Tapp H, Stotzky G
Department of Biology, New York University, New York 10003, USA.
Can J Microbiol. 1997 Nov;43(11):1074-8. doi: 10.1139/m97-153.
The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle-toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC-Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstak. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC-Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.
苏云金芽孢杆菌产生的杀虫毒素在土壤及其他自然栖息地中的积累和持久性可能会导致环境危害，如对非靶标物种的毒性以及对毒素抗性靶标物种的选择。我们描述了使用流式细胞术作为一种检测和追踪这些杀虫毒素在土壤中归宿的方法，该方法无需对毒素进行提取和纯化。苏云金芽孢杆菌亚种tenebrionis和kurstaki产生的毒素结合在从基察万土壤中分离出的黏土或粉砂大小的颗粒上，该土壤未改良（天然主要含有高岭石）或用黏土矿物蒙脱石或高岭石改良至6% v/v（作为内部对照）。将颗粒 - 毒素混合物悬浮于含有3%脱脂奶粉的0.1 M磷酸盐缓冲液（pH 7）中以阻断抗体的非特异性结合，再悬浮于针对苏云金芽孢杆菌亚种tenebrionis毒素的抗体溶液中，然后悬浮于与异硫氰酸荧光素偶联的抗兔抗体（FITC - Ab）溶液中。对照包括单独的颗粒以及颗粒与苏云金芽孢杆菌亚种kurstaki毒素的结合复合物。与在无毒素情况下对照FITC - Ab复合物与颗粒的非特异性荧光相比，所有结合了苏云金芽孢杆菌亚种tenebrionis毒素的颗粒在x轴上的荧光峰值均显著右移。对于一些结合了苏云金芽孢杆菌亚种tenebrionis毒素的颗粒，其峰值也有轻微右移，因为苏云金芽孢杆菌亚种tenebrionis和kurstaki产生的毒素之间以及它们诱导产生的抗体之间存在一些交叉反应。该方法比斑点印迹酶联免疫吸附测定更灵敏、快速，且易于完成多个样品的处理。
