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- All Subjects: Waterborne infection
- Creators: Chao, Shih-hui
Description
Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and power hungry.
In this dissertation project, an inexpensive, portable, low-energy consuming, and highly quantitative microbiological genomic sensor has been developed for in situ ocean-observing systems. A novel real-time colorimetric loop-mediated isothermal amplification (LAMP) technology has been developed for quantitative detection of microbial nucleic acids. This technology was implemented on a chip-level device with an embedded inexpensive imaging device and temperature controller to achieve quantitative detection within one hour. A bubble-free liquid handling approach was introduced to avoid bubble trapping during liquid loading, a common problem in microfluidic devices. An algorithm was developed to reject the effect of bubbles generated during the reaction process, to enable more accurate nucleic acid analysis. This genomic sensor has been validated at gene and gene expression levels using Synechocystis sp. PCC 6803 genomic DNA and total RNA. Results suggest that the detection limits reached 10 copies/μL and 100 fg/μL, respectively. This approach was highly quantitative, with linear standard curves down to 103 copies/μL and 1 pg/μL, respectively. In addition to environmental microbe characterization, this genomic sensor has been employed for viral RNA quantification during an infectious disease outbreak. As the Zika fever was spreading in America, a quantitative detection of Zika virus has been performed. The results show that the genomic sensor is highly quantitative from 10 copies/μL to 105 copies/μL. This suggests that the novel nucleic acid quantification technology is sensitive, quantitative, and robust. It is a promising candidate for rapid microbe detection and quantification in routine laboratories.
In the future, this genomic sensor will be implemented in in situ platforms as a core analytical module with minor modifications, and could be easily accessible by oceanographers. Deployment of this microbial genomic sensor in the field will enable new scientific advances in oceanography and provide a possible solution for infectious disease detection.
In this dissertation project, an inexpensive, portable, low-energy consuming, and highly quantitative microbiological genomic sensor has been developed for in situ ocean-observing systems. A novel real-time colorimetric loop-mediated isothermal amplification (LAMP) technology has been developed for quantitative detection of microbial nucleic acids. This technology was implemented on a chip-level device with an embedded inexpensive imaging device and temperature controller to achieve quantitative detection within one hour. A bubble-free liquid handling approach was introduced to avoid bubble trapping during liquid loading, a common problem in microfluidic devices. An algorithm was developed to reject the effect of bubbles generated during the reaction process, to enable more accurate nucleic acid analysis. This genomic sensor has been validated at gene and gene expression levels using Synechocystis sp. PCC 6803 genomic DNA and total RNA. Results suggest that the detection limits reached 10 copies/μL and 100 fg/μL, respectively. This approach was highly quantitative, with linear standard curves down to 103 copies/μL and 1 pg/μL, respectively. In addition to environmental microbe characterization, this genomic sensor has been employed for viral RNA quantification during an infectious disease outbreak. As the Zika fever was spreading in America, a quantitative detection of Zika virus has been performed. The results show that the genomic sensor is highly quantitative from 10 copies/μL to 105 copies/μL. This suggests that the novel nucleic acid quantification technology is sensitive, quantitative, and robust. It is a promising candidate for rapid microbe detection and quantification in routine laboratories.
In the future, this genomic sensor will be implemented in in situ platforms as a core analytical module with minor modifications, and could be easily accessible by oceanographers. Deployment of this microbial genomic sensor in the field will enable new scientific advances in oceanography and provide a possible solution for infectious disease detection.
ContributorsCi, Shufang (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2017