| Brucellosis(Brucellosis for short)is a zoonotic infectious disease caused by Brucella,which has caused serious economic losses and hazards to animal husbandry and human health all over the world.At present,the disease can not be completely cured.People infected with Brucella will have different degrees of fever,sweating,arthralgia and other symptoms.The diagnosis of brucellosis plays a key role in the treatment and prognosis.Early diagnosis and early detection is a key step to cure brucellosis.Developing a rapid diagnostic kit suitable for grass-roots use in agricultural and pastoral areas is needed in areas with high incidence of brucellosis.Objective:(1)To extract and purify lipopolysaccharides(LPS)from Brucella S2 and S19 strains and to detect their antigenicity;(2)to determine the its sensitivity and specificity of Brucella LPS antigen colloidal gold antibody detection kit;(3)To screen patients with fever and community populations in different areas of Xinjiang with this kit;(4)To construct Brucella Omp2 a expression vector and to express and purify the recombinant protein in E.coli and to determine its diagnostic value;Methods:(1)LPS was extracted from Brucella S-19 and S-2 strain cultured in liquid medium by LPS purification kit through lysis,purification and washing.The extracted LPS was used for dot ELISA test to react with Brucellosis negative and positive sera,and the reaction color was observed by adding chromogenic liquid;(2)Positive sera(n=139)were collected from brucellosis epidemic areas in Xinjiang comfirmed by SAT and RBPT.The above positive sera were tested again with colloidal gold kit,and the sensitivity of colloidal gold kit was tested;(3)Brucellosis negative sera(n=354)from these patients with similar clinical symptoms were collected from Xinjiang brucellosis epidemic areas for determining,the specificity of colloidal gold kit.The colloidal gold rapid diagnostic kit was used to screen fever patients from 8 counties(cities)and community population in three counties cities of Xinjiang.Finally,the endemic risk was predicted by the difference of positive rates of the three epidemic areas and analysized by SPSS17.0 software;(4)Brucella outer membrane protein(Omp2a)gene was synthesized and inserted into a prokaryotic expression vector.The construction was transformed into E.coli BL21.The size of inserted fragment and open-reading frame was confirmed by PCR and sequencing.After induction and purification,Omp2 a was separated by SDS-PAGE gel electrophoresis.The antigen and diagnostic value of Omp2 a were detected by dot ELISA and Western Blotting;Results:(1)The dot ELISA test showed that LPS extracted from S-19 and S-2 strain could strongly react with brucellosis positive serum;(2)The sensitivity and specificity of colloidal gold kit for Brucella were 100% and 98.6% respectively;(3)Test of 358 serum samples from fever patients by the colloidal gold kit.The positive rate in three areas of Xinjiang brucellosis epidemic area was 19.3% in Yili,16.5% in Tacheng,19.3% in Bazhou,.There was no significantly difference among the three areas(P>0.05);(4)Population screening showed that of villagers were positive against brucellosis LPS in epidemic areas and None was positive in non-epidemic areas;(5)Omp2a was successfully expressed in E.coli BL21.The affinite purified Omp2 a could be weakly recognized by dot ELISA test;Conclusion:(1)LPS extracted from S-19 and S-2 strain can react with brucellosis positive sera;(2)Brucella colloidal gold kit formatted with brucellosis LPS showed high sensitivity and specificity;(3)From the statistical analysis,we can see that the positive rate of farming and pastoral areas in Tacheng,Yili and Bazhou area are at high risk of brucellosis in Xinjiang;(4)The second outer membrane protein Omp2 a of Brucella has weak immunogenicity in term of recognition of brucellosis serum;... |
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