New Method Research About Colorimetric Biosensor For Detection Of Pathogenic Bacteria Based On DNA Catalyzed Hairpin Assembly

In recent years, bacterial infection has become a challenging issue of the concern of the whole society. Wide and even unreasonable using of antibiotics is quite serious, which results in drug resistance. The generation of superbugs has aroused extensive attention in the medical community. In addition, the clinical microbiology test has a special important role in the diagnosis and monitoring of bacterial infection, the epidemiological investigation of nosocomial infection, and effect evaluation of sterilization. Therefore, developing a versatile, simple, rapid, low-cost, sensitive and specific detection method is particularly of importance. This dissertation is divided into two parts:1. A sensitive colorimetric biosensor for specific detection of Streptococcus pneumoniae based on DNA catalyzed hairpin assembly signal amplificationStreptococcus pneumoniae, one of the most common bacterial respiratory pathogens worldwide, causes several infectious diseases including community acquired pneumonia, otitis media, meningitis and septicemia. In this study, a novel DNAzyme colorimetric sensing strategy by combining the signal transducer and CHA is developed for detecting the part of lytA gene of S. pneumoniae. The target DNA binds with the hairpin DNA to form a new nucleic acid sequence and creates a toehold in the transducer for initiating the recycle amplification reaction of CHA. The catalyzed assembly process produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex (DNAzyme). DNAzyme can catalyze the conversion of a colorless 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to a green ABTS. Under optimal conditions, the calibration curve of synthetic target DNA has good linearity from 50 pM to 200 nM with a detection limit of 32 pM. This strategy has been successfully applied to detect S. pneumoniae as low as 156 CFU mL-1, and shows a good specificity against closely related streptococci and major pathogenic bacteria. In addition, the developed method enables successful visual analysis of S. pneumoniae in clinical samples by the naked eye. Therefore, this colorimetric biosensor will provide powerful tool for clinical diagnosis of pathogenic microorganisms.2. A simple and sensitive versatile colorimetric biosensor for direct detection Salmonella Typhimurium.Salmonella Typhimurium, the gram-negative enteric bacilli, is one of the main pathogenic bacteria in acute gastroenteritis. Conventional identification methods for bacteria include culture and colony counting, enzyme-linked immunosorbant assay (ELISA), and polymerase chain reaction (PCR). Unfortunately, although these approaches are powerful and error-proof, most of them are time-consuming, labour-intensive, high-cost, and requiring for high trained personnel and expensive equipment. Thus, point-of-care, rapid, sensitive and accurate diagnosis of pathogenic bacteria is very important.In this study, a versatile aptamer-based colorimetric biosensor has been developed by using the CHA circuit’s module to detect various inputs through the use of a transducer. In the presence of Salmonella Typhimurium, the anti-Salmonella Typhimurium aptamer can specifically recognize Salmonella Typhimurium, and expose the toehold for initiating the amplification reaction. The catalyzed assembly process produces a large amount of G-rich DNA. The G-rich DNA with hemin forms G-quadruplex/hemin complex, demonstrating mimic horseradish peroxidase activity and catalyzing a colorimetric reaction. The absorbance can be adopted to quantitatively detect target bacteria. The developed method was applied to analysis of Salmonella Typhimurium in PBS buffer, and the detection limit of 103 CFU mL-1 within 2 h. In addition, the method shows a great potential to detect different targets by means of changing the corresponding complementary target DNA or aptamer strands, revealing versatile and potential colorimetric sensing platform. Thus, the developed method might provide a new strategy for future practical salmonella detection, and a powerful tool for the detection of pathogenic microorganisms in clinical diagnosis.