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Article ; Online: Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

Myungsun Park / Joungha Won / Byung Yoon Choi / C. Justin Lee

Experimental and Molecular Medicine, Vol 52, Iss 6, Pp 963-

2020 Volume 977

Abstract: Diagnostic microbiology: Boosting confidence in COVID-19 detection A design strategy for virus detection tests could lead to improved detection of SARS-CoV-2 coronavirus. Laboratories currently perform SARS-CoV-2 diagnostic assays based on a technique ... ...

Abstract	Diagnostic microbiology: Boosting confidence in COVID-19 detection A design strategy for virus detection tests could lead to improved detection of SARS-CoV-2 coronavirus. Laboratories currently perform SARS-CoV-2 diagnostic assays based on a technique called the polymerase chain reaction (PCR), which enables targeted amplification of viral nucleic acids. PCR assays are sensitive but can be plagued by false positives or false negatives. C. Justin Lee of the Institute for Basic Science, Daejon, Republic of Korea, and coworkers have devised a protocol for optimizing assay performance. The success of a PCR assay is determined by the selection of ‘primers’, short DNA strands that determine which genomic sequence gets amplified. The researchers identify a strategy for designing effective primer sets, and selecting reaction conditions that make best use of those primers. Although the focus here is on SARS-CoV-2, this approach should be applicable to other viruses.
Keywords	Medicine ; R ; Biochemistry ; QD415-436
Language	English
Publishing date	2020-06-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR.

Park, Myungsun / Won, Joungha / Choi, Byung Yoon / Lee, C Justin

Experimental & molecular medicine

2020 Volume 52, Issue 6, Page(s) 963–977

Abstract: SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose ... ...

Abstract	SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.
MeSH term(s)	Betacoronavirus/genetics ; COVID-19 ; COVID-19 Testing ; Clinical Laboratory Techniques/methods ; Coronavirus Infections/diagnosis ; Coronavirus Infections/virology ; Coronavirus RNA-Dependent RNA Polymerase ; DNA Primers ; HEK293 Cells ; Humans ; Multiplex Polymerase Chain Reaction/methods ; Pandemics ; Pharynx/virology ; Pneumonia, Viral/diagnosis ; Pneumonia, Viral/virology ; Polymerase Chain Reaction/methods ; RNA-Dependent RNA Polymerase/genetics ; Real-Time Polymerase Chain Reaction/methods ; SARS Virus/genetics ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/genetics ; Viral Nonstructural Proteins/genetics ; Viral Proteins/genetics
Chemical Substances	DNA Primers ; Spike Glycoprotein, Coronavirus ; Viral Nonstructural Proteins ; Viral Proteins ; spike protein, SARS-CoV-2 ; Coronavirus RNA-Dependent RNA Polymerase (EC 2.7.7.48) ; NSP12 protein, SARS-CoV-2 (EC 2.7.7.48) ; RNA-Dependent RNA Polymerase (EC 2.7.7.48)
Keywords	covid19
Language	English
Publishing date	2020-06-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1328915-9
ISSN	2092-6413 ; 1226-3613 ; 0378-8512
ISSN (online)	2092-6413
ISSN	1226-3613 ; 0378-8512
DOI	10.1038/s12276-020-0452-7
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

Park, Myungsun / Won, Joungha / Choi, Byung Yoon / Lee, C. Justin

Experimental & Molecular Medicine

2020 Volume 52, Issue 6, Page(s) 963–977

Keywords	Clinical Biochemistry ; Molecular Medicine ; Biochemistry ; Molecular Biology ; covid19
Language	English
Publisher	Springer Science and Business Media LLC
Publishing country	us
Document type	Article ; Online
ZDB-ID	1328915-9
ISSN	1226-3613 ; 0378-8512
ISSN	1226-3613 ; 0378-8512
DOI	10.1038/s12276-020-0452-7
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes.

Kwon, Jea / An, Heeyoung / Sa, Moonsun / Won, Joungha / Shin, Jeong Im / Lee, C Justin

Experimental neurobiology

2017 Volume 26, Issue 1, Page(s) 42–54

Abstract: Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium ( ... ...

