基于免疫磁珠快速富集分离土壤中炭疽芽孢方法的建立
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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.
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.
引文
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[8]LIANG B,ZHANG Y J,JI X,et al.Preparation of immunomagnetic beads of enterohemorrhagic E.coli O157[J].Chin J Biologicals,2017,30(3):278-282.(in Chinese)梁冰,张雅洁,纪雪,等.肠出血性大肠杆菌O157免疫磁珠的制备[J].中国生物制品学杂志,2017,30(3):278-282.
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[10]DOU X F,WU K,LIU Y,et al.The control and management of the imported case of cutaneous anthrax in Beijing[J].Capital J Pub Health,2016,10(5):208-211.(in Chinese)窦相峰,吴钶,刘园,等.北京市输入皮肤炭疽疫情处置及相关问题探讨[J].首都公共卫生学,2016,10(5):208-211.
[11]ZHANG M J,LI J Y,ZUO X W,et al.An outbreak investigation on sheep anthrax in Tongxin county of Ningxia[J].Chin Anim Health Insp,2017,34(2):5-8.(in Chinese)张淼洁,李靖宁,左兴文,等.宁夏同心县一起羊炭疽疫情的暴发调查[J].中国动物检疫,2017,34(2):5-8.
[12]World Organization for Animal Health(OIE).Manual of diagnostic tests and vaccines for terrestrial animals(chapter 2.1.1 Anthrax)[S].7th ed.2012.
[13]FENG S Z,ZHU L W,LIU J,et al.Agriculture industry standard-Diagnostic techniques for animal anthrax(NY/T 561-2015)[S].Beijing:China Agriculture Press,2015.(in Chinese)冯书章,祝令伟,刘军,等.中华人民共和国农业行业标准-动物炭疽诊断技术(NY/T 561-2015)[S].北京:中国农业出版社,2015.
[2]BEYER W,TURNBULL P C.Anthrax in animals[J].Mol Aspects Med,2009,30(6):481-489.
[3]PILO P,FREY J.Bacillus anthracis:molecular taxonomy,population genetics,phylogeny and patho-evolution[J].Infect Genet Evol,2011,11(6):1218-1224.
[4]KNISELY R F.Selective medium for Bacillus anthracis[J].J Bacteriol,1966,92(3):784-786.
[5]LUNA V A,GULLEDGE J,CANNOUS A C,et al.Improvement of a selective media for the isolation of B.anthracis from soils[J].J Microbiol Methods,2009,79(3):301-306.
[6]DRAGON D C,RENNIE R P.Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis spores[J].Lett Appl Microbiol,2001,33(2):100-105.
[7]FASAMELLA A,DI TARANTO P,GAROFOLO G,et al.Ground anthrax bacillus refined isolation(GABRI)method for analyzing environmental samples with low levels of Bacillus anthracis contamination[J].BMC Microbiol,2013,13:167.
[8]LIANG B,ZHANG Y J,JI X,et al.Preparation of immunomagnetic beads of enterohemorrhagic E.coli O157[J].Chin J Biologicals,2017,30(3):278-282.(in Chinese)梁冰,张雅洁,纪雪,等.肠出血性大肠杆菌O157免疫磁珠的制备[J].中国生物制品学杂志,2017,30(3):278-282.
[9]LIANG B,ZHU L W,JI X,et al.Identification of Bacillus anthracis strain by PCR method[J].Chin J Vet Sci,2016,36(8):1363-1366,1370.(in Chinese)梁冰,祝令伟,纪雪,等.炭疽芽孢杆菌PCR鉴定方法的建立[J].中国兽医学报,2016,36(8):1363-1366,1370.
[10]DOU X F,WU K,LIU Y,et al.The control and management of the imported case of cutaneous anthrax in Beijing[J].Capital J Pub Health,2016,10(5):208-211.(in Chinese)窦相峰,吴钶,刘园,等.北京市输入皮肤炭疽疫情处置及相关问题探讨[J].首都公共卫生学,2016,10(5):208-211.
[11]ZHANG M J,LI J Y,ZUO X W,et al.An outbreak investigation on sheep anthrax in Tongxin county of Ningxia[J].Chin Anim Health Insp,2017,34(2):5-8.(in Chinese)张淼洁,李靖宁,左兴文,等.宁夏同心县一起羊炭疽疫情的暴发调查[J].中国动物检疫,2017,34(2):5-8.
[12]World Organization for Animal Health(OIE).Manual of diagnostic tests and vaccines for terrestrial animals(chapter 2.1.1 Anthrax)[S].7th ed.2012.
[13]FENG S Z,ZHU L W,LIU J,et al.Agriculture industry standard-Diagnostic techniques for animal anthrax(NY/T 561-2015)[S].Beijing:China Agriculture Press,2015.(in Chinese)冯书章,祝令伟,刘军,等.中华人民共和国农业行业标准-动物炭疽诊断技术(NY/T 561-2015)[S].北京:中国农业出版社,2015.