基于免疫磁珠快速富集分离土壤中炭疽芽孢方法的建立  

Development of an immunomagnetic beads-based method for rapid enrichment of Bacillus anthracis spores in soil

作　　者：梁冰[1] 纪雪[1] 孙洋[1] 周伟[1] 孙诗雯 刘军[1] 郭学军[1] 冯书章[1] 祝令伟[1] LIANG Bing;JI Xue;SUN Yang;ZHOU Wei;SUN Shi-wen;LIU Jun;GUO Xue-jun;FENG Shu-zhang;ZHU Ling-wei(Institute of Military Veterinary Science,The Academy of Military Medical Science of PLA,Key Laboratory of Jilin Province for Zoonosis Prevention and Control,Changchun 130122,Jilin Province,China)

机构地区：[1]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2018年第7期763-766,770,共5页Chinese Journal of Biologicals

基　　金：吉林省青年科研基金(20150520130JH);武汉市科技计划项目(2014060101010051)

摘　　要：目的建立基于免疫磁珠快速富集分离土壤中炭疽芽孢的方法。方法将炭疽芽孢杆菌疫苗株培养形成芽孢,灭活后免疫家兔,获得多克隆抗体,与磁珠偶联制备免疫磁珠,并检测其在纯炭疽芽孢稀释液中对炭疽芽孢富集分离的敏感性。通过对模拟土壤样品中炭疽芽孢抽提效果的比较,选择最优的芽孢抽提液及抽提条件。采用多重PCR法评价免疫磁珠对土壤中的炭疽芽孢在PLET平板培养基上富集分离的效果。结果 10 mg磁珠经活化后与500μL抗体偶联效果最佳,制备的免疫磁珠在纯炭疽芽孢稀释液中,富集分离的敏感性可达19 cfu/mL。以0.5%Triton X-100蔗糖PBS溶液作为芽孢抽提液,经1 000×g离心5 min后芽孢提取率最高。在模拟土壤样品中,检测敏感性可达100 cfu/g土壤,PLET平板培养基检测的阳性率达30%。结论本研究建立的免疫磁珠法简单实用,适用于土壤等各种类型环境样品中炭疽芽孢的检测。Objective To develop an immunomagnetic beads-based method for enrichment of of Bacillus anthracis spores in soil. Methods B. anthracis vaccine strain was cultured until formation of spores, inactivated and immunized to rabbits,and the obtained polyclonal antibody was coupled to magnetic beads to prepare immunomagnetic beads. The sensitivity of immunomagnetic beads in enrichment of spores in diluted B. anthracis spore solution was evaluated. The extracted liquid and extraction condition of spores were optimized by comparison of extraction effects in mimic soil samples. The enrichment efficacy of B. anthracis spores on PLET plate was evaluated by multiplex PCR. Results The condition for coupling reaction was optimized as that 10 mg of activated carboxylate-modified magnetic beads were coupled with 500 μL antibody at 37 ℃ for 2 h. The sensitivity of prepared immunomagnetic beads in enrichment of spores in diluted B. anthracis spore solution was 19 cfu/mL. The optimal extraction liquid was sucrose plus 0. 5% Triton X-100 PBS solution, while the optimal condition for extraction was centrifugation at 1 000 × g for 5 min. The sensitivity of the method for spores in mimic soil samples was 100 cfu/g soil. The positive rate of spores on PLET plate was 30%. Conclusion The developed immunomagnetic beads-based method was simple and practical, which was suitable for determination of B. anthracis spores in various environment samples including soil.

关 键 词：免疫磁珠 炭疽芽孢 多克隆抗体 土壤 

分 类 号：R378.82[医药卫生—病原生物学]