Abstract	Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium (Ca
Language	English
Publishing date	2017-02-09
Publishing country	Korea (South)
Document type	Journal Article
ZDB-ID	2639017-6
ISSN	2093-8144 ; 1226-2560
ISSN (online)	2093-8144
ISSN	1226-2560
DOI	10.5607/en.2017.26.1.42
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

Park, Myungsun / Won, Joungha / Choi, Byung Yoon / Lee, C Justin

Exp Mol Med

Abstract	SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.
Keywords	covid19
Publisher	WHO
Document type	Article
Note	WHO #Covidence: #601243
Database	COVID19

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Article ; Online: Opto-vTrap, an optogenetic trap for reversible inhibition of vesicular release, synaptic transmission, and behavior.

Won, Joungha / Pankratov, Yuriy / Jang, Minwoo Wendy / Kim, Sunpil / Ju, Yeon Ha / Lee, Sangkyu / Lee, Seung Eun / Kim, Arie / Park, Soowon / Lee, C Justin / Heo, Won Do

Neuron

2021 Volume 110, Issue 3, Page(s) 423–435.e4

Abstract: Spatiotemporal control of brain activity by optogenetics has emerged as an essential tool to study brain function. For silencing brain activity, optogenetic probes, such as halorhodopsin and archaerhodopsin, inhibit transmitter release indirectly by ... ...

Abstract	Spatiotemporal control of brain activity by optogenetics has emerged as an essential tool to study brain function. For silencing brain activity, optogenetic probes, such as halorhodopsin and archaerhodopsin, inhibit transmitter release indirectly by hyperpolarizing membrane potentials. However, these probes cause an undesirable ionic imbalance and rebound spikes. Moreover, they are not applicable to use in non-excitable glial cells. Here we engineered Opto-vTrap, a light-inducible and reversible inhibition system to temporarily trap the transmitter-containing vesicles from exocytotic release. Light activation of Opto-vTrap caused full vesicle clusterization and complete inhibition of exocytosis within 1 min, which recovered within 30 min after light off. We found a significant reduction in synaptic and gliotransmission upon activation of Opto-vTrap in acute brain slices. Opto-vTrap significantly inhibited hippocampus-dependent memory retrieval with full recovery within an hour. We propose Opto-vTrap as a next-generation optogenetic silencer to control brain activity and behavior with minimal confounding effects.
MeSH term(s)	Brain ; Exocytosis ; Hippocampus ; Optogenetics ; Synaptic Transmission/physiology
Language	English
Publishing date	2021-11-30
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	808167-0
ISSN	1097-4199 ; 0896-6273
ISSN (online)	1097-4199
ISSN	0896-6273
DOI	10.1016/j.neuron.2021.11.003
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Zs.A 2496: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 92: Show issues

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Article: Ultimate COVID-19 Detection Protocol Based on Saliva Sampling and qRT-PCR with Risk Probability Assessment.

Won, Joungha / Kazan, Hasan Hüseyin / Kwon, Jea / Park, Myungsun / Ergun, Mehmet Ali / Ozcan, Sureyya / Choi, Byung Yoon / Heo, Won Do / Lee, C Justin

Experimental neurobiology

2021 Volume 30, Issue 1, Page(s) 13–31

Abstract: In the era of COVID-19 outbreak, various efforts are undertaken to develop a quick, easy, inexpensive, and accurate way for diagnosis. Although many commercial diagnostic kits are available, detailed scientific evaluation is lacking, making the public ... ...

Abstract	In the era of COVID-19 outbreak, various efforts are undertaken to develop a quick, easy, inexpensive, and accurate way for diagnosis. Although many commercial diagnostic kits are available, detailed scientific evaluation is lacking, making the public vulnerable to fear of false-positive results. Moreover, current tissue sampling method from respiratory tract requires personal contact of medical staff with a potential asymptomatic SARSCOV-2 carrier and calls for safe and less invasive sampling method. Here, we have developed a convenient detection protocol for SARS-COV-2 based on a non-invasive saliva self-sampling method by extending our previous studies on development of a laboratory-safe and low-cost detection protocol based on qRT-PCR. We tested and compared various self-sampling methods of self-pharyngeal swab and self-saliva sampling from non-carrier volunteers. We found that the self-saliva sampling procedure gave expected negative results from all of the non-carrier volunteers within 2 hours, indicating cost-effectiveness, speed and reliability of the saliva-based method. For an automated assessment of the sampling quality and degree of positivity for COVID-19, we developed scalable formulae based on a logistic classification model using both cycle threshold and melting temperature from the qRT-PCR results. Our newly developed protocol will allow easy sampling and spatial-separation between patient and experimenter for guaranteed safety. Furthermore, our newly established risk assessment formula can be applied to a large-scale diagnosis in health institutions and agencies around the world.
Language	English
Publishing date	2021-02-06
Publishing country	Korea (South)
Document type	Journal Article
ZDB-ID	2639017-6
ISSN	2093-8144 ; 1226-2560
ISSN (online)	2093-8144
ISSN	1226-2560
DOI	10.5607/en20063
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Platycodin D, a natural component of Platycodon grandiflorum, prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion.

