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Brucellosis (Brucellosis, referred to as the cloth disease) is caused by Brucella beast of a man suffering from infectious diseases. Hazards to human and animal health and causing huge losses to the development of animal husbandry. The disease is widely popular in the world, China is also a high incidence of brucellosis country, Xinjiang, Qinghai, Tibet, Inner Mongolia and other areas is particularly acute, the effective quarantine is an important means for the prevention and control of brucellosis, traditional detection methods easily cross-react, complicated operation, sensitivity and low research sensitive, specific and rapid detection methods suitable for clinical applications has caused a high degree of attention at home and abroad, the experiment to establish the Brucella real-time quantitative PCR and serum rapid diagnostic the dipstick detection methods, will be to provide technical support for grassroots Brucellosis quarantine and prevention and control. The experimental work done and the main results are as follows: 1. Brucella the BP26 protein expression, purification. Bp26 cloned into pGM-T vector after digestion, PCR and sequencing, to construct the recombinant plasmid pET-28a-BP26 bacteria of expression, and induction of the expression, purification, Western-blot analysis of its immunogenicity. The results show that properly constructed the pGM-T-BP26, successfully expressed BP26 fusion protein by SDS-PAGE analysis showed that to obtain a high level of expression, a protein of high purity, and Brucella positive serum specificity and BP26 protein combination, show that the immunogenicity, laid the foundation for subsequent immunological testing. Build serum Brucella BP26 protein-based rapid detection of colloidal gold immunochromatographic strip. Immunochromatographic strip for the preparation of colloidal gold standard SPA and Brucella BP26 protein were detected, and their specificity, sensitivity, accuracy and stability. Results strip for detection of Brucella positive serum into two lines, detecting other bacteria antiserum only the control line chromogenic ,5-10min can be observed to the results, with good specificity; With this method, amber-red plates, tubes the agglutination were detected 45 Brucella M5 immune serum, the positive rate of 95.6% (43/45), 86.7% (39/45) and 93.3% (42/45), test strip and amber red Tablet found was 90.6%, and the tube agglutination found was 97.9%, the test strip can be detected in a 1:100 dilution of the sheep positive serum; 1:128-fold dilution of the positive serum of mice, indicating that has better sensitivity; present test strip can be stored for 6 months at 4 ℃, showed that this method has a high stability. 3 double real-time fluorescence quantitative PCR method to establish the differential diagnosis of Brucella and Mycobacterium tuberculosis. Best primer combinations to establish screening Brucella omp10, omp19 omp17 omp25 omp31 and Mycobacterium tuberculosis cfp10 gene double real-time quantitative PCR differential diagnosis method, the method of stability, specificity and sensitivity of the test, and detection of clinical samples and analog samples. The omp25 gene by screening Brucella with the Mycobacterium tuberculosis cfp10 gene primer combinations, the weight of real-time fluorescent quantitative PCR method Brucella Tm value of 88 ° C to -89 ° C, a Mycobacterium tuberculosis Tm value of 90 ° C -92 ℃, other strains tested was negative; the Brucella minimum detectable amount of DNA 20 copies / ul, Mycobacterium tuberculosis 50 copies / ul, two pathogens are present 100copies/ul than Conventional PCR is 100 times higher. Use of the double fluorescent established quantitative PCR and conventional PCR, 11 Brucella genomic DNA and 30 analog milk samples to detect TB sputum samples of 34, was 100%. The results prove that this experiment to establish the method can be used for the simultaneous detection of Brucella and Mycobacterium tuberculosis, and has good specificity, sensitivity and accuracy.
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