Study Of Detection Methods For Sulfate-reducing Bacteria By DNA Sensor

With Sulfate-reducing bacteria(SRB) as target, this thesis focus on the study for fabrication, optimization and application of DNA sensor. Detection methods including DNA sensor based on nuclease cycling amplification, bio-enzyme signal probe and DNA nanobarcode-fluorescence system were developed. All these methods are sensitive and selective, which has great scientific significance and potential practical application. The main results are show as follows:1, By employing specific region amplicons of bacterial genome as target, genetic fragment detecting system is to design capture probe and signal probe complemented with specific target DNA. This study proposed a novel detecting method for bacterial DNA detection based on nuclease aided cycling amplification. This system has high selectivity, specificity and stability, and can be potentially applied in bacterial detection and cell analysis.2, We described a simple and novel system for bacteria detection by using lysozyme as sensitive reporter. Coupled with target bacterial DNA binding with capture probe immobilized on MBs and lysozyme acted as signal reporter, this method is highly sensitive, with a minimum response concentration of 50 cfu m L-1 bacteria. In addition, the method exhibited excellent selectivity and applicability, being able to differentiate different bacteria target and used in complex buffer like human serum.3, We designed a method of bacteria detection based on DNA nanobarcodes-based fluorescence system by flow cytometry. Couple with capture probe immobilized on MBs, lysozyme acted as signal reporter, fluorescencently-labeled DNA nanobarcode as novel signal labels, this method is both specific for bacteria and highly sensitive(down to 500 cfu m L-1 bacteria). Compared with traditional bacteria detecting methods mostly relied on the use of either small molecule affinity ligands or antibodies with some drawbacks like low sensitivity and limited bacteria application. This proposed nucleic acid-based approach allow specific quantifying of various bacteria species with 16 S r RNA sequence information, emerging as a preferred technique for microbial identification over traditional biochemical assays...