiration: 2040-08-27
Also published as: CN111983240B

Abstract

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

Description

Visual detection method for clostridium perfringens alpha toxin

Technical Field

The invention belongs to the technical field of biology, and relates to a detection method of a food-borne pathogenic bacterium, namely clostridium perfringens alpha toxin.

Background

Clostridium perfringens (Clostridium perfringens), also known as food-borne gastroenteritis and other infectious diseases. As a opportunistic pathogen, it can contaminate many types of retail meat products and produce alpha toxin 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 a variety of hosts due to the production of a variety of toxins and extracellular enzymes^[2]. To date, at least 20 exotoxins have been discovered, with the major lethal toxins being α, β, and iota. Because all types of toxins can produce alpha toxin and cause 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 muscle necrosis^[5]Production of granulocytes^[6]Inhibition of erythroid differentiation^[7]Platelet aggregation and increased vascular permeability.

Currently, the detection of clostridium perfringens is mainly based on molecular biological examination PCR or immunological examination ELISA and the like. Oisvik et al established a four-layer sandwich method for the detection of Clostridium perfringens enterotoxin. This method can detect enterotoxin at 0.1. mu.g/mL, but the entire experiment takes 4 days to complete. McClane et al^[8]The established indirect ELISA method can detect enterotoxin with the concentration of 25ng/mL and shorten the experimental time. Hale et al^[9]An antigen capture ELISA method is established for detecting the clostridium perfringens toxin, the method has good specificity and sensitivity, and the minimum detection amount of alpha toxin is 19 ng/mL. These methods can be used to diagnose Clostridium perfringens using PCR techniques instead of serum neutralization assays^[10]The goat feces and the toxin content of the gastrointestinal tract were successfully tested.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 major toxins of clostridium perfringens, but this approach requires extraction and purification of the bacterial genome. Baums CG, etc^[12]A multiplex PCR method capable of typing Clostridium perfringens is established, which can directly detect and distinguish cpa (alpha), cpb (beta 1), etx (), iap (iota), cpe (enterotoxin) and cpb2 (beta 2) and other toxin genes, and has good specificity and repeatability. Furthermore, Gurjar et al^[13]Fluorescent quantitative PCR was established. The major toxin gene of clostridium perfringens can be detected directly from fecal samples and can be used for epidemiological investigation of diseases. In conclusion, the sensitivity of detecting clostridium perfringens by ELISA in the prior art is higher, and the lowest detection limit can reach 19ng/mL, but the general problem is that the operation process is time-consuming and tedious; and a PCR method can be used for simultaneously detecting various clostridium perfringens toxins, but the toxins need to be purified in the detection process, the sensitivity is not high, and the method is time-consuming and tedious. Therefore, it is necessary to establish a detection system for detecting clostridial toxins that is efficient, sensitive, and convenient.

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, single detection technology deficiency and the like in the prior art.

The purpose of the invention is realized by 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 injecting CPA truncated protein expressed by pronucleus into an animal to prepare an antibody, labeling the antibody with Fluorescein Isothiocyanate (FITC), coupling the silica microspheres to capture the alpha toxin in food, uploading a detected fluorescence image 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 truncation CPA_NThe construction of (2) and the preparation of antibodies comprising two mutation sites of CPA protein (D56G, D130G), the CPA_NThe protein sequence of truncated body SEQ ID No. 1.

The method comprises the following main steps:

(1) CPA protein truncation CPA_NThe construction of (1): because of the toxicity and lethality of CPA proteins, the use of CPA mutants in experiments to eliminate the toxic effects on immunized animals is convenient for subsequent antiserum preparations. Plasmid containing mutant of amino acid at position 4 (pET30 a-CPA) given by professor of the aged cloud_m4) And a C-terminal triple-copy tandem plasmid (pET30 a-CPA)_C3)^[14,15]. At CPA_m4Design of primers (CPA)_m4Del TM-F, SEQ ID No.2 and CPA_m4Dele TM-R, SEQ ID No.3) deletion transmembrane domain to obtain N-terminal truncated plasmid pET30a-CPA_NThe plasmid contained only two amino acid mutations (D56G, D130G).

