CN111323602B - iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep - Google Patents
iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep Download PDFInfo
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Abstract
The invention discloses an iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep, which comprises the following steps: (1) inoculating the mycoplasma capricolum goat pneumonia subspecies 1801 strain to MTB culture medium, performing amplification culture, centrifuging the bacterial liquid, sterilizing, performing PBS ultrasonic resuspension, and separating to obtain antigen; (2) coating an enzyme label plate with the antigen, and standing overnight at 4 ℃ after 1h in a 37 ℃ incubator; adding sealing liquid, and sealing at 37 deg.C for 2 hr; adding 200 times diluted serum sample, and incubating for 1h at 37 ℃; adding a 5000-time rabbit anti-sheep secondary antibody diluted by 5% of skimmed milk powder, and incubating at 37 ℃ for 1 h; (3) washing the plate with PBST, adding TMB color development solution, and incubating at 37 deg.C for 10 min; adding a stop solution to terminate the reaction; enzyme-linked immunosorbent assay (OD) reading450nmThe value is obtained. The sensitivity of the iELISA method can reach 98%, the operation is simple, the sample treatment capacity is large, and the method can be used for screening two serological negative sheep with Mcp and Mo pathogens and monitoring the Mcp antibody level in experiments.
Description
Technical Field
The invention belongs to the technical field of veterinary detection, and particularly relates to an iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep.
Background
China is the country with the most sheep and goat feeding amount and most goat marketing amount in the world, the stock quantity in 2017 reaches 30231.7 thousands, but sheep are easy to have various respiratory diseases in the breeding process, wherein sheep pneumonia is the most common disease in clinic, and the influence on the sheep industry is great. The sheep pneumonia can be divided into four types of viral, bacterial, mycoplasma and parasitic, and the mycoplasma which clinically causes sheep pneumonia in ChinaThere are two main types: mycoplasma ovipneumoniae (Mycoplasma ovipneumoniaeMo) and Mycoplasma capricolum subspecies pneumonia: (Mo)Mycoplasma capricolum subsp. capripneumoniaeMccp), both of these pathogens can infect both goats and sheep.
In the research work of vaccines and diagnostic techniques aiming at two pathogens of Mo and Mccp, the preparation work of screening negative sheep in the early stage of an experiment and the monitoring of the antibody level change in the later stage need to use a serological detection method, and besides, some clinical samples also need to be subjected to serological primary detection. Currently, alternative common serological methods include: ELISA, IHA and CFT, etc., but the prior ELISA, IHA and CFT have the following defects in the detection of Mo or Mcpcp antibody: the cELISA kit for detecting the Mccp antibody has good sensitivity and specificity, but is expensive, and the detection cost reaches 40 yuan per sample; IHA needs sensitized sheep red blood cells each time, which often leads to reduced batch-to-batch repeatability; the specificity and the sensitivity of CFT are poor, and even the detection result of infected sheep is negative. In addition, the methods can only detect serological antibodies of Mo or Mccp, which means that two rounds of detection are required when Mccp and Mo negative sheep are screened, which costs a lot of manpower and material resources and is not suitable for screening a large number of samples.
Disclosure of Invention
Aiming at the defects pointed out in the background technology, the invention provides the iELISA method for screening the sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies serological negative sheep, the sensitivity of the iELISA method can reach 98 percent, and the method has the advantages of simple operation and large sample handling capacity.
In order to achieve the purpose, the invention adopts the technical scheme that:
an iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma hyopneumoniae subspecies serological negative sheep, comprising the following steps:
(1) inoculating the mycoplasma capricolum goat pneumonia subspecies 1801 strain to a Modified Taiya Broth (MTB) culture medium for propagation, centrifuging the bacterial liquid, sterilizing, ultrasonically resuspending by PBS, and separating to obtain an antigen;
(2) coating an enzyme label plate with the antigen, wherein each hole is 100 mu l, and the enzyme label plate is incubated at 37 ℃ for 1h and then kept at 4 ℃ overnight; discarding the coating solution, washing with washing solution, adding 100 μ l of sealing solution into each well, and sealing at 37 deg.C for 2 hr; adding 100 μ l of 200-fold diluted serum sample, and incubating at 37 ℃ for 1 h; after washing the plate with PBST, adding 100 mul of rabbit anti-sheep secondary antibody diluted 5000 times with 5% skimmed milk powder into each hole, and incubating for 1h at 37 ℃;
(3) after incubation, washing the plate with PBST, adding 100 μ l of TMB into each well for color development, and incubating at 37 ℃ for 10 min; adding a stop solution to stop the reaction; placing the reacted enzyme label plate on an enzyme label instrument to read OD450 nmThe value is obtained.
Preferably, the antigen coating is 60 ng/well.
Preferably, the confining liquid is 5% skimmed milk powder.
The invention further provides application of the iELISA method in screening two seronegative sheep with pathogens of mycoplasma ovipneumoniae and mycoplasma capricolum.
