Cloning and characterization of ribosomal RNA genes from three species of Haemonchus (Nematoda: Trichostrongyloidea) and identification of PCR primers for rapid differentiation.
Cloning and characterization of ribosomal RNA genes from three species of Haemonchus (Nematoda: Trichostrongyloidea) and identification of PCR primers for rapid differentiation.
复制标题
三种血矛线虫属（线虫纲：毛圆线虫纲）核糖体 RNA 基因的克隆和表征以及快速分化 PCR 引物的鉴定。
DOI：
10.1006/expr.1994.1003
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发表时间：
1994
影响因子：
2.1
通讯作者：
J. Lichtenfels
中科院分区：
文献类型：
--
作者：
D. Zarlenga;F. Stringfellow;M. Nobary;J. Lichtenfels
利用螺旋血吸虫、胎盘血吸虫和相似血吸虫的成虫基因组DNA，克隆和定位了每个物种的核糖体RNA基因重复序列。重复单元的长度估计分别为7.2、7.2和7.4kb，其中第二个和独特的重复单元长度为6.9kb，仅从反转毛虫中鉴定。所有核糖体RNA基因中的限制性内切酶识别位点都是完全保守的，除了6.9-kb扭转螺旋体重复序列中大亚基rRNA基因中的SalI位点丢失外，在外部间隔区DNA中的限制性内切酶识别序列只有很小的差异。亚克隆的每个物种的小亚基核糖体DNA的序列分析表明，在1758bp的片段内100%保守，在相邻的5‘外转录间隔区只观察到有限的序列变异。利用rDNA大亚基3‘端和小亚基5’端的3‘端和5’端互补的引物对外部间隔区序列进行酶促扩增，可以利用该重复区域内的大小变异性快速区分螺旋体和胎盘毛滴虫。
Genomic DNAs prepared from adult worms of Haemonchus contortus, Haemonchus placei, and Haemonchus similis were used to clone and map complete ribosomal RNA gene repeats from each species. The lengths of the repeating units were estimated at 7.2, 7.2, and 7.4 kb, respectively, with a second and distinct repeat 6.9 kb in length identified from H. contortus only. Restriction enzyme recognition sites within all ribosomal RNA genes were fully conserved except for the loss of a SalI site within the large subunit rRNA gene of the 6.9-kb H. contortus repeat where only minor differences were observed in the restriction enzyme recognition sequences within the external spacer DNAs. Sequence analysis of the subcloned small subunit ribosomal DNAs from each species demonstrated 100% conservation within the 1758-bp fragments with only limited sequence variability observed within the adjacent 5' external-transcribed spacer. Enzymatic amplification of external spacer sequences using primers complementary and proximal to the 3'-end of the large subunit and the 5'-end of the small subunit rDNAs enabled rapid differentiation of individual worms of H. contortus from H. placei by utilizing the size variability within this region of the repeat.