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

本研究旨在评估一种新开发的竞争性PCR检测方法的有效性，用于追踪重组瘤胃细菌。该试验针对的是该细菌特有的16S rRNA基因序列。实验结果如下：1.实验结果表明：利用该菌株特有的16S rRNA基因序列，建立了一种用于基因工程或重组宿主细菌的高定量竞争性PCR检测方法。最小可量化水平为100-200个细胞。选择培养基的活菌计数表明，重组菌接种于新鲜瘤胃液后3h，其水平下降。定量PCR显示了类似的趋势，尽管在减少中观察到几个小时的滞后时间。这是由于PCR在测定值中涉及来自死亡重组细胞的DNA。将重组菌接种于混合瘤胃细菌洗涤细胞悬液中，重组菌的活菌数也有所下降，但48h内PCR检测值保持不变。这表明重组DNA在洗涤悬浮液中的降解速度比在新鲜瘤胃液中慢得多。重组蛋白在绵羊瘤胃中接种后，其数量迅速下降，接种144h后无法检测到。竞争性聚合酶链反应的跟踪显示了几乎相似的变化，没有任何时间滞后。从以上结果可以看出，所建立的PCR方法适用于瘤胃中上述组合菌的追踪。然后，从死亡重组体中释放的DNA在实验中可以忽略不计，因为它在瘤胃中迅速降解。可能有未知的因素抑制了接种重组蛋白的建立，需要进一步澄清。

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.