(2) Preparing an antibody: the constructed pET30a-CPA_NAnd pET30a-CPA for benefit of professor_C3Prokaryotic expression vector is transformed into competent cell of Escherichia coli BL21(DE3), and recombinant protein is induced and purified by IPTG. anti-CPA was obtained by immunizing rabbits three times with a protein amount of 1mg/mL_C3And CPA_NThe polyclonal antibody is purified by rabbit serum and a Saturated Ammonium Sulfate (SAS) precipitation method, and the antibody titer is detected by Western-blot and ELISA.

(3) CPA Using semi-permeable Membrane method_NThe labeling of Fluorescein Isothiocyanate (FITC) of the antibody is convenient for the coupling and fluorescence detection of the subsequent silica microspheres, the general principle of the detection is shown in fig. 7, and the fluorescence intensity of the microspheres detected by a fluorescence microscope is the concentration of the corresponding clostridial alpha toxin by utilizing the antigen-antibody combined double antibody sandwich principle.

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

(1) the toxicity substitution experiment, namely the lecithin hydrolysis experiment, is used for quantifying the clostridium perfringens alpha toxin, and the finally measured natural CPA concentration is 10LD₅₀。

(2) Silica microspheresActivation of (2): CPA coupled with surface of carboxylated silicon dioxide microsphere_NPolyclonal antibody, adding coupling agent N-hydroxysuccinimide (NHS) and carbodiimide (EDC) with final concentration of 10mg/mL, and finally blocking.

(3) Optimizing the conditions of the silica microsphere detection experiment: the optimal conditions for the fluorescence detection experiment were determined from the experimental data, namely capture antibody (CPA) at 0.1mg/ml at 25 deg.C_C3) After 2h interaction with activated silica microspheres, blocking was performed with 5% BSA overnight at 4 deg.C, the next day at 37 deg.C, alpha toxin and 0.2mg/ml detection antibody (FITC-CPA)_N) And (3) interacting with the silica microspheres coupled with the capture antibody for 1h, and detecting by a fluorescence microscope.

Establishment of alpha toxin capture standard curve, detection line sensitivity is 0.625LD₅₀The detection time is about 90 min.

The method comprises the following steps:

(1) establishment of a standard curve: and carrying out double gradient dilution on natural clostridium perfringens alpha toxin (CPA) for 5 dilutions, wherein each dilution is 3 biological replicates, and then detecting the fluorescence intensity of the silica microspheres coupled with the FITC labeled antibody to obtain a group of fluorescence pictures.

(2) The fluorescence intensity in the picture is subjected to gray value analysis (conventional image analysis software such as Photoshop and ImageJ is used), the research uses the ImageJ software to perform gray value analysis, firstly, the picture is converted into an 8-bit gray value picture, background interference is eliminated, then, brightness values in all image areas are selected to perform averaging analysis, finally, the average gray value of a corresponding image is obtained, and data are sorted to obtain a regression equation Y which is 0.01915X +0.002875 (R)²0.9972). Wherein X represents LD₅₀And Y represents an average gray value.

(3) The image processing technology is programmed into an intelligent mobile phone APP, the general principle is that RGB value analysis is carried out on a picture, then RGB color values are converted into hexadecimal color codes (gray), accordingly the gray value of the picture is obtained, and finally different gray value ranges correspond to CPA concentrations through an IF algorithm. After the APP is prepared, the corresponding CPA concentration can be directly obtained by uploading a fluorescence picture.

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

The invention has the beneficial effects that: the invention discloses a method for detecting food-borne clostridium perfringens alpha toxin by coupling alpha toxin antibody with FITC label and silica microspheres according to a double-antibody sandwich method. And analyzing and processing the fluorescence micrographs through APP software universal for android smart phones to determine the content of the alpha toxin. The lowest detection limit of the method is 0.625LD₅₀And the whole detection time is less than or equal to 90 min. The whole data processing time is less than 5 seconds. The method has the advantages of good stability, high sensitivity, convenience and rapidness in operation and the like, and provides a new idea and direction for detecting the food-borne clostridium perfringens alpha toxin.

