Chen K, Neimark H, Rumore P, Steinman C R
Department of Medicine, State University of New York, Health Science Center at Brooklyn 11203.
FEMS Microbiol Lett. 1989 Jan 1;48(1):19-24. doi: 10.1016/0378-1097(89)90139-0.
In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.
在本报告中，我们描述并鉴定了两种寡聚体探针，它们与人类18 rRNA或人类线粒体12S rRNA中不存在的保守真细菌16S核糖体RNA（rRNA）序列具有广泛的同源性。通过斑点印迹杂交，其中一种或两种探针能够检测所有23种系统发育上不同的真细菌核酸。检测灵敏度约为每10个真核细胞中有1个细菌。通过在聚合酶链反应（PCR）中使用这些寡聚体序列或其互补序列作为引物，在存在大量过量真核DNA的情况下，检测到了相当于1 pg的大肠杆菌DNA。通过对扩增DNA进行直接序列分析并与已知序列或目录进行比较，可以获得有助于对检测到的生物体进行部分系统发育分类的信息。与更具特异性的探针相比，这种广泛同源的探针在检测身份未知的生物体方面具有优势。因此，只要探针不与宿主核酸杂交，它们就可用于识别无法培养的生物体的感染，例如在组织培养或病因不明的植物或动物疾病中可能出现的情况。