The invention further provides application of the iELISA method in monitoring the antibody level of mycoplasma capricolum subsp pneumoniae.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
the Indirect ELISA (iELISA) method for screening the mycoplasma ovipneumoniae and the mycoplasma capricolum subspecies capricolumnae seronegative sheep, which is established by the invention, has the sensitivity of 98 percent, and has the advantages of simple operation and large sample handling capacity. The iELISA method has high sensitivity, and is suitable for screening of serological negative sheep with two pathogens of Mccp and Mo; analysis also finds that the method can show the change of the antibody level in the experimental process in time, and is consistent with the detection result and experimental fact of the conventional cELISA kit, which shows that the iELISA method can be applied to the research work of screening Mo and Mccp negative sheep and the monitoring of the Mccp antibody level in the experiment.
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FIG. 1 is a graph showing the results of monitoring the changes in Mccp antibody levels by the iELISA and the conventional cELISA method used in the present invention. In the figure: the cselisa 077 represents the cselisa assay result (SPI%) for sheep serum No. 077, indicated by the solid line, while the iiiisa 077 represents the elisa assay result (OD 450) for sheep serum No. 077, indicated by the dashed line, and so on.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Primary reagent
96-well enzyme-linked immunosorbent assay (ELISA) plate, Casein, BSA (bovine serum albumin), skimmed milk powder, secondary antibodies (rabbit anti-sheep and sigma), a Mccp antibody detection cELISA kit (IDEXX company), an enzyme-linked immunosorbent assay (ELISA) instrument, a BCA kit and the like. Strain: mccp 1801 strain, stored at the lanzhou veterinary institute of chinese academy of agricultural sciences. Serum: the total amount of the vaccine is 550 parts at different time points in a vaccine/infection experiment.
2. Antigen preparation
Inoculating a mycoplasma capricolum goat pneumonia subspecies (Mcpcp) 1801 strain stored in a laboratory into an MTB culture medium, performing amplification culture at a ratio of 37 ℃ and 1/10 to finally obtain 500ml of bacterial liquid, centrifuging at 10000 Xg for 20 min to collect the bacterial liquid, sterilizing, washing for 5 times with 0.01M PBS, finally resuspending with 20 ml of 0.01M PBS, ultrasonically resuspending until the bacterial liquid becomes clear, centrifuging at 10000 Xg for 2min to separate supernatant precipitate, packaging and storing the supernatant at-40 ℃, and performing BCA quantification.
3. Selection of optimal working concentration of antigen antibody
The optimal working concentration of the antigen and the antibody is determined by adopting a chessboard titration method, and the specific process is as follows:
the coated antigen is prepared in the step 2, the coating concentration of the 1 st column of the 96-well enzyme label plate is 10 mug/hole, each hole is 100 mug, 2 times of gradient dilution is sequentially carried out by using carbonate coating buffer solution with 0.05M PH9.6, the antigen is not coated in the last column (12 th column), the incubation is carried out for 1h in an incubator at 37 ℃, and the overnight at 4 ℃ is carried out. Washing the plate 3 times with 0.01M PBST, 5% skimmed milk powder 100 μ l per well, and sealing at 37 deg.C for 2 h; washing the plate 3 times with 0.01M PBST, sequentially performing 50, 100, 200, and 400-fold gradient dilution on positive serum, respectively adding into lines 1 to 4, and performing the same dilution on negative serumAdding 100 μ l of the mixture in each well on lines 5 to 8, and incubating at 37 ℃ for 1 h; washing the plate 4 times with 0.01M PBST, diluting rabbit anti-sheep secondary antibody with 5% skimmed milk powder 5000 times, incubating at 37 deg.C for 1h in each well of 100 μ l; washing the plate 4 times with 0.01M PBST, adding 100 μ l TMB per well for color development, and incubating at 37 deg.C for 10 min; 100 μ l of 2M H2SO4Terminating the reaction per well; placing a 96-hole enzyme label plate on an enzyme label instrument to read OD450 nmThe value, then the P/N value (OD) at the same dilution was calculatedPositive serum/ODNegative serum) And taking the antigen-antibody dilution with larger P/N value and positive serum hole OD value close to '1'.
The results show that the optimal antigen coating was 60 ng/well, conditions: the temperature is kept in an incubator at 37 ℃ for 1h, and then the temperature is kept overnight at 4 ℃; the optimal serum dilution was 1/200, conditions: incubate at 37 ℃ for 1 h.
4. Selection of optimal confining liquid
Under the condition of the selected optimal antigen-antibody working concentration, 5% skimmed milk powder, 2% BSA and Casein are respectively used as blocking solutions, 0.01M PBST is used as an unblocked control, a serum blank hole is simultaneously formed, namely only a secondary antibody is incubated, and the serum and the secondary antibody are diluted by the blocking solutions, and the step is the same as the step 3. The blocking conditions were selected to have higher P/N values and negative serum well OD values close to those of serum blank wells.
The results show that the optimal confining liquid is 5% skimmed milk powder, and the conditions are as follows: incubate at 37 ℃ for 2 h.