Drawings

FIG. 1: CPA recombinant protein CPA_C3And CPA_NSDS-PAGE results of expression and purification; (a) CPA_C3Expression electrophoretogram of recombinant protein. Lanes 1-2 are purified CPA _C32 samples of recombinant protein; (b) CPA_NElectrophoresis result of recombinant protein M: protein molecular mass Standard (Blue Plus) lanes 3-4 are purified CPA_NTwo parallel samples of recombinant protein;

FIG. 2: clostridium perfringens alpha toxin (CPA)_C3And CPA_N) (iii) determination of polyclonal antibody concentration and titer;

(a) standard curve of antibody concentration by Bradford method;

(b) recombinant protein CPA_C3Western blot results of (M: protein marker (GenStar M221) lanes 1-2: CPA)_C3Recombinant proteins);

(c) recombinant protein CPA_C3And CPA_NWestern blot results of (M: protein marker (GenStar M221) lanes 3-4: CPA)_NRecombinant proteins);

(d) recombinant protein CPA_C3Indirect ELISA results of (1): detected CPA_C3The titer of the antibody was 1: 3200;

(e) recombinant protein CPA_NIndirect ELISA results of (1): detected CPA_NThe titer of the antibody was 1: 6400;

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

FIG. 4: optimal conditions of a silica microsphere fluorescence detection experiment;

(a) determining the optimal antibody coating conditions (fixing other conditions, selecting toxin concentration as 10LD50, antibody dilution concentration as 0.02, 0.1, 0.5mg/ml, experiment temperature and time as 37 ℃ for 2h, 25 ℃ for 2h, 4 ℃ for 12h, and 3 biological repetitions of each group of experiments);

(b) determining the optimal blocking conditions (fixing other conditions, selecting toxin concentration as 10LD50, selecting blocking agent as 5% milk and 5% BSA, selecting experiment temperature and time as 37 deg.C for 2h, 25 deg.C for 2h, 4 deg.C for 12h, and 3 biological repetitions per group);

(c) the optimal incubation time and the dilution (fixation) of the enzyme-labeled antibody are unchanged, the toxin concentration is 10LD50, the dilution concentration of the fluorescence-labeled antibody is 0.05, 0.1 and 0.2mg/mL, and the experimental temperature and time are respectively 0.5h at 37 ℃, 1h at 37 ℃ and 2h at 37 ℃; 3 biological replicates per set of experiments);

FIG. 5: CPA detected fluorescence map and established standard regression equation;

(a) fluorescence pictures at different toxin concentrations: from left to right, the concentration is respectively 10LD₅₀，5LD₅₀，2.5LD₅₀， 1.25LD₅₀，0.625LD₅₀Three biological replicates per concentration gradient (i.e., corresponding to three columns in the figure);

(b) standard regression equation established based on fluorescence intensity and toxin concentration, Y0.01915X +0.002875 (R)²＝0.9972)。

FIG. 6: an APP detection interface of the smart phone;

FIG. 7: principle of silica microsphere coupling fluorescent antibody combined with smartphone APP detection system.

Detailed Description

The invention is further illustrated with reference to the figures and the specific embodiments.

Example 1 basic preparation of the detection method

Step one, the A-type standard strain (C57-1) is purchased from a Chinese veterinary drug inspection institute. Alpha toxin 4 amino acid mutant plasmid (CPA)_m4) And in alpha toxin (CPA)_C3) The C-terminal three-copy tandem prokaryotic expression vector (pET30a) was constructed by professor cloud Cheng of the Chinese veterinary drug research institute^[14,15]。

Because site-directed mutagenesis of toxins can greatly reduce the toxicity and lethality of CPA, subsequent preparation of rabbit serum antibodies without lethality is facilitated.

We then used the existing alpha toxin 4 amino acid mutant plasmid (CPA)_m4) The N-terminal truncation construction was performed, and thus the constructed expression vector contained two mutation sites (D56G, D130G). Construction of N-terminal expression vector of CPA PET30a-CPA Using two primers_NAccording to Genbank, the alpha toxin gene of clostridium perfringens (Genbank accession number: DQ202275) is searched, and N-terminal truncation body primers (CPA) are designed by removing the transmembrane domain_m4Del TM-F, SEQ ID No.2 and CPA_m4Del TM-R, SEQ ID No.3), upstream and downstream containing BamHI and Xho I restriction sites, respectively.

