A Quantitative MRNA Assay Based On Molecular Beacon And The Application

In the thesis, based on conventional monitoring parameters of microbial fermentation mainly about environmental parameters and biological parameters, we proposed the new idea that product yield is increased through monitoring intracellular activities during microbial fermentation process. And mRNA was chosen as the first research object, because transcriptional regulation is the major mechanism to regulate the product synthesis in bacterial cell.We summarized a lot of methods that were reported for mRNA quantification, including real-time RT-PCR and molecular beacon. After comparison, the unique feature of molecular beacon was clear: one-step reaction, short hybridization time and no requirement of enzyme. One-step liquid hybridization is easy for the engineering of detection technology. phzC gene is one component of the PCA biosynthesis operon phzABCDEFG, and its sequence was not reported before. In order to get that, we designed PCR primer according to the sequence of Paeruginosa PAO1 that was likely with M18 in biological characters as much as 99%. Based on the sequence of phzC gene we got, phzC MB was designed and synthesized.According to the reported papers of RNA quantification with molecular beacon (MB) hybridization, we found that MB hybridization conditions such as hybridization buffer and temperature were all arbitrarily chosen and have not been systematically optimized. For the determination which buffer was the best for our MB hybridization, seven typical hybridization buffers were systematically examined with complementary DNA oligonucleotide. And the thermal denaturation profiles of phzC MB with cDNA oligonucleotide were studied. And a optimized buffer and temperature were gained.To validate the results from phzC MB and cDNA, a further research on phzC MB and RNA was processed.Formamide is often used to protect RNA from RNase degradation and to decrease the hybridization temperature. The effect of Formamide on MB system was also studied. And that was the first report since now.Then, the process of hybridization between phzC MB and mRNA was established: RNA extraction—directly dissolved in the hybridization buffer—heated at 95°C for 5 min—incubated and assayed in the Rotor-Gene PCR. At last, the PCA and mRNA were monitored during M18G fermentation process, and a relationship between them was established.The method for monitoring mRNA based on molecular beacon has the characteristics as: simple, low-cost, multi-samples, and fast. The whole work can be finished within 30 min, which give a big support for online monitoring of fermentation process.