Purchase H G
College of Veterinary Medicine, Mississippi State University, Mississippi 39762.
Avian Dis. 1989 Oct-Dec;33(4):609-14.
A workshop in which 17 practicing scientists participated was intended to address primarily people who use or could use biotechnology in their work and was confined to five techniques. Endonuclease fingerprinting and mapping involved cleaving nucleic acid with a specific restriction enzyme and separating the nucleic acid fragments by electrophoresis. Field and vaccine isolates of Pasteurella multocida could be distinguished; Salmonella enteritidis could be divided into three groups; chlamydia could be grouped into seven groups; and vaccinia, quail pox, and fowl pox could be clearly distinguished. Preparation of nucleic acid probes involved producing large amounts of labeled oligonucleotides, usually of unknown sequence. Successful probes had been made for infectious bursal disease virus, avian influenza virus, Newcastle disease virus, and infectious bronchitis virus. In Southern, Northern, and dot blotting, either DNA or RNA fragments were placed on or transferred to a solid substrate and probed. The procedure was able to detect infectious bursal disease virus, infectious bronchitis virus, Mycoplasma gallisepticum, and Marek's disease virus. In situ hybridization involved applying a labeled probe to frozen or fixed sections or to intact cells. In Polymerase chain reaction, two primers, some distance apart, were annealed to a denatured target DNA. Repeated cycles of DNA synthesis with a thermostable polymerase, denaturing, and reannealing resulted in great amplification of a rare sequence. After 30 cycles, a rare gene sequence could be amplified more than 10(6) times. It was used successfully to detect minute quantities of influenza virus and infectious bursal disease virus, and the process was used to facilitate DNA sequencing of coccidiosis gene segments.
一个有17位在职科学家参加的研讨会，主要面向那些在工作中使用或可能使用生物技术的人员，且仅限于五种技术。核酸内切酶指纹图谱和图谱分析涉及用特定的限制性酶切割核酸，并通过电泳分离核酸片段。多杀性巴氏杆菌的田间分离株和疫苗株能够被区分；肠炎沙门氏菌可分为三组；衣原体可分为七组；牛痘、鹌鹑痘和鸡痘能够被清楚地区分。核酸探针的制备涉及大量生产通常序列未知的标记寡核苷酸。已经成功制备了针对传染性法氏囊病病毒、禽流感病毒、新城疫病毒和传染性支气管炎病毒的探针。在Southern印迹、Northern印迹和斑点印迹中，DNA或RNA片段被放置或转移到固体基质上并进行探测。该程序能够检测传染性法氏囊病病毒、传染性支气管炎病毒、鸡毒支原体和马立克氏病病毒。原位杂交涉及将标记探针应用于冷冻或固定切片或完整细胞。在聚合酶链反应中，两个相距一定距离的引物与变性的靶DNA退火。用热稳定聚合酶进行DNA合成、变性和重新退火的重复循环导致稀有序列的大量扩增。经过30个循环后，一个稀有基因序列可以被扩增超过10⁶倍。它成功地用于检测微量的流感病毒和传染性法氏囊病病毒，并且该过程用于促进球虫病基因片段的DNA测序。
