Guillou J P, Hénault S, Ostyn A, Thorel M F
Centre national d'études vétérinaires et alimentaires, Laboratoire central de recherches vétérinaires, Maisons-Alfort, France.
Rev Sci Tech. 1993 Jun;12(2):605-15.
A polymerase chain reaction was developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for Mycobacterium paratuberculosis (15-20 copies per genome). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA), from faeces stored at +4 degrees C, -20 degrees C, in 70% ethanol or in a buffer solution; (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridisation with an internal labelled oligonucleotide of digoxigenin. Reproducible results were obtained with DNA extracted from faeces stored at -20 degrees C or in 70% ethanol. The sensitivity and specificity of the method used, particularly double amplification and hybridisation, are discussed by comparing the results obtained by bacterial culture from faeces.
开发了一种聚合酶链反应，其使用1451个碱基对的插入元件（IS 900）作为靶序列，该元件对副结核分枝杆菌具有特异性（每个基因组有15 - 20个拷贝）。该测试分三个阶段进行：（1）从保存在+4℃、-20℃、70%乙醇或缓冲溶液中的粪便中提取细菌脱氧核糖核酸（DNA）；（2）通过耐热DNA聚合酶扩增靶DNA；（3）通过电泳检测扩增的DNA，并在与地高辛标记的内部寡核苷酸杂交后通过斑点印迹分析进行确认。从保存在-20℃或70%乙醇中的粪便中提取的DNA获得了可重复的结果。通过比较从粪便中进行细菌培养获得的结果，讨论了所使用方法的灵敏度和特异性，特别是双重扩增和杂交。