Step two, constructing a prokaryotic expression vector (CPA)_C3And CPA_N) Expressing and purifying prokaryotic recombinant protein, and immunizing rabbit with 1mg/ml protein amount for three times to obtain anti-CPA_C3And CPA_NThe polyclonal antibody is purified by rabbit serum and a Saturated Ammonium Sulfate (SAS) precipitation method, and the antibody titer is detected by Western-blot and ELISA. The results are shown in FIG. 1, where CPA is a recombinant protein CPA_C3And CPA_NCan be expressed normally. As can be seen from FIG. 2, CPA of Clostridium perfringens alpha toxin was determined by Bradford method_C3And CPA_NThe polyclonal antibody concentration of (2) was 7.02mg/mL and 7.47mg/mL, respectively. Western blot results of FIGS. 2-b, c show that the recombinant protein CPA_C3And CPA_NHas good specificity; FIG. 2-d, e Indirect ELISA test results show that the recombinant protein CPA_C3And CPA_NThe potency of (A) was 1:3200 and 1:6400, respectively.

Step three, CPA is carried out by using a semipermeable membrane method_NLabeling of Fluorescein Isothiocyanate (FITC) of the antibody was performed using a 0.025M carbonate buffer, pH 9.0The wash corrected the protein concentration of the antibody solution to 10mg/mL and placed in the dialysis bag. Simultaneously weighing the amount of the antibody protein 1/20FITC, namely the required amount (mg) of FITC (Beijing Solel technologies, Inc.) is equal to the protein content (mg/mL) multiplied by the amount of the antibody solution (mL) multiplied by 1/20; dissolving FITC in 10 times of 0.025M antibody solution, pH 9.0 carbon in saline buffer, and stirring gently to avoid air bubbles; the antibodies in the dialysis bag were immersed in FITC solution and left at 4 ℃ for 16-18 hours with periodic stirring 1-2 times per hour with a magnetic stirrer for a total stirring time of about 6 hours. The dialysis bag was removed and dialyzed against 0.01M PBS pH7.2 for 4 hours.

And step four, quantifying the clostridium perfringens alpha toxin by using a virulence replacement experiment, namely a lecithin hydrolysis experiment. First, freshly cooked egg yolk is mashed and mixed, then 1% CaCl is added₂Physiological saline 40 mg/g and 6% Al (OH)₃Gel 3ml/g, mix and place in a refrigerator at 4 ℃ overnight. The following day, the supernatant was collected as lecithin after centrifugation at 720g for 20 minutes. With physiological saline (2)⁰、2^-1、2^-2、 2^-3、2^-4、2^-5) The alpha toxin was diluted, 1mL of lecithin was added for each dilution, and then allowed to stand 1 time at 37 ℃. And (4) hours. Negative medium and 1mL lecithin as controls at OD₅₅₀The absorbance (turbidity) was measured, and the dilution of each hydrolysis reaction was equivalent to 1/6.4LD₅₀Toxicity, i.e. the toxicity of the reaction after 2-fold dilution 2x/6.4 LD₅₀. The final measured native CPA concentration was 10LD₅₀. The results are shown in figure 3, the hydrolysis method of lecithin is used for measuring the hydrolysis degree of CPA, each concentration gradient is subjected to three biological repetitions, a series of light absorption values are obtained, the toxicity and the hydrolysis turbidity degree have stable correlation, the corresponding relation between the hydrolysis degree and the concentration of CPA is calibrated by a curve, and finally CPA with the lowest detectable dilution factor is compared with formula 2x/6.4 LD by the comparison₅₀The clostridial toxin (undiluted) extracted in this experiment was calculated to be 10LD₅₀。

Coupling the carboxyl modified silicon dioxide microspheres with the fluorescent antibody: weighing a certain amount of silica microspheres (particle size 10 μm, Tianjin Yijia science and technology Co., Ltd.) in a ratio of 0.5: 1 EDC was added in a ratio of 1: NHS was added at ratio 1, mixed well and shaken well at room temperature. After 20 minutes, the cells were washed with PBS and centrifuged to remove the washing solution (repeated three times). The optimal experimental conditions for coupling the antibody to the silica microspheres were then determined.

