土壤样本中炭疽芽孢杆菌检测的免疫磁珠富集前处理研究

张益铜; 徐健皓; 袁兵; 王妍; 王景林; 袁媛

doi:10.3784/jbjc.202411130636

土壤样本中炭疽芽孢杆菌检测的免疫磁珠富集前处理研究

Pre-treatment method for immunomagnetic beads enrichment for detection of Bacillus anthracis in soil samples

摘要

摘要:
目的建立基于免疫磁珠分离（IMS）的土壤样本中炭疽芽孢杆菌的快速富集技术。
方法培养炭疽疫苗株芽孢并制备兔多克隆抗体与免疫磁珠（IMBs），通过菌落计数法检测不同种类IMBs对芽孢稀释液与模拟土壤样本中炭疽疫苗株芽孢的富集灵敏度；通过比较模拟土壤样品中炭疽疫苗株芽孢提取效率，确定芽孢抽提液最佳浓度与体积；采用实时荧光定量聚合酶链式反应（qPCR）评价IMS技术对模拟土壤样本与炭疽芽孢阳性土壤样本中炭疽芽孢的富集与检测效果。
结果使用含 0.5% Triton X-100 的质量体积百分比（W/V）为 200% 的蔗糖–磷酸盐缓冲液（PBS）作为芽孢抽提液，抽提液与土壤样本悬液体积比为 4∶1 时，采用差速离心法对芽孢的提取效率最高；Dynabeads™ 抗体偶联试剂盒制备的IMBs对稀释液与模拟土壤样本中炭疽疫苗株芽孢的富集灵敏度可达到 10² 菌落形成单位（CFU）/mL；qPCR法对炭疽疫苗株芽孢杆菌染色体特异基因BA_5345检测结果表明，模拟土壤样本经IMS法处理后，IMS处理组（K+）Ct值比两个磁分离对照组（K−、K0）提前 4 ～ 8 个循环；将该方法应用于炭疽芽孢阳性土壤样本的检测，结果显示IMS处理组其3个靶标基因 BA_5345、pagA 和capC 的检测 Ct 值比对应的磁分离对照组（K−、K0）提前约 5 ～ 8 个循环。
结论芽孢抽提液预处理结合 IMS技术能够快速有效富集纯化土壤样本中的炭疽芽孢。该方法简单有效，可为应对炭疽疫情的快速检测与溯源分析提供有效的技术支持。

Abstract:
Objective To establish a rapid enrichment technique for Bacillus anthracis in soil samples based on immunomagnetic beads separation techniques (IMS).
Methods B. anthracis vaccine strain spores were cultured and used to immunize rabbits for the preparation of rabbit polyclonal antibodies for immunomagnetic beads (IMBs). The sensitivity evaluation of different IMBs in enriching B. anthracis vaccine strain spores was evaluated using the colony counting method, with tests conducted on both spores dilutions and simulated soil samples. The optimal concentration and volume of the extraction liquid for spore recovery were identified by comparing the extraction efficiency of B. anthracis vaccine spores from simulated soil samples. Quantitative real-time polymerase chain reaction (qPCR) was used to evaluate the enrichment and detection effects of IMS on B. anthracis vaccine strain spores in simulated soil samples and B. anthracis virulent strain spores in positive soil samples.
Results The optimal extraction liquid was 0.5% Triton X-100 phosphate-buffered saline (PBS) solution with 200% (W/V) sucrose. The highest spore extraction efficiency was achieved via differential centrifugation when the volume ratio of extraction solution to the soil sample suspension was 4∶1. IMBs prepared using Dynabeads antibody coupling kit exhibited a sensitivity of 10² colony forming units(CFU)/mL for B. anthracis vaccine strain spores in both diluent and simulated soil samples. The detection of target gene BA_5345 with qPCR indicated that in simulated soil samples, IMS improved the qPCR cycle threshold (Ct) values by 4−8 cycles compared with control samples. This method was further applied to detect B. anthracis virulent strain in spores-positive soil samples, the results indicated that the Ct values for three target genes (BA_5345, pagA and capC) in the IMS-treated group were detected approximately 5− 8 cycles earlier than those in the control group (K−, K0).
Conclusions The combination of spore extraction pretreatment with IMBs enrichment can rapidly and effectively concentrates B. anthracis spores in soil samples while improving detection sensitivity. This simple and practical method provides effective technical support for the rapid detection and traceability analysis of anthrax epidemics.

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