Dataset Information

Real-Time Fluorescence Measurement for Droplet Generation and Signal Detection in a Cylindrical Tube.

ABSTRACT: This study provides an approach for generating droplets in a cylindrical tube. This creative design utilizes a single tube to generate droplets for the emulsion polymerase chain reaction (PCR). We fabricated the multi-layered tube and detected samples in 1 mm microchannel length for the minimum disposable material used for droplet signal analysis. In this research, we verify the validity of the non-planar droplet chip with a single driving pump by experimentally analyzing the light intensity of the liquids during droplet processing in real time. Experimental observation shows that droplet diameter from 150 to 285 μm was obtained by the cylindrical tube with different dispersed liquid and gas pressure settings. Average size of droplets decreased by approximately 37% as the dispersed phase liquids change at the same flow pressure of 50 mbar. In this study, the effects of the laser site, 6-carboxyfluorescein (6-FAM) dye buffer, DNA reagent, and driving pressure are analyzed directly by the signal recorded during droplet generation in the cylindrical tube. Finally, we have developed a real-time detection method to count exon 19 deletion mutations of epidermal growth factor receptor (EGFR) in a droplet using a cylindrical tube. Two digital droplet PCR methods were compared in measuring the copy numbers of EGFR with the same target DNA concentration of 10⁵ copies/μL.

SUBMITTER: Parng SH

PROVIDER: S-EPMC9996802 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Real-Time Fluorescence Measurement for Droplet Generation and Signal Detection in a Cylindrical Tube.

Parng Shaw-Hwa SH Wu Li-An LA Kuo Chien-Chih CC Wu Ping-Jung PJ Tsai Yu-Yin YY Lee Chih-Hung CH

ACS omega 20230222 9

This study provides an approach for generating droplets in a cylindrical tube. This creative design utilizes a single tube to generate droplets for the emulsion polymerase chain reaction (PCR). We fabricated the multi-layered tube and detected samples in 1 mm microchannel length for the minimum disposable material used for droplet signal analysis. In this research, we verify the validity of the non-planar droplet chip with a single driving pump by experimentally analyzing the light intensity of ...[more]

PMID: 36910981

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Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Dataset Information

Real-Time Fluorescence Measurement for Droplet Generation and Signal Detection in a Cylindrical Tube.

Publications

Real-Time Fluorescence Measurement for Droplet Generation and Signal Detection in a Cylindrical Tube.

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