Example 2: condition optimization of detection methods

Step one, determining an optimal coating condition: the variable is the coating antibody (bound silica microsphere antibody CPA)_C3) At a concentration of 10LD, under fixed conditions, and at a time and temperature, the toxin concentration is selected to be₅₀(each condition was repeated 3 times) and then the fluorescence image was observed by fluorescence microscope observation, and finally the ratio was obtained by gray value analysis. Finally, the optimal coating conditions were determined to be 0.1mg/mL capture antibody and the silica microspheres were mixed for 2 hours at 25 ℃. The results are shown in figure 4: optimal conditions for microsphere fluorescence detection experiments; (a) determining the optimal antibody coating condition.

Step two, determining the optimal sealing condition: the variables were selected blocking agents (5% skim milk powder or 5% BSA), other conditions were fixed, and the selected toxin concentration was 10LD₅₀(3 replicates per condition) and the grey values of the fluorescence picture analysis were obtained. Finally, the optimal sealing conditions were determined to be 5% BSA at 4 ℃ for 12 hours. The results are shown in figure 4: optimal conditions for microsphere fluorescence detection experiments; (b) the optimal sealing conditions are determined.

Step three, determining the optimal incubation time and the optimal enzyme labeled antibody (CPA)_N) Dilution of (c): the variables are the dilution factor (1: 50, 1: 100, 1: 200) and time of the enzyme-labeled antibody and the temperature is fixed, and when other conditions are fixed, the selected toxin concentration is 10LD₅₀(3 biological replicates per condition) and the grey values of the fluorescence images were analyzed. Finally, the enzyme-labeled antibody was optimally diluted at a concentration of 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 experiment are that the capture antibody (CPA) is 0.1mg/ml at 25 deg.C_C3) After 2h interaction with activated silica microspheres, blocking with 5% BSA overnight at 4 deg.C the following dayAlpha toxin and 0.2mg/ml detection antibody (FITC-CPA) were added at 37 deg.C_N) And (3) interacting with the silica microspheres coupled with the capture antibody for 1h, and detecting by a fluorescence microscope.

Example 3: establishment of detection method

Step one, performing concentration gradient dilution of toxin CPA, and preliminarily quantitatively determining that the undiluted protoxin concentration is 10LD₅₀Then we serially diluted it 2 times, i.e. 10LD was obtained₅₀，5LD₅₀， 2.5LD₅₀，1.25LD₅₀，0.625LD ₅₀3 biological replicates were performed for each concentration gradient and the corresponding fluorescence pictures were obtained by fluorescence microscopy. The results are shown in FIG. 5, and CPA was diluted by 10LD gradient₅₀，5LD₅₀，2.5LD₅₀，1.25LD₅₀，0.625LD₅₀Fluorescence intensity was then measured, and three biological replicates were set up for each concentration gradient. The equation stability is proved to be good.

Step two, using ImageJ software to analyze the gray value, and finally drawing the obtained gray value into a regression curve, wherein the regression equation is as follows: Y0.01915X +0.002875 (R)²0.9972). The method is adopted to determine the mouse LD with the minimum detection limit of 0.625 times of the clostridium perfringens alpha toxin₅₀And has high sensitivity. The specific result is shown in figure 5 in the specification.

And step three, dividing five dilutions of the detected toxin into five groups, performing three biological repetitions on each dilution, and analyzing the variation coefficient of the gray value result obtained by detection. The analysis shows that the results are all 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. The specific results are shown in the specification and attached table 1.

TABLE 1 repeatability test for fluorescence detection of silica microspheres (n ═ 3)

Example 4: silicon dioxide microsphere detection of CPA concentration in food

The effect of the detection technology established in the experiment is evaluated by carrying out actual detection on the milk which is determined according to the GB 4789.13-2012 detection and does not contain the clostridium welchii, namely, the applicability and the interference resistance of the detection system to actual food are tested. For milk, we took low, medium and high concentration (0.625 LD) of clostridium perfringens toxin respectively₅₀， 2.5LD₅₀，10LD₅₀) And mixing them with 1mL of milk, respectively, and then mixing the mixture with the antibody CPA_C3With the well-linked fluorescent FITC antibody CPA_NThe silica microspheres interact for one hour, milk is removed by washing, then the silica microsphere fluorescence detection experiment introduced by us is carried out, the obtained fluorescence image is uploaded to APP, the fluorescence intensity of the APP is analyzed, the corresponding toxin concentration is obtained by conversion according to a standard regression equation, the recovery rate is satisfactory, and the range is 80% -108.8%. The specific results are shown in the specification and attached table 2.

