CN112986560A - Bovine pathogenic bacillus cereus indirect ELISA detection kit - Google Patents
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Abstract
The invention relates to a microbial detection technology, in particular to an indirect ELISA detection kit for bovine pathogenic bacillus cereus. The method is characterized in that: the indirect ELISA detection kit takes bovine pathogenic bacillus cereus suspension as a coating antigen. Has the advantages that: the prepared detection kit is suitable for detecting bovine-derived pathogenic bacillus cereus, has the characteristics of strong detection specificity, high sensitivity, quick detection process, convenient operation and the like, and provides an effective technology and tool for preventing the infection of the bovine pathogenic bacillus cereus.
Description
Technical Field
The invention relates to a microbial detection technology, in particular to an indirect ELISA detection kit for bovine pathogenic bacillus cereus.
Background
Bacillus cereus (frankland) belongs to the family of bacillaceae, and the Bacillus cereus is similar to Bacillus thuringiensis, Bacillus anthracis and other biological characteristics, and widely exists in natural environment and food. Can produce antibacterial substances, inhibit the propagation of harmful microorganisms, degrade the nutrient components in the soil and improve the ecological environment. Part of the bacillus cereus can be used as a plant growth promoter and a microbial additive in animal feed. The bacteria are conditional pathogenic bacteria, which cause vomiting or diarrhea of human beings or animals clinically and are one of common food-borne pathogenic bacteria. However, no report on the lethality of the bacterium to bovine infection has been found before. In 2018, sudden death cases of cattle occur in Tongliao city in Mongolia, after infectious diseases such as bacillus anthracis and foot and mouth disease virus disease are investigated on two dead cattle, the blood is subjected to bacterial culture and colony growth is caused. Separating and identifying the growing bacteria, and finally determining the growing bacteria as bacillus cereus. In order to rapidly and effectively detect the diseases caused by the bovine pathogenic bacillus cereus, a sensitive, specific and efficient serological detection technology of the bacillus cereus needs to be established clinically.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for bovine pathogenic bacillus cereus.
The above purpose is realized by the following technical scheme:
provides an indirect ELISA detection kit for bovine pathogenic bacillus cereus, wherein the indirect ELISA detection kit takes bovine pathogenic bacillus cereus suspension as a coating antigen.
The bovine pathogenic bacillus cereus suspension is prepared by the following method:
streaking and inoculating bovine pathogenic bacillus cereus on a common agar culture medium, carrying out overnight culture at 37 ℃, selecting a single colony, inoculating the single colony to an LB liquid culture medium, and carrying out culture at 37 ℃ for 12-24 h; and centrifuging the cultured bacillus cereus culture, collecting the thallus, washing, and suspending the thallus by using a carbonate buffer solution to prepare the bacillus cereus suspension.
The common agar culture medium is prepared from the following raw materials by weight:
10g of peptone, 14g of agar, 5g of sodium chloride, 3g of beef extract powder and 1000ml of distilled water, boiling and dissolving, subpackaging, and autoclaving at 121 ℃ for 15 minutes for later use.
The raw materials of the LB liquid culture medium comprise 10g of peptone, 5g of yeast extract powder, 5g of sodium chloride, 2-3g of glucose and 1000ml of distilled water, the PH value is adjusted to 7.2-7.4, the raw materials are heated, boiled and dissolved, and the raw materials are subpackaged and sterilized for 15 minutes at 121 ℃ for standby.
The indirect ELISA detection kit further comprises: the kit comprises an ELISA plate, a confining liquid, bovine pathogenic bacillus cereus positive serum, bovine pathogenic bacillus cereus negative serum, an ELISA secondary antibody, a substrate color developing liquid and a stop solution.
The confining liquid is 5% skimmed milk powder.
The enzyme-labeled secondary antibody is HRP-goat anti-bovine IgG.
