Dataset Information

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

ABSTRACT: Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

SUBMITTER: Kang BH

PROVIDER: S-EPMC10081571 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Publications

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Kang Byoung-Hoon BH Jang Kyung-Won KW Yu Eun-Sil ES Na Hamin H Lee Yun-Jae YJ Ko Woong-Yeol WY Bae NamHo N Rho Donggee D Jeong Ki-Hun KH

ACS nano 20230327 7

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlen ...[more]

PMID: 36971140

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Project description:BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Project description:Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental understanding of single-molecule reactions and providing advancements in practical applications such as medical diagnostics, forensics and environmental sampling.

Dataset Information

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Publications

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

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