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Method for detecting mycoplasma gallisepticum antibody of enzyme-linked nucleic acid aptamer and kit special for mycoplasma gallisepticum antibody

A nucleic acid aptamer and mycoplasma gallisepticum technology, which can be applied to measuring devices, DNA/RNA fragments, instruments, etc., can solve the problems of high detection cost and narrow promotion range

Active Publication Date: 2019-05-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, my country usually uses detection kits from companies such as IDEXX in the United States to carry out MG serological monitoring, which has high detection costs and narrow promotion scope

Method used

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  • Method for detecting mycoplasma gallisepticum antibody of enzyme-linked nucleic acid aptamer and kit special for mycoplasma gallisepticum antibody
  • Method for detecting mycoplasma gallisepticum antibody of enzyme-linked nucleic acid aptamer and kit special for mycoplasma gallisepticum antibody
  • Method for detecting mycoplasma gallisepticum antibody of enzyme-linked nucleic acid aptamer and kit special for mycoplasma gallisepticum antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Embodiment 1, the preparation of recombinant PVPA protein

[0117] PVPA protein is a species-specific protein that exists on the surface of MG cell membrane and can be recognized by the chicken's immune system. The amino acid sequence of the PVPA protein is shown in sequence 2 of the sequence listing, and its corresponding gene is shown in sequence 1 of the sequence listing.

[0118] 1. Replace the fragment between the BamHI-HF and XhoI restriction sites of the pET-28a(+) vector with the DNA molecule shown in Sequence 1 of the Sequence Listing to obtain a recombinant expression vector (sequencing verification).

[0119] 2. Introduce the recombinant expression vector obtained in step 1 into BL21(DE3) competent cells to obtain recombinant bacteria.

[0120] 3. Inoculate the recombinant bacteria obtained in step 2 in LB liquid medium, shake and culture at 220r / min, 37°C for 8-12h.

[0121] 4. After completing step 3, transfer the bacterial liquid to 50ml LB liquid medium...

Embodiment 2

[0132] Embodiment 2, the screening of the nucleic acid aptamer that can specifically bind PVPA protein

[0133] Through library construction, microwell plate screening, and high-throughput sequencing, several candidate nucleic acid aptamers that bind to PVPA protein were screened out, biotin-labeled at the 5' end, and the binding force of each aptamer was detected by indirect ELAA. Specific steps are as follows:

[0134] 1. Coating: Dilute the recombinant PVPA protein prepared in Example 1 with PBS (pH 7.2-7.4) to a concentration of 1 μg / ml, 100 μl per well, and coat overnight at 4°C.

[0135] 2. Washing: Shake off the liquid in the wells, add 300 μl PBST to each well, and wash three times.

[0136] 3. Blocking: 200 μl of 5% BSA was added to each well, and blocked at 37° C. for 2 hours.

[0137] 4. Washing: Shake off the liquid in the wells, add 300 μl PBST to each well, and wash three times.

[0138] 5. Add the pretreated aptamer solution (the 5' end of the aptamer is labe...

Embodiment 3

[0148] Example 3. Establishment of a detection method for Mycoplasma gallisepticum antibodies based on nucleic acid aptamers

[0149] The nucleic acid aptamer Apt-236 used in this example was biotinylated at the 5' end.

[0150] 1. Preparation of MG standard negative and positive serum

[0151] 1. Feed 4-week-old SPF white legion chickens, collect blood to separate serum, and test with IDEXX Mycoplasma avian septica antibody detection kit to confirm that there is no MG infection.

[0152] 2. Take the chickens that have been identified as having no MG infection in step 1, collect blood and separate the serum, and obtain the standard negative control serum.

[0153] 3, get step 1 through identification and do not have the chicken that MG infects, adopt MG strain Mycoplasma gallisepticum BG44T strain (CVCC350) immunization, concrete steps are as follows:

[0154] (1) Set the concentration to 1x10 8 The CCU / ml MG strain Mycoplasma gallisepticum BG44T strain was concentrated 20 ...

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Abstract

The invention discloses a method for detecting a mycoplasma gallisepticum antibody of an enzyme-linked nucleic acid aptamer and a kit special for the mycoplasma gallisepticum antibody. The nucleic acid aptamer is shown as sequence 3 of a sequence list. The method for detecting the mycoplasma gallisepticum antibody of the enzyme-linked nucleic acid aptamer applies the nucleic acid aptamer. The method for detecting the mycoplasma gallisepticum antibody of the enzyme-linked nucleic acid aptamer has good sensitivity and specificity and is suitable for rapid detection of a large number of samples in the field. The method can effectively detect the antibody infected by MG, the reliable detection method is provided for identifying MG infection, and scientific guidance is also provided for prevention and control of MG epidemic situations.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enzyme-linked nucleic acid aptamer-based detection method for Mycoplasma gallisepticum antibody and a special kit thereof. Background technique [0002] Mycoplasma gallisepticum (MG) is the pathogen of chronic respiratory disease (CRD) in chickens. It can cause damage to the respiratory tract, air sacs and infraorbital sinuses of chickens. The egg production rate of laying hens decreases, the quality of broilers decreases, and the abandonment rate increases. It is a serious hazard to the poultry industry. one of the infectious diseases. MG can be transmitted vertically through eggs, resulting in decreased hatching rate and fertilization rate, increased chick mortality and growth and development disorders, and making the offspring of breeders become MG-positive flocks. This is an important reason why the disease is very common. It is very difficult to control and purify the pathog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/569G01N33/543
Inventor 吴文学王丰
Owner CHINA AGRI UNIV
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