The method for detecting the bacillus cereus antibody by using the indirect ELISA detection kit comprises the following steps:
1. and (3) using an antigen coating solution to mix the bacillus cereus suspension 1: diluting with 60 ℃, coating an enzyme label plate, and coating overnight at 4 ℃;
2. taking out the enzyme label plate, washing with PBST solution, adding a sealing solution, and sealing for 2h at 37 ℃;
3. adding 1:200 diluted bovine pathogenic bacillus cereus positive serum, bovine pathogenic bacillus cereus negative serum and serum to be detected; incubating at 37 ℃ for 1 h;
4. adding enzyme-labeled secondary antibody diluted according to the ratio of 1:8000, and incubating for 1h at 37 ℃;
5. adding substrate color development solution, and reacting for 15min at 37 ℃ in a dark place;
6. adding stop solution to stop reaction, reading light absorption value at 450nm, determining detection result, and determining OD of sample to be detected450nm>0.525, the result is judged to be positive, otherwise, the result is negative.
The antigen coating solution is prepared by the following method: 1.465g of NaHCO3And 0.795g Na2CO3Dissolving in 400mL of deionized water, diluting to 500mL, and adjusting the pH value to 9.5 with NaOH.
Has the advantages that: the prepared detection kit is suitable for detecting bovine-derived pathogenic bacillus cereus, has the characteristics of strong detection specificity, high sensitivity, quick detection process, convenient operation and the like, and provides effective technology and equipment for preventing the infection of the bovine pathogenic bacillus cereus.
Detailed Description
The indirect ELISA detection kit provided by the invention takes bovine pathogenic bacillus cereus as a coating antigen, and finally establishes and assembles the bovine pathogenic bacillus cereus indirect ELISA detection kit by determining the concentration of the coating antigen, the dilution of serum to be detected and critical values of positive serum and negative serum.
1. Optimization of an indirect ELISA detection method:
taking standard positive serum and negative serum, performing orthogonal experiment by taking the purified and cultured bacillus cereus as a coating antigen, and optimizing the reaction conditions of indirect ELISA: respectively setting antigen coating concentration gradients, namely diluting bacterial liquid with the concentration of 1/20, 1/40, 1/60, 1/80 and 1/100, and coating overnight at 4 ℃; serum dilutions 1/50, 1/100, 1/200 and 1/400, incubated for 1h at 37 ℃; the blocking liquid type comprises 5% skimmed milk powder, 1% BSA and 1% gelatin; the secondary antibody is diluted at 1/2000, 1/4000, 1/6000, 1/8000 and 1/10000, and is incubated for 1h at 37 ℃; the color development time is 5min, 10min, 15min and 20min respectively. 2mol/L of H is used2SO4After termination of the reaction, the OD was detected450nmUsually, the positive OD value is 1, and the P/N value is the maximum, which is the optimal reaction condition. The specific test results are shown in Table 1.
TABLE 1 reaction condition optimization results of indirect ELISA detection methods
The bacteria liquid has complicated components, and the substances contained in the culture medium are difficult to completely remove, so that the OD value has errors, but the initial concentrations of the bacteria liquid under the same culture conditions are not greatly different and can be considered to be consistent, namely, the concentration of the coating antigen can be limited according to the dilution ratio of the bacteria liquid.
2. The indirect ELISA detection method comprises the following operation steps:
(1) preparation of coating antigen: streaking and inoculating bovine pathogenic bacillus cereus on a common agar culture medium, carrying out overnight culture at 37 ℃, selecting a single colony, inoculating the single colony to an LB liquid culture medium, and carrying out culture at 37 ℃ for 12-24 h; centrifuging at 12000r/min for 3-5min, collecting thalli, and re-suspending the thalli by using a carbonate buffer solution to prepare a bacterial suspension, namely the coating antigen.
(2) Coating of ELISA plate: the bacterial suspension is added to the ELISA plate at 100 mu L/well and stays overnight at 4 ℃. After coating, adding PBST to the ELISA plate, vibrating for 3-5min at 200 μ L/hole, discarding PBST in the ELISA plate, washing for 3 times, and patting off residual PBST in the hole after the last washing.
