Freeze-dried hemagglutination inhibition test antigen for detecting mycoplasma synoviae, antigen combination and preparation method
A technique for mycoplasma synovial bursae and hemagglutination inhibition test, applied in the field of freeze-dried hemagglutination inhibition test antigen and antigen combination for detection of mycoplasma synovial bursa Less difficult to operate, stable antigen properties, and high sensitivity
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[0055] Example 1: Isolation and screening of MS JS2018-4 strain
[0056] 1. Experimental materials
[0057] Mycoplasma broth basal medium (product code: CM0403B) and Mycoplasma agar basal medium (product code: CM0401B) were purchased from OXOID; arginine, cysteine, and coenzyme Ⅰ were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; pig serum was purchased from Jinyuankang Biological Engineering Co., Ltd.
[0058] Improved Frey's liquid medium preparation method: Mycoplasma broth basal medium (OXOID, product number: CM0403B) 25.5g / L, glucose 3.3g / L, coenzyme Ⅰ 100mg / L, arginine 400mg / L, cysteine 100mg / L, phenol red indicator 0.02%, add each component to ultrapure water, and adjust the pH to 7.8. Use 0.22μm bacteria filter to filter and sterilize, add 15%-20% pig serum.
[0059] Improved Frey's solid medium preparation method: Mycoplasma broth basal medium (OXOID, product number: CM0403B) 35.5g / L, autoclaved at 121°C for 15min, cooled to 50°C, add glucose 3.3g / L, coen...
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[0067] Example 2: Preparation and application of freeze-dried MS hemagglutination inhibition test antigen
[0068] 1 Experimental materials
[0069] 1.1 strain
[0070] MS JS2018-4 strain was isolated, identified and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University, and was preserved in the China Center for Type Culture Collection (Wuhan University, Wuhan, China) with the deposit number: CCTCC M20191100.
[0071] 2 Experimental methods
[0072] 2.1 Strain culture
[0073] 2.1.1 Obtainment of seed bacterial liquid
[0074] Take the strain of MS JS2018-4 isolate, inoculate it into fresh modified Frey's liquid medium, place it in an incubator at 37°C, culture at 220r / min for 24h, until the color of the medium changes from purple-red to orange-yellow. Then suck the bacteria liquid to inoculate fresh modified Frey's liquid medium at a ratio of 1:10, culture for 24 hours, and perform 3 consecutive subculture...
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[0121] Example 3: Preparation and application of freeze-dried MG hemagglutination inhibition test antigen
[0122] 3.1 Mass preparation of MG hemagglutination inhibition test antigen
[0123] A batch of MG hemagglutination inhibition test antigen was prepared in large quantities according to the preparation conditions of the freeze-dried MS hemagglutination inhibition test antigen determined in 2.1 to 2.4 in Example 2.
[0124] 3.2 MG hemagglutination inhibition test antigen test
[0125] 3.2.1 HA titer determination
[0126] Use 2ml PBS solution to dissolve the hemagglutination inhibition test antigen and determine the HA titer. The results showed that the HA titer of 10 randomly selected vials of freeze-dried antigen were all 2. 8 .
[0127] 3.2.2 Specificity test
[0128] The HI test method was used to detect MG, MS, NDV, AIV, IBV positive sera and SPF chicken sera using the prepared MG hemagglutination inhibition test antigen. The results are shown in Table 8. The antigen does not c...
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