TABLE 2 recovery of different alpha toxin concentrations in milk samples

Example 5: combination of detection method and smart phone

By means of combination of an image processing technology and a smart phone, mobile phone APP application software is further manufactured through programming, and the software program is suitable for all android smart phones. The method comprises the following steps of setting basic parameters: image name, mean gray value and LD₅₀The value is obtained. The specific operation is that the corresponding clostridial toxin concentration is obtained in real time immediately after a fluorescence image obtained by detection is uploaded to a mobile phone APP. The specific result is shown in figure 6 in the specification.

In summary, in the developed system, the whole process is less than 90 minutes, and the detection limit is 0.625LD₅₀And (4) CPA. The result shows that the silicon dioxide microsphere is combined with the CPA detection system of the smart phone, so that the method is sensitive, stable and convenient, and a new method is provided for detecting the natural CPA in the food.

Although the method and the preparation technology of the present invention have been described by way of preferred embodiments, it is obvious to those skilled in the art that the method and the preparation technology described herein can be modified or recombined to realize the final preparation technology without departing from the content, spirit and scope of the present invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be 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. Dugege, et al, evaluation of the expression and immunoprotection of Clostridium perfringens alpha toxin mutants Chinese veterinary bulletin 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> Tianjin university

<120> method for visually detecting alpha toxin of clostridium perfringens

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 247

<212> PRT

<213> Artificial sequence ()

<400> 1

His Met Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr His Ala Met Ile

1 5 10 15

Val Thr Gln Gly Val Ser Ile Leu Glu Asn Asp Leu Ser Lys Asn Glu

20 25 30

Pro Glu Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Glu Asn Met His

35 40 45

Glu Leu Gln Leu Gly Ser Thr Tyr Pro Gly Tyr Asp Lys Asn Ala Tyr

50 55 60

Asp Leu Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe

65 70 75 80

Ser Lys Asp Asn Ser Trp Tyr Leu Ala Tyr Ser Ile Pro Asp Thr Gly

85 90 95

Glu Ser Gln Ile Arg Lys Phe Ser Ala Leu Ala Arg Tyr Glu Trp Gln

100 105 110

Arg Gly Asn Tyr Lys Gln Ala Thr Phe Tyr Leu Gly Glu Ala Met His

115 120 125

Tyr Phe Gly Gly Ile Asp Thr Pro Tyr His Pro Ala Asn Val Thr Ala

130 135 140

Val Asp Ser Ala Gly His Val Lys Phe Glu Thr Phe Ala Glu Glu Arg

145 150 155 160

Lys Glu Gln Tyr Lys Ile Asn Thr Ala Gly Cys Lys Thr Asn Glu Asp

165 170 175

Phe Tyr Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala Trp Ser Lys

180 185 190

Glu Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile Tyr Tyr Ser

195 200 205

His Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr Ala Ala Lys

210 215 220

Val Thr Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr Ile Tyr Arg

225 230 235 240

Phe Leu His Asp Val Ser Glu

245

<210> 2

<211> 27

<212> DNA

<213> CPAm4

<400> 2

ggatcccata tgtggggggg ggggagg 27

<210> 3

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ctcgagggat gggtcattac cctcgc 26

Claims

1. A visual detection method for clostridium perfringens alpha toxin is characterized in that CPA truncation protein expressed by pronucleus is injected into animals to prepare antibodies, the antibodies are labeled with fluorescein isothiocyanate and then are coupled with silica microspheres to capture the alpha toxin in food, and the concentration of the alpha toxin of a sample to be detected is obtained in real time after a detected fluorescence picture is uploaded to a mobile phone APP.

2. The method for visual detection of clostridium perfringens alpha toxin according to claim 1, wherein said prokaryotic expression CPA protein truncation CPA_NThe construction of (2) and the preparation of antibodies comprising two mutation sites of CPA protein (D56G, D130G), the CPA_NThe truncated body is the protein sequence of SEQ ID No. 1.

