scholarly journals Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Scott Sherrill-Mix ◽  
Young Hwang ◽  
Aoife M. Roche ◽  
Abigail Glascock ◽  
Susan R. Weiss ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Dna Amplification ◽  
Molecular Beacons ◽  
Locked Nucleic Acid ◽  
Melting Temperatures ◽  
Rapid Assays ◽  
Global Pandemic ◽  
Rapid Spread ◽  
Good Concordance ◽  
Reaction Volumes

Abstract Background Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. Results Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. Conclusions LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

scholarly journals LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons

2020 ◽  
Author(s):  
Scott Sherrill-Mix ◽  
Young Hwang ◽  
Aoife M. Roche ◽  
Abigail Glascock ◽  
Susan R. Weiss ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Dna Amplification ◽  
Molecular Beacons ◽  
Locked Nucleic Acid ◽  
Melting Temperatures ◽  
Rapid Assays ◽  
Global Pandemic ◽  
Rapid Spread ◽  
Assay Methods ◽  
Good Concordance

AbstractBackgroundRapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse Transcription and Loop-Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however a common artifact is nonspecific DNA amplification, which complicates detection.ResultsHere we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. We also describe custom polymerases for LAMP-BEAC and inexpensive purification procedures.ConclusionsLAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening.

scholarly journals Wellness Constraints Among International University Students Transitioning Through Covid 19

2021 ◽  
Vol 58 (3) ◽  
pp. 3444-3456
Author(s):  
Mr J Dorasamy, Et. al.
Keyword(s):  
World Health Organization ◽  
Social Economic ◽  
World Health ◽  
Social Emotional ◽  
Global Pandemic ◽  
Rapid Spread ◽  
The World ◽  
International University ◽  
Novel Coronavirus ◽  
Health Organization

The World Health Organization (Who) In March 2020 Declared Covid 19 A Pandemic, Due To The  Global And Rapid Spread Of A Novel Coronavirus (Who, 2020). The Covid 19 Pandemic Being Highly Infectious And Unpredictable, Has  Disrupted  Social, Economic, Environmental And Political Spheres Of Life. Globally, People Have Ventured Into A “Lockdown World”, Increasing Uncertainty About Their Future Amidst The Covid 19 Pandemic. As A Result Of The Pandemic, Social Alteration Has Taken The Form Of Social Distancing, Self-Isolation And Self-Quarantine.  Many Were Unprepared For The Shift From The “Normal”, Propelling  Undue  Stress Under The New Normal Way Of Doing Things During The Current Global Pandemic Crisis. This Has Been Accompanied By Social, Emotional And Mental Effects, As The Ongoing And Fluid Nature Of The Pandemic Has Created Uncertainty For Many People. The Covid 19 Pandemic, As A Multidimensional Stressor Affecting Wellbeing, Has Affected Individuals, Families, Educational, Occupational, And Broader Societal Systems.  

scholarly journals Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2

2021 ◽  
Author(s):  
Scott Sherrill-Mix ◽  
Gregory D. Van Duyne ◽  
Frederic D. Bushman
Keyword(s):  
Genetic Variants ◽  
Molecular Beacons ◽  
Spike Protein ◽  
Detection Methods ◽  
Gene Encoding ◽  
Detection And Identification ◽  
Rapid Spread ◽  
The World ◽  
Primer Sets ◽  
Current Detection

AbstractOver the course of the COVID-19 pandemic, several SARS-CoV-2 genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants is important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive qPCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons paired with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants which contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific, affordable and allows multiplexing with other SARS-CoV-2 LAMP primer sets.

scholarly journals Duration of viral detection in throat and rectum of a patient with COVID-19

2020 ◽  
Author(s):  
Le Van Tan ◽  
Nghiem My Ngoc ◽  
Bui Thi Ton That ◽  
Le Thi Tam Uyen ◽  
Nguyen Thi Thu Hong ◽  
...  
Keyword(s):  
Infection Control ◽  
Patient Management ◽  
Ho Chi Minh City ◽  
Rt Pcr ◽  
Viral Detection ◽  
Oral Transmission ◽  
Viral Shedding ◽  
Clinical Recovery ◽  
Global Pandemic ◽  
Rapid Spread

