PCR
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Ultrafast, on-Chip PCR Could Speed Up Diagnoses during Pandemics
A rapid point-of-care diagnostic plasmofluidic chip can deliver result in only 8 minutes
Reverse transcription-polymerase chain reaction (RT-PCR) has been the gold standard for diagnosis during the COVID-19 pandemic. However, the PCR portion of the test requires bulky, expensive machines and takes about an hour to complete, making it difficult to quickly diagnose someone at a testing site. Now, researchers at KAIST have developed a plasmofluidic chip that can perform PCR in only about 8 minutes, which could speed up diagnoses during current and future pandemics.
The rapid diagnosis of COVID-19 and other highly contagious viral diseases is important for timely medical care, quarantining and contact tracing. Currently, RT-PCR uses enzymes to reverse transcribe tiny amounts of viral RNA to DNA, and then amplifies the DNA so that it can be detected by a fluorescent probe. It is the most sensitive and reliable diagnostic method.
But because the PCR portion of the test requires 30-40 cycles of heating and cooling in special machines, it takes about an hour to perform, and samples must typically be sent away to a lab, meaning that a patient usually has to wait a day or two to receive their diagnosis.
Professor Ki-Hun Jeong at the Department of Bio and Brain Engineering and his colleagues wanted to develop a plasmofluidic PCR chip that could quickly heat and cool miniscule volumes of liquids, allowing accurate point-of-care diagnoses in a fraction of the time. The research was reported in ACS Nano on May 19.
The researchers devised a postage stamp-sized polydimethylsiloxane chip with a microchamber array for the PCR reactions. When a drop of a sample is added to the chip, a vacuum pulls the liquid into the microchambers, which are positioned above glass nanopillars with gold nanoislands. Any microbubbles, which could interfere with the PCR reaction, diffuse out through an air-permeable wall. When a white LED is turned on beneath the chip, the gold nanoislands on the nanopillars quickly convert light to heat, and then rapidly cool when the light is switched off.
The researchers tested the device on a piece of DNA containing a SARS-CoV-2 gene, accomplishing 40 heating and cooling cycles and fluorescence detection in only 5 minutes, with an additional 3 minutes for sample loading. The amplification efficiency was 91%, whereas a comparable conventional PCR process has an efficiency of 98%. With the reverse transcriptase step added prior to sample loading, the entire testing time with the new method could take 10-13 minutes, as opposed to about an hour for typical RT-PCR testing. The new device could provide many opportunities for rapid point-of-care diagnostics during a pandemic, the researchers say.
-Publication
Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics
ACS Nano (https://doi.org/10.1021/acsnano.1c02154)
-Professor
Ki-Hun Jeong
Biophotonics Laboratory
https://biophotonics.kaist.ac.kr/
Department of Bio and Brain Engineeinrg
KAIST
2021.06.08 View 8352 -
Universal Virus Detection Platform to Expedite Viral Diagnosis
Reactive polymer-based tester pre-screens dsRNAs of a wide range of viruses without their genome sequences
The prompt, precise, and massive detection of a virus is the key to combat infectious diseases such as Covid-19. A new viral diagnostic strategy using reactive polymer-grafted, double-stranded RNAs will serve as a pre-screening tester for a wide range of viruses with enhanced sensitivity.
Currently, the most widely using viral detection methodology is polymerase chain reaction (PCR) diagnosis, which amplifies and detects a piece of the viral genome. Prior knowledge of the relevant primer nucleic acids of the virus is quintessential for this test.
The detection platform developed by KAIST researchers identifies viral activities without amplifying specific nucleic acid targets. The research team, co-led by Professor Sheng Li and Professor Yoosik Kim from the Department of Chemical and Biomolecular Engineering, constructed a universal virus detection platform by utilizing the distinct features of the PPFPA-grafted surface and double-stranded RNAs.
The key principle of this platform is utilizing the distinct feature of reactive polymer-grafted surfaces, which serve as a versatile platform for the immobilization of functional molecules. These activated surfaces can be used in a wide range of applications including separation, delivery, and detection. As long double-stranded RNAs are common byproducts of viral transcription and replication, these PPFPA-grafted surfaces can detect the presence of different kinds of viruses without prior knowledge of their genomic sequences.
“We employed the PPFPA-grafted silicon surface to develop a universal virus detection platform by immobilizing antibodies that recognize double-stranded RNAs,” said Professor Kim.
To increase detection sensitivity, the research team devised two-step detection process analogues to sandwich enzyme-linked immunosorbent assay where the bound double-stranded RNAs are then visualized using fluorophore-tagged antibodies that also recognize the RNAs’ double-stranded secondary structure.
By utilizing the developed platform, long double-stranded RNAs can be detected and visualized from an RNA mixture as well as from total cell lysates, which contain a mixture of various abundant contaminants such as DNAs and proteins.
The research team successfully detected elevated levels of hepatitis C and A viruses with this tool.
“This new technology allows us to take on virus detection from a new perspective. By targeting a common biomarker, viral double-stranded RNAs, we can develop a pre-screening platform that can quickly differentiate infected populations from non-infected ones,” said Professor Li.
“This detection platform provides new perspectives for diagnosing infectious diseases. This will provide fast and accurate diagnoses for an infected population and prevent the influx of massive outbreaks,” said Professor Kim.
This work is featured in Biomacromolecules. This work was supported by the Agency for Defense Development (Grant UD170039ID), the Ministry of Science and ICT (NRF-2017R1D1A1B03034660, NRF-2019R1C1C1006672), and the KAIST Future Systems Healthcare Project from the Ministry of Science and ICT (KAISTHEALTHCARE42).
Profile:-Professor Yoosik KimDepartment of Chemical and Biomolecular Engineeringhttps://qcbio.kaist.ac.kr
KAIST-Professor Sheng LiDepartment of Chemical and Biomolecular Engineeringhttps://bcpolymer.kaist.ac.kr
KAIST
Publication:Ku et al., 2020. Reactive Polymer Targeting dsRNA as Universal Virus Detection Platform with Enhanced Sensitivity. Biomacromolecules (https://doi.org/10.1021/acs.biomac.0c00379).
2020.06.01 View 17108 -
Low Cost and Simple Gene Analysis Technology Developed
Professor Park Hyun Kyu of the Department of Biology and Chemical Engineering has developed a ‘real time CPR’ using Methylene Blue (nucleic acid bonding molecule with Electro-Chemical property).
The current gene analysis being used in the field is the real time PCR (Polymerase Chain Reaction) which takes advantage of the luminescent property of the gene and therefore requires expensive machines and chemicals to run.
By contrast, the electro-chemical method is easy to use and low cost and, most importantly, it allows the machine to become small and portable.
Professor Park’s research team used the decrease in the electro-chemical signal when the Methylene Blue reacts with nucleic acid and applied this to PCR which allowed for the real time analysis of the nucleic acid amplification process.
With the result of the experiment as the basis, the team was able to perform a trial with Chlamydia trachomatis, a pathogen that causes sexually transmitted disease.
The result showed that the electro-chemical method showed the same performance level as the real time PCR, which proved that the technology can be applied to diagnosing various diseases and gene research.
2011.04.30 View 9080