| Summary: | 碩士 === 東海大學 === 化學系 === 105 === Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of morbidity and mortality around the world. It is noteworthy that the bacteria may evolve into the strains with drug-resistance gene during spreading among mankind, which is now bringing great challenge to disease prevention and treatment. These features underscore the significance of an economic, quick and accurate diagnostic method which is capable of identifying the drug- resistant bacteria, so as to take control the epidemic efficiently.
In this work, we present an analytical method for detecting both specific nucleic acid fragment and single nucleotide substitution (SNS) at the same time. The method was inherent with a construction of Holliday junction formed by the hybridization of four probes which harnessed one molecular beacon and the designed assistant strand as the core. To further understand the condition for junction formation, we discussed association and dissociation of the junction thermodynamically and kinetically.
To identify M.tb and determine its drug resistance simultaneously, we regulated the length of the analyte-binding arm to yield tiered fluorescence output which interpreted not only the sole presence of either specific fragment or point mutation, but also concurrent existence of both targets of interest. To achieve higher sensitivity, we designed the signal amplification mechanism driven by strand displacement polymerase (KF) and endonuclease (Nt.A1wI). The amplification driven by the biocatalytic reactions resulted in a 64-fold signal enhancement, pushing limit of detection down to 300 pM.
The sensing system was also successfully demonstrated to implement accurate detection of M.tb in the sample containing amplicons templated with DNA extract and boiling broth cultures. Influence caused by nontuberculous mycobacteria is minimal. The tiered-output mechanism worked out at bacterial concentration ranging 2.5*10^2~2.5*10^6 CFU/mL. The results showed integrity of the sensing method as an alternative strategy in rapid screening of M.tb and its rifampin-resistance.
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