Indirect ELISA method for detecting duck tembusu virus antibody
A duck Tembusu virus and antibody technology, applied in the field of animal infectious disease detection, can solve the problems of difficult to reach production level, long-term production performance, etc., and achieve the effects of long duration, high IFA compliance rate and strong sensitivity
Inactive Publication Date: 2019-04-05
ANHUI AGRICULTURAL UNIVERSITY
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It takes a long time for ducks that have survived infection to recover, and it is difficult to reach the production level before infection
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[0031] Below in conjunction with accompanying drawing, the present invention is described in further detail:
[0032] The specific implementation plan is as follows:
[0033] 1.1 Design of NS1 gene primers
[0034] Referring to the full gene sequence of DTMUV AH-F10 strain (GenBank accession number: KM102539), a pair of specific primers for amplifying the DTMUVNS1 gene was designed using Oligo7. The primer sequence is:
[0035] NS1-up: 5'-CGGGATCCATGGACACGGGGTGCTCAATC-3';
[0036] NS1-dw: 5'-CCCAAGCTTTCAAGCCATGACCTTTGATTTG-3';
[0037] The size of the target gene is 1056bp, and BamH I and Hind III restriction sites are added to both ends of the upstream and downstream primers respectively.
[0038] 1.2 Extraction of viral RNA
[0039] The extraction of viral RNA was carried out according to routine laboratory methods, and the steps were as follows:
[0040] 1. Take a small amount of frozen-thawed duck Tembusu virus liquid, centrifuge at 8000r / min for 3min, and take the su...
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The invention discloses an indirect ELISA method for detecting a duck tembusu virus antibody. The method comprises the steps of designing a pair of primers by using the complete genome sequence of a duck tembusu virus to amplify an NS1 gene; building a protein expression carrier; acquiring a recombinant expressed protein inclusion body via inducible expression of IPTG (isopropyl thioglycoside); and building the indirect ELISA method for detecting the duck tembusu virus antibody in the serum of duck blood by using the inclusion body that is subjected to purification, renaturation and concentration as an envelope antigen. According to the method provided by the invention, the antibody is the NS1 protein in non-structural protein, in detection results, the false positives are fewer and the IFA coincidence rate is high, the detection results are good in repeatability, strong in sensitivity and fast in detection time, and the result can be basically detected within 5 hours. An animal test result shows that the after the antibody infects with the duck tembusu virus, the positive time is early, and the duration is long.
Description
technical field [0001] The invention belongs to the technical field of animal infectious disease detection, and in particular relates to an indirect ELISA method for detecting duck Tembusu virus antibody. Background technique [0002] Duck Tembusu virus disease is a new infectious disease caused by DTMUV. The disease mainly occurs in ducks, which can cause a decrease in laying duck laying rate, growth retardation and death of ducklings. The incidence rate in duck flocks can reach 80%, and the egg production rate of laying ducks can be reduced from 90% to 10%. It takes a long time for the ducks that have survived the infection to recover their production performance, and it is difficult to reach the production level before the infection. After the first case of Duck Tembusu virus disease appeared in my country in 2010, there were reports of ducks infected with DTMUV in most of the domestic duck breeding areas. The healthy development of the duck industry has also brought a s...
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IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2469/20
Inventor 王桂军周祺徐前明刘红梅宋祥军苏观志许泽军顾香雪黄荣
Owner ANHUI AGRICULTURAL UNIVERSITY