AbstractThe rapid spread of coronavirus disease 2019 (COVID-19) raises concern about a global pandemic. Knowledge about the duration of viral shedding remains important for patient management and infection control. We report the duration of viral detection in throat and rectum of a COVID-19 patient treated at the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam. Despite clinical recovery, SARS-CoV-2 RNA remained detectable by real time RT-PCR in throat and rectal swabs until day 11 and 18 of hospitalization, respectively. Because live SARS-CoV-2 has been successfully isolated from a stool sample from a COVID-19 patient in China, the results demonstrate that COVID-19 patients may remain infectious for long periods, and fecal-oral transmission may be possible. Therefore, our finding has important implications for infection control.

scholarly journals Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

Reverse transcription and subsequent DNA amplification of rubella virus RNA

1989 ◽  
Vol 25 (1) ◽  
pp. 21-29 ◽  
Author(s):  
William F. Carman ◽  
Carolyn Williamson ◽  
Beverley A. Cunliffe ◽  
Alistair H. Kidd
Keyword(s):  
Reverse Transcription ◽  
Rubella Virus ◽  
Dna Amplification ◽  
Virus Rna

scholarly journals Computational Determination of Toxicity Risks Associated with a Selection of Approved Drugs having Demonstrated Activity Against COVID- 19

2021 ◽  
Author(s):  
Maral Aminpour ◽  
Williams Miranda-Delgado ◽  
Soren Wacker ◽  
Sergey Noskov ◽  
Michael Houghton ◽  
...  
Keyword(s):  
Late Phase ◽  
Severe Pneumonia ◽  
Toxicity Assessment ◽  
Global Pandemic ◽  
Rapid Spread ◽  
Essential Knowledge ◽  
Approved Drugs ◽  
In Silico Models ◽  
Selection Of

Abstract BackgroundThe emergence and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) at late 2019 has caused a devastating global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19). Although vaccines have been and are being developed, they are not accessible to everyone and not everyone can receive these vaccines. Also, it typically takes more than 10 years until a new therapeutic agent is approved for usage. Therefore, repurposing of known drugs can lend itself well as a key approach for significantly expediting the development of new therapies for COVID-19.MethodsWe have been incorporated machine learning-based computational tools and in silico models into the drug discovery process to predict Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiles of 90 potential drugs for COVID-19 treatment identified from two independent studies mainly with the purpose of mitigating late-phase failures because of inferior pharmacokinetics and toxicity. ResultsHere, summarized the cardiotoxicity and general toxicity profiles of 90 potential drugs for COVID-19 treatment and summarize the risks of repurposing and propose a stratification of patients accordingly. We shortlist a total of five compounds based on their non-toxic properties.ConclusionIn summary, this manuscript aims to provide a potentially useful source of essential knowledge on toxicity assessment of 90 compounds for health care practitioners and researchers to find off-label alternatives for the treatment for COVID-19. The majority of the molecules discussed in this manuscript have already moved into clinical trials and thus their known pharmacological and human safety profiles are expected to facilitate a fast track preclinical and clinical assessment for treating COVID-19.

scholarly journals Validation of Half-reaction Volumes of the Promega PowerPlex® Forensic Amplification Kits (PowerPlex® 18D Systems, PowerPlex ® 21System, PowerPlex® Fusion System and PowerPlex® Y23 System) in STR Analysis

2020 ◽  
Vol 2 (2) ◽  
pp. 164-171
Author(s):  
Hanan K. Mahmood ◽  
Nadia F. Salman ◽  
Dhurgham H. Hasan ◽  
Khaleefah M. Salih ◽  
Maryam A. Sadiq ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Dna Amplification ◽  
Sensitivity Study ◽  
Fusion System ◽  
Reaction Volume ◽  
Forensic Dna Analysis ◽  
Str Analysis ◽  
Amplification Protocol ◽  
Reaction Amplification ◽  
Reaction Volumes

