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

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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.