布鲁氏菌多重PCR及间接ELISA检测方法的建立与应用
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摘要
布鲁氏菌病是由布鲁氏菌(Brucella)引起的人畜共患传染病,该病是世界范围内严重危害公共卫生安全的人畜共患传染病传染病之一。布鲁氏菌病对畜牧业生产和人类健康造成非常大的潜在威胁。本试验主要以布鲁氏菌的部分外膜蛋白为研究对象,建立起布鲁氏菌病原的多重PCR检测方法和ELISA抗体检测方法,并进行初步的应用,目的是期望能够应用于临床中快速检测和诊断布鲁氏菌病,为布鲁氏菌的检验检疫和净化提供技术支持。
1.根据GenBank上发表的布鲁氏菌omp31(FJ984569)、bp26(AY166766)、omp10(BRUOMP10A)基因序列,设计合成三对特异性引物,通过对反应条件的优化,建立了布鲁氏菌多重PCR快速检测方法。结果显示,该方法从布鲁氏菌中最低可检出1.1×10-3 ng的DNA,在牛奶感染布鲁氏菌模拟标本中最低可检出细菌数为80 cfu/ml。应用该方法对采自陕西省部分地区奶牛场的484份奶牛乳样进行了检测,检出阳性6份,阳性检出率约为1.24 %。检测结果表明,该方法具有特异、敏感、重复性好等优点,可用于奶牛布鲁氏菌病的早期临床诊断及流行病学监测等。
2.将用PCR扩增出的布鲁氏菌omp10、omp25和bp26基因片段分别克隆到原核表达载体pET-32a中,构建原核表达质粒。将其转入大肠杆菌BL21(DE3) PlysS中,用IPTG诱导表达,经HisTrap HP亲和层析柱分离纯化。基因测序及酶切鉴定证明原核表达载体构建成功;SDS-PAGE表明,omp10、omp25融合蛋白均以包涵体的形式在大肠杆菌中高效表达,bp26融合蛋白以可溶性的形式表达。经His亲和层析纯化,成功获得了大小分别为34 ku、44 ku和48 ku的融合蛋白,与预测的蛋白分子量一致。Westen blot证明纯化的omp10、omp25和bp26融合蛋白能被免疫的牛布鲁氏菌阳性血清所识别。
3.以纯化的布鲁氏菌omp10、omp25、bp26融合蛋白为抗原,分别包被酶标板,对反应条件进行优化,成功建立起布鲁氏菌ELISA抗体检测方法。应用该方法对采自陕西省部分地区的432份牛血清进行检测,并与试管凝集试验(SAT)和虎红平板凝集试验(RBPT)进行比较,结果显示,对于SAT和RBPT检测为阳性的样品,ELISA检测均为阳性,而对于ELISA检测为阳性样品,SAT和RBPT检测为部分阳性,说明ELISA方法比SAT和RBPT较敏感。试验结果显示,建立的ELISA检测方法具有特异、敏感,快速的特点,适合应用于临床布鲁氏菌病的快速检测。
Brucellosis is a zoonotic disease caused by Brucella. It is one of the zoonotic diseases which could seriously endanger the safety of public sanitation around the world. Brucellosis poses immensely potential threaten to the production of animal husbandry and the human health. The research primarily focuses on the parts of Brucella’s outer membrane proteins (omp), aiming at developing the multiplex PCR pathogen method and ELISA antibody method for the detection of brucellosis and carrying out their preliminary application. Our purpose is to hopefully utilize the methods in the rapid detection and diagnosis of brucellosis in clinic.
1. Three specific pairs of primers for the same pathogen were designed according to the omp31(FJ984569), bp26(AY166766) and omp10(BRUOMP10A) gene sequences published on the National Center for Biotechnology Information (NCBI) Genbank database. Through the optimization of the experiment conditions, a rapid multiplex PCR detection method was developed. The results showed that the lowest amount of DNA which could be detected was 1.1×10-3ng. The minimum number of bacterium detected from the modeling samples of milk infected with Brucella was 80 cfu/ml. 484 milk samples collected from several dairy farms in Shaanxi province were detected by the method. The result showed that there were 6 positive samples and the positive detective rate was 1.24 %. The detection result indicates that this method has the advantages of specificity, sensitivity and repeatability, so it can be used in the clinical detection of dairy cattle brucellosis and its epidemiological surveillance.
