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Tracking of recombinant rumen bacterium by indecular method using target 16S rRNA sequence

使用目标 16S rRNA 序列通过内嵌法追踪重组瘤胃细菌

基本信息

批准号：
09660302
负责人：
KOBAYASHI Yasuo
金额：
$ 1.15万
依托单位：
MIE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660302/
关键词：
16S rRNA PCR Rumen Bacteria Genetic Engineering Fiber Digestion Sheep Target sequence DNA 16S rRNA

项目摘要

This study is aimed at assessing availability of a newly developed competitive PCR assay for tracking a recombinant rumen bacterium. The assay was targeted to 16S rRNA gene sequences specific to the bacterium. The results obtained are follows :1. A highly quantitative competitive PCR assay for a host bacterium for genetic engineering, or its recombinant, was developed by using 16S rRNA gene sequence specific to the bacterial strain. Minimal quantifiable level was 100-200 cells.2. Viable counting with a selective medium demonstrated that the recombinant inoculated into fresh rumen fluid decreased its level 3h after the inoculation. The quantitative PCR gave a similar trend even though a few hours' lag time in the decrease was observed. This is due to the fact that the PCR involves DNA from dead recombinant cells in the assay value. When recombinant was inoculatedinto washed cell suspension of mixed rumen bacteria, viable number of the recombinant also decreased, though the PCR assay value remained constant during 48h. These suggest much slower degradation of the recombinant DNA in the washed suspension than in fresh rumen fluid.3. Recombinant inoculated in sheep rumen was rapidly depressed in its number and became undetectable 144h after the inoculation. Tracking by the competitive PCR showed an almost similar change without any time lag.From all the above results, it is apparent that the developed PCR assay is applicable to tracking of there combinant bacterium in the rumen. Then, DNA released from the dead recombinant is negligible in the assay since it is rapidly degraded in the rumen. There might be unknown factors inhibitory to establishment of the inoculated recombinant that need to be clarified in the future.

本研究旨在评估一种新开发的竞争性PCR检测方法用于跟踪重组瘤胃细菌的可用性。该测定针对细菌特异性的16 S rRNA基因序列。主要研究结果如下：1.利用16 SrRNA基因序列建立了一种高效的基因工程宿主菌及其重组体的竞争PCR检测方法。最小可定量水平为100-200个细胞。用选择性培养基进行活菌计数表明，重组体接种到新鲜瘤胃液中，接种后3 h其水平下降。定量PCR给出了类似的趋势，即使观察到几个小时的下降滞后时间。这是由于PCR在测定值中涉及来自死亡重组细胞的DNA。将重组体接种到混合瘤胃液中，48 h内PCR检测值基本不变，但活菌数也有所下降。这表明重组DNA在洗涤过的悬浮液中的降解比在新鲜瘤胃液中慢得多。在绵羊瘤胃中接种重组体后，重组体的数量迅速下降，144 h后检测不到重组体。竞争性PCR的追踪结果显示，两者的变化趋势基本一致，无时间滞后，表明所建立的PCR方法适用于瘤胃内3种复合菌的追踪。然后，从死亡重组体释放的DNA在测定中可忽略不计，因为它在瘤胃中快速降解。可能存在未知的因素抑制接种重组体的建立，需要在将来澄清。