5. Cut-off value determination
According to the experimental steps, an iELISA system is initially established, 276 parts of known negative serum stored in a laboratory is detected by using an iELISA method, a 95% confidence interval is taken, and the mean value (M) +2SD of negative serum OD450 readings is taken as a critical value, namely ODCut-off=MNegative serum OD+1.96 × SD. According to SPSS analysis, the sample data conforms to a normal distribution according to the formula M +1.96 × SD: 0.191+1.96 × 0.085=0.357, and a Cut-off value of 0.36 was determined, and a negative was judged when the OD value was less than 0.36.
6. Evaluation of negative serum screening Capacity
The screening ability of the iELISA negative serum is evaluated by using 550 parts of serum in total, the screening ability is compared with the detection results of a commonly used commercial Mccp antibody detection cELISA kit (Mccp-cELISA) and a Mo antibody detection Mo-iELISA kit (Mo-iELISA), and the specificity and sensitivity of the iELISA method are calculated by taking the results as reference, so that whether the iELISA method can be used as screening negative serum or not is judged.
306 parts of serum in 550 parts of serum is collected from a Mccp vaccine experiment, and all the serum is detected by Mccp-cELISA; another 244 sera were taken from the Mo infection experiment and tested by Mo-iELISA. When the iELISA of the invention detects serum collected in an Mcp vaccine experiment, the sensitivity of the iELISA of the invention for detecting Mcp is 99.02 percent, the specificity is 76.47 percent, and the result is shown in Table 1 by taking the cELISA result as reference.
TABLE 1 detection of Mccp antibodies by Mccp-cELISA and the iELISA of the invention
Similarly, when serum collected in Mo vaccine experiments is detected, the sensitivity and specificity of Mo detection by the iELISA of the invention reach 98.71% and 65.17% respectively by taking the currently commonly used Mo-iELISA detection result as reference, and the results are shown in Table 2.
TABLE 2 detection of Mo antibodies by Mo-iELISA and the iELISA of the present invention
7. Mccp antibody level monitoring
Serum at different time points in the vaccine experiment period is taken to carry out Mcp antibody level detection, the serum at four time points of immunization 0 day, immunization 7 days, challenge 0 day (namely 1 month after immunization) and killing/death (namely 1 month after challenge) is selected in the experiment, 5 parts of serum are selected at each time point, 20 parts of serum are selected in total, the existing commercial cELISA kit with good specificity and sensitivity and the iELISA established by the invention are respectively used for detection, and whether antibody change trends reflected by two groups of results are consistent or not is observed.
The results are shown in FIG. 1, with cELISA results expressed as percent inhibition (SPI%) and more than 55% being positive, while iELISA results expressed as OD450 values and less than 0.36 being negative. The result shows that compared with the existing cELISA detection result, the iELISA can reflect the antibody level at different time points in the experimental period, the change trends of the iELISA and the cELISA are similar, and the antibody level is increased after one week of immunization, even the serum turns positive. This shows that the elisa can also present changes in Mccp antibody levels in a timely and accurate manner.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (4)
1. An iELISA method for screening sheep mycoplasma pneumoniae and goat mycoplasma goat pneumonia subspecies seronegative sheep is characterized by comprising the following steps:
(1) inoculating the mycoplasma capricolum goat pneumonia subspecies 1801 strain to an improved Taya broth culture medium for propagation, centrifuging the bacterial liquid, sterilizing, ultrasonically resuspending by PBS, and separating to obtain an antigen;
(2) coating an enzyme label plate with the antigen, wherein each hole is 100 mu l, and the enzyme label plate is incubated at 37 ℃ for 1h and then kept at 4 ℃ overnight; discarding the coating solution, washing with washing solution, adding 100 μ l of sealing solution into each well, and sealing at 37 deg.C for 2 hr; adding 100 μ l of 200-fold diluted serum sample, and incubating at 37 ℃ for 1 h; after washing the plate with PBST, adding 100 mul of rabbit anti-sheep secondary antibody diluted 5000 times with 5% skimmed milk powder into each hole, and incubating for 1h at 37 ℃;
(3) after incubation, washing the plate with PBST, adding 100 μ l of TMB into each well for color development, and incubating at 37 ℃ for 10 min; adding a stop solution to stop the reaction; placing the reacted enzyme label plate on an enzyme label instrument to read OD450 nmThe value is obtained.
2. The iiisa method for screening seronegative sheep for mycoplasma ovipneumoniae and mycoplasma capricolum subsp pneumoniae as claimed in claim 1, wherein said antigen is coated in an amount of 60 ng/well.
3. The iiisa method for screening seronegative sheep of mycoplasma ovipneumoniae and mycoplasma capricolum subsp pneumoniae as claimed in claim 1, wherein the blocking solution is 5% skimmed milk powder.
4. An application of the antigen separated from the strain 1801 of mycoplasma capricolum and the subspecies pneumonia of goat in screening the seronegative sheep with two pathogens of mycoplasma ovicolum and mycoplasma capricolum and the subspecies pneumonia of goat.
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