2. A polymerase chain reaction (PCR) method was used to amplify the omp10, omp25 and bp26 gene regions, which were ligated with pET-32a prokaryotic expression vector into recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) PlysS whose expression was induced by IPTG and then separated and purified through HisTrap HP affinity column. The results of gene sequencing and restriction enzyme digestion identification both proved that the pET-32a-omp10/omp25 vectors were successfully constructed. The SDS-PAGE electrophoresis showed that the omp10 and omp25 recombinant proteins were both highly expressed in the form of inclusion bodies in E. coli and the bp26 recombinant protein was expressed in the soluble way. After the purification of HisTrap HP affinity column, recombinant proteins in size of 34ku, 44ku and 48ku were successfully obtained, which correspond to the expected protein molecular weight. The detection of western-blot proved that purified omp10, omp25 and bp26 recombinant proteins could be identified by the Brucella positive serum from immunized cattle.
3. ELISA plates were coated respectively by the purified Brucella omp10, omp25 and bp26 recombinant proteins that act as antigens. The ELISA antibody method for the detection of Brucella was developed through the optimization of the reaction conditions. The results showed that the concentration of the coated omp10, omp25 and bp26 antigens was 10μg/mL, 5μg/mL and 5μg/mL and the best condition was under 4℃overnight. The dilution multiple of serum was all 1:100. The optimal dilution multiple of HRP secondary antibody was all 1:5 000. The positive and negative judgment of S/P critical value of omp10, omp25 and bp26 was 0.261, 0.192 and 0.191 respectively. Based on the above results, the indirect ELISA method, tube agglutination test and rose bengal plate agglutination test were compared to detect 432 bovine serum samples. The results were that those samples detected positive by SAT and RBPT also showed positive by ELISA, while those samples detected positive by ELISA showed partially positive by SAT and RBPT. Therefore, the ELISA method was more sensitive than SAT and RBPT. The experiment results prove that the established ELISA detection method own the characteristics of specificity, sensitivity and rapidity, which are suitable for the rapid diagnosis of brucellosis in clinic.
1.根据GenBank上发表的布鲁氏菌omp31(FJ984569)、bp26(AY166766)、omp10(BRUOMP10A)基因序列,设计合成三对特异性引物,通过对反应条件的优化,建立了布鲁氏菌多重PCR快速检测方法。结果显示,该方法从布鲁氏菌中最低可检出1.1×10-3 ng的DNA,在牛奶感染布鲁氏菌模拟标本中最低可检出细菌数为80 cfu/ml。应用该方法对采自陕西省部分地区奶牛场的484份奶牛乳样进行了检测,检出阳性6份,阳性检出率约为1.24 %。检测结果表明,该方法具有特异、敏感、重复性好等优点,可用于奶牛布鲁氏菌病的早期临床诊断及流行病学监测等。
2.将用PCR扩增出的布鲁氏菌omp10、omp25和bp26基因片段分别克隆到原核表达载体pET-32a中,构建原核表达质粒。将其转入大肠杆菌BL21(DE3) PlysS中,用IPTG诱导表达,经HisTrap HP亲和层析柱分离纯化。基因测序及酶切鉴定证明原核表达载体构建成功;SDS-PAGE表明,omp10、omp25融合蛋白均以包涵体的形式在大肠杆菌中高效表达,bp26融合蛋白以可溶性的形式表达。经His亲和层析纯化,成功获得了大小分别为34 ku、44 ku和48 ku的融合蛋白,与预测的蛋白分子量一致。Westen blot证明纯化的omp10、omp25和bp26融合蛋白能被免疫的牛布鲁氏菌阳性血清所识别。
3.以纯化的布鲁氏菌omp10、omp25、bp26融合蛋白为抗原,分别包被酶标板,对反应条件进行优化,成功建立起布鲁氏菌ELISA抗体检测方法。应用该方法对采自陕西省部分地区的432份牛血清进行检测,并与试管凝集试验(SAT)和虎红平板凝集试验(RBPT)进行比较,结果显示,对于SAT和RBPT检测为阳性的样品,ELISA检测均为阳性,而对于ELISA检测为阳性样品,SAT和RBPT检测为部分阳性,说明ELISA方法比SAT和RBPT较敏感。试验结果显示,建立的ELISA检测方法具有特异、敏感,快速的特点,适合应用于临床布鲁氏菌病的快速检测。
Brucellosis is a zoonotic disease caused by Brucella. It is one of the zoonotic diseases which could seriously endanger the safety of public sanitation around the world. Brucellosis poses immensely potential threaten to the production of animal husbandry and the human health. The research primarily focuses on the parts of Brucella’s outer membrane proteins (omp), aiming at developing the multiplex PCR pathogen method and ELISA antibody method for the detection of brucellosis and carrying out their preliminary application. Our purpose is to hopefully utilize the methods in the rapid detection and diagnosis of brucellosis in clinic.
