研究目的
To develop a Fast Light-Activated Substrate cHromogenic (FLASH) PCR platform for colorimetric nucleic acid detection and quantification, enabling point-of-care (POC) and field-based applications.
研究成果
The FLASH PCR platform successfully enables colorimetric nucleic acid detection and quantification with simplicity and flexibility, demonstrated through a portable electronic reader and a paper-based FLASH strip. The technology holds potential for rapid, robust, and sensitive nucleic acid detection at decentralized settings, opening new avenues for POC diagnosis and field-based applications.
研究不足
The study primarily focuses on endpoint readout for PCR, with real-time FLASH PCR being a potential future development. The compatibility of FLASH reactants and conditions with real-time PCR and the need for equipping FLASH readers with heating modules are areas for optimization.
1：Experimental Design and Method Selection:
The FLASH PCR system was designed based on the principle that DNA intercalating dyes (DIDs) can promote the rapid photooxidation of chromogenic substrates through light-induced production of singlet oxygen. This principle was used to convert DID-based fluorescent PCR assays into colorimetric FLASH PCR.
2：Sample Selection and Data Sources:
Synthetic DNA standards corresponding to subsequences of the surface protein gene of Hepatitis B virus (HBV-S) and clinical samples including soil-transmitted helminth (STH) infections were used.
3：List of Experimental Equipment and Materials:
SYBR Green I dye, 3,3’5,5’-tetramethylbenzidine (TMB), phosphate buffer saline (PBS), TWEEN 20, sodium citrate, citrate acid solution, hydrochloric acid, herring sperm DNA, Taq 2× PCR Master Mix, iTaqTM Universal SYBR Green Supermix, TEMED, ammonium persulfate, acrylamide/bis-acrylamide solution, DNA loading buffer, 20 bp DNA ladder, Luna Universal qPCR Master Mix, Monarch PCR & DNA Cleanup Kit, Phusion Blood Direct PCR kit, QIAamp Circulating Nucleic Acid Kit, QIAprep Spin Miniprep Kit, QuntiFluor dsDNA quantification kit.
4：Experimental Procedures and Operational Workflow:
PCR amplicons were mixed with SG-I and TMB in citrate buffer and irradiated using a 96 FLASH array or a portable FLASH reader. Absorbance at 650 nm or colorimetric signal at the Red channel was measured after each 5s irradiation.
5：Data Analysis Methods:
The analytical performance of the FLASH assay was evaluated by comparing its limit of detection (LOD) with that of conventional DID-based fluorescent turn-on assay and commercial quantitative PCR.
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SYBR Green I
Sigma
DNA intercalating dye used for nucleic acid staining and as a photosensitizer in the FLASH assay.
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3,3’5,5’-tetramethylbenzidine
TMB
Sigma
Chromogenic substrate used in the FLASH assay for colorimetric detection of nucleic acids.
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Taq 2× PCR Master Mix
Bio-Rad Laboratories, Inc.
Used for PCR amplification of nucleic acids.
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iTaqTM Universal SYBR Green Supermix
Bio-Rad Laboratories, Inc.
Used for real-time PCR with SYBR Green I dye.
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Luna Universal qPCR Master Mix
New England BioLabs
Used for quantitative PCR (qPCR) applications.
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Monarch PCR & DNA Cleanup Kit
New England BioLabs
Used for purification of PCR products and DNA cleanup.
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Phusion Blood Direct PCR kit
Thermo Fisher Scientific
Used for direct PCR from blood samples without DNA extraction.
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QIAamp Circulating Nucleic Acid Kit
Qiagen Inc.
Used for the extraction of circulating nucleic acids from plasma, serum, and other body fluids.
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QIAprep Spin Miniprep Kit
Qiagen Inc.
Used for plasmid DNA purification from bacterial cultures.
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QuntiFluor dsDNA quantification kit
Promega
Used for quantification of double-stranded DNA.
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