Kim, Tai Young / Jeon, Sangeun / Jang, Youngho / Gotina, Lizaveta / Won, Joungha / Ju, Yeon Ha / Kim, Sunpil / Jang, Minwoo Wendy / Won, Woojin / Park, Mingu Gordon / Pae, Ae Nim / Han, Sunkyu / Kim, Seungtaek / Lee, C Justin

Experimental & molecular medicine

2021 Volume 53, Issue 5, Page(s) 956–972

Abstract: An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by ... ...

Abstract	An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.
MeSH term(s)	Antiviral Agents/chemistry ; Antiviral Agents/pharmacology ; COVID-19/metabolism ; Cell Line ; Humans ; Lysosomes/drug effects ; Lysosomes/metabolism ; Models, Molecular ; Platycodon/chemistry ; SARS-CoV-2/drug effects ; SARS-CoV-2/physiology ; Saponins/chemistry ; Saponins/pharmacology ; Serine Endopeptidases/metabolism ; Triterpenes/chemistry ; Triterpenes/pharmacology ; Virus Internalization/drug effects ; COVID-19 Drug Treatment
Chemical Substances	Antiviral Agents ; Saponins ; Triterpenes ; platycodin D (CWJ06TA2GI) ; Serine Endopeptidases (EC 3.4.21.-) ; TMPRSS2 protein, human (EC 3.4.21.-)
Language	English
Publishing date	2021-05-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1328915-9
ISSN	2092-6413 ; 1226-3613 ; 0378-8512
ISSN (online)	2092-6413
ISSN	1226-3613 ; 0378-8512
DOI	10.1038/s12276-021-00624-9
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Zs.A 4775: Show issues

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Jg. 1995 - 2021: Lesesall (2.OG)
ab Jg. 2022: Lesesaal (EG)

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Article: Pharmacological Dissection of Intrinsic Optical Signal Reveals a Functional Coupling between Synaptic Activity and Astrocytic Volume Transient.

Woo, Junsung / Han, Young-Eun / Koh, Wuhyun / Won, Joungha / Park, Min Gu / An, Heeyoung / Lee, C Justin

Experimental neurobiology

2019 Volume 28, Issue 1, Page(s) 30–42

Abstract: The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for ... ...

Abstract	The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K
Language	English
Publishing date	2019-02-28
Publishing country	Korea (South)
Document type	Journal Article
ZDB-ID	2639017-6
ISSN	2093-8144 ; 1226-2560
ISSN (online)	2093-8144
ISSN	1226-2560
DOI	10.5607/en.2019.28.1.30
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Tweety-homolog (

Han, Young-Eun / Kwon, Jea / Won, Joungha / An, Heeyoung / Jang, Minwoo Wendy / Woo, Junsung / Lee, Je Sun / Park, Min Gu / Yoon, Bo-Eun / Lee, Seung Eun / Hwang, Eun Mi / Jung, Jae-Young / Park, Hyungju / Oh, Soo-Jin / Lee, C Justin

Experimental neurobiology

2019 Volume 28, Issue 2, Page(s) 183–215

Abstract: In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers ... ...

Abstract	In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl
Language	English
Publishing date	2019-04-30
Publishing country	Korea (South)
Document type	Journal Article
ZDB-ID	2639017-6
ISSN	2093-8144 ; 1226-2560
ISSN (online)	2093-8144
ISSN	1226-2560
DOI	10.5607/en.2019.28.2.183
Database	MEDical Literature Analysis and Retrieval System OnLINE

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