van der Giessen J W, Eger A, Haagsma J, Haring R M, Gaastra W, van der Zeijst B A
Department of Bacteriology, School of Veterinary Medicine, University of Utrecht, The Netherlands.
J Med Microbiol. 1992 Apr;36(4):255-63. doi: 10.1099/00222615-36-4-255.
A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.
开发了一种基于16S核糖体RNA（rRNA）序列的探针，用于检测副结核分枝杆菌，它是牛约翰氏病的病原体。使用三种通用引物对各种分枝杆菌物种的16S rRNA基因的扩增片段进行测序。分析核苷酸序列时，在快速生长物种的序列中检测到一个缺失。合成了针对该区域的寡核苷酸探针（P），并在斑点试验中与各种分枝杆菌菌株的总RNA直接杂交。该探针检测到副结核分枝杆菌、鸟分枝杆菌-胞内分枝杆菌（MAI）复合体的其他一些生长缓慢的分枝杆菌以及一株非典型菌株戈登分枝杆菌。为了提高探针的灵敏度，扩增了副结核分枝杆菌16S rRNA基因在P和第二个寡核苷酸引物之间的413 bp片段，并与副结核分枝杆菌/鸟分枝杆菌特异性探针杂交。对牛的粪便样本进行检测时，所有培养阳性样本在PCR检测中均为阳性。