(3) And (3) sealing: 5% of skimmed milk powder is taken, 100 mu L of skimmed milk powder is added to an enzyme label plate, and the mixture is sealed for 2h at 37 ℃. And (3) washing after the closing is finished, wherein the washing operation is as shown in (2).
(4) Incubation of primary antiserum: the serum to be detected was diluted at 1:200, added to the microplate at 100. mu.L/well, and incubated at 37 ℃ for 1 h. After the incubation, the washing was performed as shown in (2).
(5) Incubation of enzyme-labeled secondary antibody: the enzyme-labeled secondary antibody was diluted 1:8000 with PBST, added to the plate at 100. mu.L/well, and incubated at 37 ℃ for 1 h. After the incubation, the washing was performed as shown in (2).
(6) Color development: TMB developing solution stored at 4 ℃ in a dark place is added to an ELISA plate at a concentration of 100. mu.L/well, and incubated at 37 ℃ for 15 min.
(7) Reaction termination and determination of results: taking 2mol/L H stored at room temperature2SO4100 μ L/well was added to the microplate and the results were read at 450nm with a microplate reader.
3. Determination of a threshold value
According to the above procedure, 39 standard negative sera were diluted 1:200 to be used as primary antiserum, and OD was determined450nmCalculating the mean value (QUOTE)
) And Standard Deviation (SD), and the critical value is QUOTE
+3SD, the critical value determined for this study was 0.525. The specific measurement results are shown in Table 2.
Table 2: critical value determination result
4. Specificity test
According to the operation steps, serum obtained by respectively immunizing rabbits with bacillus megaterium, bacillus amyloliquefaciens and bacillus subtilis stored in the laboratory is detected. Serum was diluted 1:200 and OD was determined450nmMeanwhile, standard positive serum and negative serum of bacillus cereus are set to be used as positive and negative controls, and detection results show that the determination results of the positive serum of bacillus megatherium, bacillus amyloliquefaciens and bacillus subtilis are all smaller than a positive critical value of 0.525, which indicates that the test method determined by the research has good specificity. The specific test results are shown in Table 3.
Table 3: specific test results
5. Repeatability test
(1) In-batch repeatability test: on the same enzyme label plate, 5 portions of positive serum and negative serum of Bacillus cereus are respectively taken, and OD is determined according to the above operation steps450nmTriplicate for each serum, the intra-batch Coefficient of Variation (CV) was calculated, and the intra-batch CV ═ SD/OD450nmAverage) × 100%. The results show that the intra-batch coefficient of variation is less than 10%. The specific test results are shown in Table 4.
Table 4: test results of in-batch repeatability tests
(2) Batch to batch repeatability test: taking 5 batches of positive serum and negative serum of bacillus cereus on enzyme label plates coated in different batches, and determining OD according to the ELISA operation steps in step 2450nmThree replicates per batch were performed and the inter-batch Coefficient of Variation (CV) was calculated, where (SD/OD)450nm) X 100%. The results show that the inter-batch coefficient of variation is less than 10%. The specific test results are shown in Table 5.
Table 5: test results of batch-to-batch repeatability tests
Assembly of ELISA kits
The bovine pathogenic bacillus cereus indirect ELISA detection kit specifically comprises the following components:
(1) one blank 96-hole enzyme label plate;
(2) one pure bovine pathogenic bacillus cereus is stored at the temperature of-20 ℃, and before use, the mixture is diluted by a coating diluent 1: 60, diluting;
(4) HRP-goat anti-bovine IgG enzyme-labeled secondary antibody is diluted by sample diluent 1:8000 before use;
(5) TMB substrate developing solution, stop solution, confining solution, sample diluent and washing solution are respectively one.
6. Detection of clinical samples
Clinical blood samples of 15 parts and 5 parts of healthy cattle blood were collected from the sick cattle farm. Separating serum and measuring OD450nm. The specific test results are shown in Table 6.
Table 6: results of clinical sample testing
The detection result shows that the pathogenic bovine serum is larger than the positive critical value and the OD of the healthy cattle450nmThe detection results are all smaller than the positive critical value, and the accuracy is 100%, which shows that the detection method established by the research is efficient and reliable.