DNA amplification is known to be the most expensive step during forensic DNA analysis. This study evaluated the half-reaction amplification protocol (12.5 µL PCR product) using DNA amplification kits from Promega PowerPlex® (PowerPlex® 18D System, PowerPlex ®21System, PowerPlex® Fusion System and PowerPlex® Y23 System), which might aid in reducing sample analysis cost by half and allow the analysis of more samples. A sensitivity study (15 samples) along with testing of various blood stain samples (n=100) that were submitted to the Medico-Legal Directorate laboratory for DNA testing was accomplished to compare the DNA profiles resulting from half-reaction volume procedure to those with full-reaction volume procedure, using three differed methods along with standard protocol to evaluate the effect of half reaction volume with some variables. Results demonstrated the use of half-reaction amplification protocol preceded by washing step for all aforementioned DNA amplification kits gave a robust and reliable amplification result that aid to increase the number of samples analyzed and decreased the test cost for each kit without compromising the quality of 3DNA profiles obtained.

scholarly journals TESTAMENTARY DISPOSITIONS IN THE CONTEXT OF GLOBAL PANDEMIC

2021 ◽  
Author(s):  
Marko Stilinović
Keyword(s):  
Comparative Analysis ◽  
Comparative Method ◽  
Legal Framework ◽  
Traditional Methods ◽  
Inheritance Law ◽  
New Normal ◽  
New Methods ◽  
Global Pandemic ◽  
Rapid Spread ◽  
Technological Developments

The outbreak and the rapid spread of global COVID-19 pandemic have put significant strains on the institutions. The need to adapt to “new normal” and contain the rapid spread of disease, while maintaining a functional society, resulted with introduction of numerous new legal mechanisms and adaptation of the existing ones. However, it seems that one area of law remains on the fringes: the regulation of wills. Even before the start of the pandemic many authors often pointed to the fact that the current legal framework does not follow modern technological developments, but no significant attempts were made to overhaul the inheritance law. Also, once the pandemic started in its full, there were no references to introduction of extraordinary mechanisms or new legal solutions to overcome the potential difficulties in forming wills. Comparative analysis yielded no better results: although some countries (such as Austria) recently completely overhauled their regulation of inheritance law, it seems that no attempts were made to introduce new types of wills or new methods of drafting wills into their regulations. Furthermore, following the spread of the pandemic, increasing number of potential testators find themselves unable to use traditional methods of drafting wills as they, or the authorized persons tasked with assistance and creation of wills, remain isolated from one another due to various reasons (lock-downs, isolation in case of experiencing symptoms, etc.). Having in mind these circumstances, it is necessary to ascertain whether there is a genuine need to introduce new types of wills into existing legal framework, or to adapt the current legal framework by facilitating the communication between citizens and the institutions. Also, it is necessary to analyze whether the interpretation of the existing legal framework enables the introduction of certain facilitating mechanisms. In order to reach these goals and clarify the possibilities within the current legal framework, interpretative and comparative method are used.

scholarly journals Reverse Transcription Lesion-Induced DNA Amplification: An Instrument-Free Isothermal Method to Detect RNA

2020 ◽  
Author(s):  
Bibi Safeenaz Alladin-Mustan ◽  
Jesse Yuzik ◽  
Daria Raquel Queiroz de Almeida ◽  
Yuning Liu ◽  
Yimeng Li ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Room Temperature ◽  
Point Of Care ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Rna Detection ◽  
Isothermal Method ◽  
Enzymatic Amplification ◽  
Point Of Care Diagnostics ◽  
Target Rna

<p>One challenge in point-of-care diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform a reverse transcription ligase chain reaction using our isothermal lesion induced DNA amplification (LIDA) technique that can be tuned to operate at any desired temperature. Using RNA-triggered LIDA, we can detect as little as ~100 attomoles target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.</p>

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