1. Three specific pairs of primers for the same pathogen were designed according to the omp31(FJ984569), bp26(AY166766) and omp10(BRUOMP10A) gene sequences published on the National Center for Biotechnology Information (NCBI) Genbank database. Through the optimization of the experiment conditions, a rapid multiplex PCR detection method was developed. The results showed that the lowest amount of DNA which could be detected was 1.1×10-3ng. The minimum number of bacterium detected from the modeling samples of milk infected with Brucella was 80 cfu/ml. 484 milk samples collected from several dairy farms in Shaanxi province were detected by the method. The result showed that there were 6 positive samples and the positive detective rate was 1.24 %. The detection result indicates that this method has the advantages of specificity, sensitivity and repeatability, so it can be used in the clinical detection of dairy cattle brucellosis and its epidemiological surveillance.
2. A polymerase chain reaction (PCR) method was used to amplify the omp10, omp25 and bp26 gene regions, which were ligated with pET-32a prokaryotic expression vector into recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) PlysS whose expression was induced by IPTG and then separated and purified through HisTrap HP affinity column. The results of gene sequencing and restriction enzyme digestion identification both proved that the pET-32a-omp10/omp25 vectors were successfully constructed. The SDS-PAGE electrophoresis showed that the omp10 and omp25 recombinant proteins were both highly expressed in the form of inclusion bodies in E. coli and the bp26 recombinant protein was expressed in the soluble way. After the purification of HisTrap HP affinity column, recombinant proteins in size of 34ku, 44ku and 48ku were successfully obtained, which correspond to the expected protein molecular weight. The detection of western-blot proved that purified omp10, omp25 and bp26 recombinant proteins could be identified by the Brucella positive serum from immunized cattle.
3. ELISA plates were coated respectively by the purified Brucella omp10, omp25 and bp26 recombinant proteins that act as antigens. The ELISA antibody method for the detection of Brucella was developed through the optimization of the reaction conditions. The results showed that the concentration of the coated omp10, omp25 and bp26 antigens was 10μg/mL, 5μg/mL and 5μg/mL and the best condition was under 4℃overnight. The dilution multiple of serum was all 1:100. The optimal dilution multiple of HRP secondary antibody was all 1:5 000. The positive and negative judgment of S/P critical value of omp10, omp25 and bp26 was 0.261, 0.192 and 0.191 respectively. Based on the above results, the indirect ELISA method, tube agglutination test and rose bengal plate agglutination test were compared to detect 432 bovine serum samples. The results were that those samples detected positive by SAT and RBPT also showed positive by ELISA, while those samples detected positive by ELISA showed partially positive by SAT and RBPT. Therefore, the ELISA method was more sensitive than SAT and RBPT. The experiment results prove that the established ELISA detection method own the characteristics of specificity, sensitivity and rapidity, which are suitable for the rapid diagnosis of brucellosis in clinic.
引文
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