Ludwig W, Dorn S, Springer N, Kirchhof G, Schleifer K H
Lehrstuhl für Mikrobiologie, Technische Universität München, Federal Republic of Germany.
Appl Environ Microbiol. 1994 Sep;60(9):3236-44. doi: 10.1128/aem.60.9.3236-3244.1994.
DNA coding for a variable region within domain III of bacterial 23S rRNA was used as the target for group-specific polynucleotide hybridization probes. The corresponding rDNA was amplified in vitro by the PCR technique in combination with a pair of primers specific for flanking conserved target sites. The amplified fragments were cloned or used directly as probes. RNA probes were generated by in vitro transcription of cloned or amplified rDNA. The probes were labeled by incorporating modified nucleotides during in vitro DNA amplification or in vitro transcription or by random priming. The use of in vitro transcribed single-stranded RNA probes instead of double-stranded DNA probes provided stronger hybridization signals. Group-specific probes were prepared from genomic DNAs or directly from cells of Acinetobacter calcoaceticus, Alcaligenes faecalis, Aeromonas hydrophila, Nannocystis exedens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri.
编码细菌23S rRNA结构域III内可变区的DNA被用作组特异性多核苷酸杂交探针的靶标。通过PCR技术结合一对针对侧翼保守靶位点的引物，在体外扩增相应的rDNA。扩增片段被克隆或直接用作探针。RNA探针通过对克隆或扩增的rDNA进行体外转录产生。探针通过在体外DNA扩增或体外转录过程中掺入修饰核苷酸或通过随机引物延伸进行标记。使用体外转录的单链RNA探针而非双链DNA探针可提供更强的杂交信号。组特异性探针由乙酸钙不动杆菌、粪产碱菌、嗜水气单胞菌、伸展纳米囊菌、铜绿假单胞菌、荧光假单胞菌和施氏假单胞菌的基因组DNA或直接从这些细菌的细胞制备。