Claims (8)
1. An indirect ELISA detection kit for bovine pathogenic bacillus cereus is characterized in that: the indirect ELISA detection kit takes bovine pathogenic bacillus cereus suspension as a coating antigen.
2. The indirect ELISA detection kit for bovine-derived pathogenic Bacillus cereus according to claim 1, wherein the kit comprises: the bovine pathogenic bacillus cereus suspension is prepared by the following method: streaking and inoculating bovine pathogenic bacillus cereus on a common agar culture medium, carrying out overnight culture at 37 ℃, selecting a single colony, inoculating the single colony to an LB liquid culture medium, and carrying out culture at 37 ℃ for 12-24 h; and centrifuging the cultured bacillus cereus culture, collecting the thallus, washing, and suspending the thallus by using a carbonate buffer solution to prepare the bacillus cereus suspension.
3. The indirect ELISA detection kit for bovine-derived pathogenic Bacillus cereus according to claim 2, wherein the kit comprises: the common agar culture medium is prepared from the following raw materials by weight: 10g of peptone, 14g of agar, 5g of sodium chloride, 3g of beef extract powder and 1000ml of distilled water, boiling and dissolving, subpackaging, and autoclaving at 121 ℃ for 15 minutes for later use.
4. The indirect ELISA detection kit for bovine-derived pathogenic Bacillus cereus according to claim 2, wherein the kit comprises: the raw materials of the LB liquid culture medium comprise 10g of peptone, 5g of yeast extract powder, 5g of sodium chloride, 2-3g of glucose and 1000ml of distilled water, the PH value is adjusted to 7.2-7.4, the raw materials are heated, boiled and dissolved, and the raw materials are subpackaged and sterilized for 15 minutes at 121 ℃ for standby.
5. The indirect ELISA detection kit for bovine-derived pathogenic Bacillus cereus according to claim 1, wherein the kit comprises: the indirect ELISA detection kit further comprises: the kit comprises an ELISA plate, a confining liquid, bovine pathogenic bacillus cereus positive serum, bovine pathogenic bacillus cereus negative serum, an ELISA secondary antibody, a substrate color developing liquid and a stop solution.
6. The indirect ELISA detection kit for bovine-derived pathogenic Bacillus cereus according to claim 1, wherein the kit comprises: the confining liquid is 5% skimmed milk powder; the enzyme-labeled secondary antibody is HRP-goat anti-bovine Ig.
7. The indirect ELISA detection kit for bovine-derived pathogenic Bacillus cereus according to claim 1, wherein the kit comprises: the method for detecting the bacillus cereus antibody by using the indirect ELISA detection kit comprises the following steps:
(1) and (3) using an antigen coating solution to mix the bacillus cereus suspension 1: diluting with 60 ℃, coating an enzyme label plate, and coating overnight at 4 ℃;
(2) taking out the enzyme label plate, washing with PBST solution, adding a sealing solution, and sealing for 2h at 37 ℃;
(3) adding 1:200 diluted bovine pathogenic bacillus cereus positive serum, bovine pathogenic bacillus cereus negative serum and serum to be detected; incubating at 37 ℃ for 1 h;
(4) adding enzyme-labeled secondary antibody diluted according to the ratio of 1:8000, and incubating for 1h at 37 ℃;
(5) adding substrate color development solution, and reacting for 15min at 37 ℃ in a dark place;
(6) adding stop solution to stop reaction, reading light absorption value at 450nm, determining detection result, and determining OD of sample to be detected450nm>0.525, the result is judged to be positive, otherwise, the result is negative.
8. The bovine-derived pathogenic bacillus cereus indirect ELISA detection kit according to claim 7, characterized in that: the preparation method of the antigen coating liquid comprises the following steps: 1.465g of NaHCO3And 0.795g Na2CO3Dissolving in 400mL of deionized water, diluting to 500mL, and adjusting the pH value to 9.5 with NaOH.
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