國家衛生研究院 NHRI:Item 3990099045/10979
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    题名: Simultaneous detections of genetic fragment and single nucleotide mutation with a three-tiered output for tuberculosis diagnosis
    作者: Huang, SY;Chang, JR;Liao, YC;Dou, HY;Chuang, MC
    贡献者: National Institute of Infectious Diseases and Vaccinology;Institute of Population Health Sciences
    摘要: Tuberculosis (TB) remains one of the major infectious diseases worldwide. The pathogenic bacterium, Mycobacterium tuberculosis (M.tb), continuously evolves strains carrying drug-resistance genes, thus posing a growing challenge to TB prevention and treatment. We report a diagnostic system that uses a molecular beacon probe and an assistant strand as the core to simultaneously interact with an M.tb-specific fragment (in IS6110) and a single nucleotide substitution (SNS)-encoded segment (in rpoB) associated with drug resistance. A single fluorescent output in three-tiered levels was produced for combinatorial interpretations based on formation of a four-way DNA junction (4WJ). The SNS caused the 4WJ to partially dissociate, thus resulting in medium-level fluorescence. By contrast, high- and low-level fluorescence, represented the complete complementary complex and absence of either targeted fragments, respectively. Manipulating the length of the analyte-binding arm realized the medium output. The thermodynamics and kinetics of 4WJ construction were investigated to maximize the tiered-output performance. Biocatalytic amplification driven by the Klenow Fragment and Nt.AlwI was incorporated into the method to enhance the signal 64-fold and ensure long-term stability of the three-tiered output. The detection accuracy of the sensing system was verified using unpurified amplicons with templates of extracted DNA and boiled bacterial solutions. The tiered-output mechanism was usable at bacterial loads ranging from 4 × 100 to 4 × 103 CFU per reaction. The interference caused by nontuberculous mycobacteria was minimal. The results demonstrated the integrity of the sensing method as an alternative strategy for rapid screening of M.tb and detecting rifampin-resistance.
    日期: 2018-05-12
    關聯: Analytica Chimica Acta. 2018 May 12;1007:1-9.
    Link to: http://dx.doi.org/10.1016/j.aca.2017.12.028
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0003-2670&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000423843500001
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=85040021151
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