3. The method for visual detection of clostridium perfringens alpha toxin according to claim 2, wherein said prokaryotic expression CPA protein truncation CPA_NThe construction and the preparation of the antibody comprise the following steps:

(1) CPA protein truncation CPA_NThe construction of (1): because the CPA protein has toxicity and lethality, in order to facilitate the subsequent antiserum preparation, the CPA mutant is used to eliminate the toxic influence on the immune animals in the experiment; plasmid containing mutant of amino acid at position 4 (pET30 a-CPA) given by professor of the aged cloud_m4) And a C-terminal triple-copy tandem plasmid (pET30 a-CPA)_C3) (ii) a At CPA_m4Design of primers (CPA)_m4Del TM-F, SEQ ID No.2 and CPA_m4Dele TM-R, SEQ ID No.3) deletion transmembrane domain to obtain N-terminal truncated plasmid pET30a-CPA_NThe plasmid contains only two amino acid mutations (D56G, D130G);

(2) preparing an antibody: the constructed pET30a-CPA_NAnd pET30a-CPA for benefit of professor_C3Transforming escherichia coli BL21(DE3) competent cells by the prokaryotic expression vector, and inducing and purifying recombinant protein by IPTG; anti-CPA was obtained by immunizing rabbits three times with a protein amount of 1mg/mL_C3And CPA_NPurifying the polyclonal antibody by a rabbit serum and Saturated Ammonium Sulfate (SAS) precipitation method, and detecting the titer of the antibody by Western-blot and ELISA;

(3) CPA Using semi-permeable Membrane method_NLabeling Fluorescein Isothiocyanate (FITC) of the antibody so as to facilitate the coupling and fluorescence detection of the subsequent silica microspheres, and detecting the fluorescence intensity of the microspheres through a fluorescence microscope by utilizing the double-antibody sandwich principle of combining antigen and antibody, namely the concentration of the corresponding clostridium alpha toxin.

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

(1) the toxicity substitution experiment, namely the lecithin hydrolysis experiment, is used for quantifying the clostridium perfringens alpha toxin, and the finally measured natural CPA concentration is 10LD₅₀；

(2) Activation of silica microspheres: CPA coupled with surface of carboxylated silicon dioxide microsphere_NAdding a polyclonal antibody, namely adding coupling agents of N-hydroxysuccinimide (NHS) and carbodiimide (EDC) with the final concentration of 10mg/mL, and finally blocking;

(3) optimizing the conditions of the silica microsphere detection experiment: including optimally coupled CPA_C3Antibody conditions, optimal blocking conditions, optimal coupling detection antibody conditions; finally, the optimal conditions for the fluorescence detection experiment were determined from the experimental data, i.e.capture antibody (CPA) at 0.1mg/ml at 25 ℃_C3) After 2h interaction with activated silica microspheres, blocking was performed overnight at 4 ℃ with 5% BSA, and the final day at 37 ℃ with alpha toxin and 0.2mg/ml detection antibody (FITC-CPA)_N) And (3) interacting with the silica microspheres coupled with the capture antibody for 1h, and detecting by a fluorescence microscope.

5. 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 0.625LD₅₀The detection time is 90 min.

6. The method for visual detection of alpha toxin of clostridium perfringens according to claim 5 wherein said establishment of said alpha toxin capture standard curve comprises the steps of:

(1) establishment of a standard curve: carrying out double gradient dilution on the natural clostridium perfringens alpha toxin, wherein the dilution is 5, each dilution is 3 biological repetitions, and then detecting the fluorescence intensity of a silica microsphere coupled to the FITC labeled antibody to obtain a group of fluorescence pictures;

(2) performing gray value analysis on the fluorescence intensity in the picture, firstly converting the picture into an 8-bit gray image and eliminating background interference, then selecting brightness values in all image areas to perform averaging analysis, finally obtaining the average gray value of the corresponding image, and sorting data to obtain a regression equation Y which is 0.01915X +0.002875(R is the sum of R and R is the sum of X and R is the sum of Y and R is²0.9972); wherein X represents LD₅₀Y represents the mean gray value;

(3) programming an image processing technology into an intelligent mobile phone APP, firstly analyzing RGB values of a picture, then converting the RGB color values into hexadecimal color code gray, thereby obtaining the gray value of the picture, and finally utilizing an IF algorithm to enable different gray value ranges to correspond to CPA concentrations; after the APP is prepared, the corresponding CPA concentration can be directly obtained by uploading a fluorescence picture.