Rapid Detection Of Pathogenic Bacteria And ATP Based On Bio-Beads/Nucleic Acid Probe

Diseases caused by pathogen infection are the major problems that threaten human health and lead to the recent social panic and the major cause of human death. The complexity of sample and the urgency of pathogenic bacteria posed a severe challenge for the traditional detection methods. But the traditional analytical tools and methods are difficult to meet the growing demands of rapid detection in the deficiencies of the sensitivity, specificity, simple operation, and rapidity. Therefore, it is very significant to develop a rapid, sensitive and accurate detection method towards pathogenic bacteria. Functional nanomaterials are well-suited for a wide range of biological applications because of their unique physical and chemical properties. As one of the most widely used molecular biology technologies, gene probe technology is a powerful tool for the qualitative or quantitative detection of specific RNA or DNA sequences. A new high sensitive and specifical detection method for pathogenic bacteria and their biomolecules was well constructed based on the nano-materials and gene probe technology in this study.This paper includes the following four reseaches: (1) Three functional immunomagnetic beads (IMBs) were constructed through the different functional groups on the surfaces of magnetic beads (MBs) covalently binding to antibodies, respectively. Their binding properties to antibody were also analysed. The results showed that all of the beads have high affinity, and ones with aldehyde groups are best among them. The effect of Group G Streptococcus (GGS) seperated by IMB was discussed. It was demonstrated that the three kinds of IMBs could separate GGS from samples, and the separation effects of IMBs with aldehydes are actually best. Continually, SPR technology based on the interaction of antibody and streptococci was used in order to quantitatively analyze GGS. It can successfully detect Strep, rapidly and its detected limit is 1.0x107CFU/ml. This method is more simple and rapid compared with traditional means.(2) A simple, efficient fluorescence detection technology for ATP was developed. Using DNA-intercalating dye Eva GreenTM to react with aptamer-ATP complex, the method can be quickly used to detect ATP based on its significant fluorescence signal changes. Some affection on the detection has been discussed, such as temperature, concentration and reaction time. It was shown that the best temperature and pH were 12℃and pH7.5, respectivily. The detection limitation of ATP could be 10-6 M under the optimized conditions.(3) Fluorescence resonance energy transfer (FRET) system has been utilized in order to find a fast and brief detection method for Staphyloccocus aureus, in which quantum dot was used as donors and organic fluorophore dyes as acceptors to inspect S. aureus specific 16s RNA. The result showed that the best fluorescent effect can been achieved when the ratio of carboxyl-modif ied QD to the amino-modified DNA is 1:80. The FRET efficiency would be steady when target DNA is 60nm and reach to its peak when target DNA is 80nm. Furthermore, this developed system is so sensitive that it can detect 20nm 16s rDNA. This work clearly indicated that FRET system is more sensitive and specific than other detect methods for S. aureus, in which quantum dot as.donors and organic fluorophore dyes as acceptors. Of course, it is possible to detect other bio-macromolecules with low density such as RNA and proteins in future.(4) A rapid, visible detection method for S. aureus was established based on the specific identification between aptamer and ligand. The results were displayed through the color changes of gold nanoparticles (GNPs) in different conditions. GNPs with uniform size and good dispersion were synthesized with chemical reduction method. The probe for S. aureus detection has been constructed based on the combination of sulphur-modified aptamer and GNPs and the batctera has been successfully detected.