• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied and Environmental Microbiology >Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism
【24h】

Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism

机译：建立基于实时PCR的方法，以沙门氏菌为模型生物，对水中细菌RNA目标进行准确定量

获取原文
       
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.
机译：定量PCR（Q-PCR）是定量目标基因的快速有效工具。在真核细胞中，还使用定量逆转录PCR（Q-RT-PCR）来量化基因表达，并以稳定表达的管家基因作为标准。在细菌中，不会出现管家基因的这种稳定表达，并且使用DNA标准品会导致广泛的低估。因此，仅通过使用适当的RNA标准品，对RNA进行准确定量是可行的。我们建立并验证了一种Q-PCR方法，该方法不仅可以定量靶基因的拷贝数（即细菌细胞的数目），还可以定量RNA拷贝的数目。选择编码肠炎沙门氏菌鼠伤寒沙门氏菌InvA和16S rRNA的基因进行方法评价。作为DNA标准，使用了靶基因的扩增片段，而在体外转录了相同的DNA标准以开发合适的RNA标准。然后将沙门氏菌培养物和接种细菌的环境水样品用于最终测试。两种实验方法均可通过Q-PCR和Q-RT-PCR对所选靶基因和RNA分子进行灵敏，准确和可重复的定量。 RNA标准首次成功用于原核生物中RNA分子数量的精确定量。这证明了这种方法在确定环境样品中病原细菌的存在和代谢活性方面的潜力。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号