Carozzi N B, Kramer V C, Warren G W, Evola S, Koziel M G
CIBA-Geigy Biotechnology Research Unit, Research Triangle Park, North Carolina 27709.
Appl Environ Microbiol. 1991 Nov;57(11):3057-61. doi: 10.1128/aem.57.11.3057-3061.1991.
A rapid analysis of Bacillus thuringiensis strains predictive of insecticidal activity was established by using polymerase chain reaction (PCR) technology. Primers specific to regions of high homology within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each insecticidal class. Predictions of insecticidal activity were made on the basis of the electrophoretic patterns of the PCR products. Included in the screen were PCR primers specific for cryI, cryIII, and cryIV genes, which are insecticidal for lepidopterans, coleopterans, and dipterans, respectively. Known B. thuringiensis strains as well as unidentified strains isolated from soil and insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a single PCR reaction containing 12 to 20 primers capable of distinguishing between the different insecticidal genes. Insecticidal activity predicted by the PCR screen was found to correspond with the insecticidal activity of insect bioassays. In addition to identifying strains with known insecticidal genes, the PCR screen can identify strains with altered electrophoretic patterns containing potentially novel genes.
通过使用聚合酶链反应（PCR）技术，建立了一种对苏云金芽孢杆菌菌株进行快速分析以预测其杀虫活性的方法。针对编码苏云金芽孢杆菌三类主要晶体蛋白的基因内高度同源区域的特异性引物，用于生成每个杀虫类别的特征性PCR产物图谱。基于PCR产物的电泳图谱对杀虫活性进行预测。筛选中包括针对cryI、cryIII和cryIV基因的特异性PCR引物，它们分别对鳞翅目、鞘翅目和双翅目昆虫具有杀虫活性。通过PCR对已知的苏云金芽孢杆菌菌株以及从土壤和昆虫尸体中分离出的未鉴定菌株进行了分析。在一个包含12至20种能够区分不同杀虫基因的引物的单一PCR反应中，对少量粗样品裂解物进行了检测。发现通过PCR筛选预测的杀虫活性与昆虫生物测定的杀虫活性相符。除了鉴定具有已知杀虫基因的菌株外，PCR筛选还可以鉴定出具有改变的电泳图谱且含有潜在新基因的菌株。
