scholarly journals Programmable low-cost DNA-based platform for viral RNA detection

2020 ◽  
Author(s):  
Lifeng Zhou ◽  
Arun Richard Chandrasekaran ◽  
Jibin Abraham Punnoose ◽  
Gaston Bonenfant ◽  
Stephon Charles ◽  
...  
Keyword(s):  
Biological Fluids ◽  
Low Cost ◽  
Rna Extraction ◽  
Human Saliva ◽  
Viral Rna ◽  
Disease Spread ◽  
Rna Detection ◽  
Preparation Step ◽  
Enzymatic Detection ◽  
The Cost

AbstractViral detection is critical for controlling disease spread and progression. Recent emerging viral threats including Zika, Ebola, and the current COVID-19 outbreak highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches designed to mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show non-enzymatic detection of viral RNA to the attomole level, with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with a sample preparation step using either RNA extraction or isothermal pre-amplification. Our assay can be performed with minimal or no lab infrastructure, and is readily adaptable to detect other viruses. We demonstrate the adaptability of our method by quickly developing and testing DNA nanoswitches for detecting a fragment of SARS-CoV-2 RNA in human saliva. Given this versatility, we expect that further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.

scholarly journals Programmable low-cost DNA-based platform for viral RNA detection

2020 ◽  
Vol 6 (39) ◽  
pp. eabc6246 ◽  
Author(s):  
Lifeng Zhou ◽  
Arun Richard Chandrasekaran ◽  
Jibin Abraham Punnoose ◽  
Gaston Bonenfant ◽  
Stephon Charles ◽  
...  
Keyword(s):  
Zika Virus ◽  
Ebola Virus ◽  
Biological Fluids ◽  
Low Cost ◽  
Rna Extraction ◽  
Viral Rna ◽  
Disease Spread ◽  
Rna Detection ◽  
The Cost ◽  
Further Development

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.

scholarly journals Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1558
Author(s):  
Zhan Qiu Mao ◽  
Mizuki Fukuta ◽  
Jean Claude Balingit ◽  
Thi Thanh Ngan Nguyen ◽  
Co Thach Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Rna Extraction ◽  
Viral Rna ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Resource Limited Settings ◽  
Rna Detection ◽  
Resource Limited ◽  
Starting Point

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

scholarly journals Wide Application of Minimally Processed Saliva on Multiple RT-qPCR Kits for SARS-CoV-2 Detection in Indonesia

2021 ◽  
Vol 11 ◽  
Author(s):  
Caroline Mahendra ◽  
Maria Mardalena Martini Kaisar ◽  
Suraj Rajan Vasandani ◽  
Sem Samuel Surja ◽  
Enty Tjoa ◽  
...  
Keyword(s):  
Target Genes ◽  
Local Population ◽  
Rna Extraction ◽  
Extraction Process ◽  
Human Saliva ◽  
Attractive Alternative ◽  
Sampling Device ◽  
Agreement Rate ◽  
Commercial Kits ◽  
The Cost

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, evaluating a proposed workflow amongst the local population is recommended. Here, we aim to validate the collection and treatment of human saliva as a direct specimen for RT-qPCR-based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimens and remained stable for five days either refrigerated or stored at room temperature. The method of processing saliva specimens described in this report bypasses the need for an RNA-extraction process, thereby reducing the cost, time, and manpower required for processing samples. The developed method was tested across nine commercial kits, including the benchmark, to demonstrate its wide applicability on multiple existing workflows. Our developed method achieved an 86% overall agreement rate compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a saliva sampling device, the collection was found to be more convenient for individuals and improved the overall agreement rate to 97%.

scholarly journals Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva

2020 ◽  
Author(s):  
Ethan Li ◽  
Adam Larson ◽  
Anesta Kothari ◽  
Manu Prakash
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Sample Collection ◽  
Rna Detection ◽  
Open Hardware ◽  
Point Of Care Diagnostics ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
The Cost

AbstractPoint of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (<10 genomes per μL) (Nagamine, Hase, and Notomi 2002). Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020).This LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply.We present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of <5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.

scholarly journals Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2

2020 ◽  
Author(s):  
Julio C. Chacón-Torres ◽  
C. Reinoso ◽  
Daniela G. Navas-Leon ◽  
S. Briceño ◽  
G. González
Keyword(s):  
Magnetic Nanoparticles ◽  
Low Cost ◽  
Rna Extraction ◽  
International Health ◽  
Private Companies ◽  
Wide Range ◽  
Chemical Availability ◽  
Scalable Synthesis ◽  
The Cost ◽  
And Control

Abstract Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT- PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA purification to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.

scholarly journals Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing

2021 ◽  
Vol 9 (1) ◽  
pp. 1
Author(s):  
Khaldoun Al-Romaih ◽  
Ibtihaj Alsharif ◽  
Razan Bakheet ◽  
Lina Mahmoud ◽  
Najla Alharbi ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Viral Rna

scholarly journals Low Cost tool for finding ventilation rates for Airborne Infection Control in the Built Environment

2020 ◽  
Author(s):  
Raja Singh
Keyword(s):  
Carbon Dioxide ◽  
Natural Ventilation ◽  
Low Cost ◽  
Ambient Air ◽  
Disease Spread ◽  
Indoor Space ◽  
Airborne Infection ◽  
New Apparatus ◽  
The Cost ◽  
Ventilation Rates

Dilution Ventilation is an accepted way of providing Natural Ventilation for reduction of Disease spread in Indoor Spaces. It is most relevant in low resource settings where the cost of advanced technologies may be a barrier. Studies have been performed in India to find a correlation between Ventilation of spaces and its role in the prevention of Tuberculosis, which is a major airborne disease with high incidence levels in India. These studies lack the measurement of the air changes in the room which is an important criterion to find out the disease spread by transmission models. This paper presents a new apparatus prepared to measure Air changes in an indoor space using Carbon Dioxide as a biomarker which acts as a surrogate for the ventilation in the space. The apparatus prepared uses a pre-existing carbon dioxide meter but adds value to it by creating a tamper-proof, vandal-resistant and a poke-resistant system. It also includes a built-in air suction and withdrawal system so that the sensor can be supplied with the ambient air in the room so that it can accurately give the carbon dioxide measurements. The study enhances the methods of investigation for Indoor Air Quality studies.

scholarly journals SARS-CoV-2 RNA detection in nasal mid-turbinate swabs

2020 ◽  
Author(s):  
Laura Dioni ◽  
Benedetta Albetti ◽  
Federica Rota ◽  
Valentina Bollati
Keyword(s):  
Rna Extraction ◽  
Positive Control ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Detection ◽  
Nasal Swabs ◽  
Internal Positive Control ◽  
Polymerase Chain ◽  
Genomic Regions

Abstract In this protocol, we describe a method to investigate the presence of SARS-CoV-2 RNA in nasal swabs, using a commercially available high-throughput Real-Time Polymerase Chain Reaction (RT-PCR) assay (TaqPath™ Covid-19 kit, ThermoFisher Scientific) in a 384-Well Plate. After Viral RNA extraction with QIAamp Viral RNA Mini kit, reverse transcription and cDNA amplification, all samples are assessed for the presence of three specific SARS-CoV-2 viral genomic regions and an internal positive control (IPC), in one Multiplex RT-PCR reaction.

scholarly journals Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Julio C. Chacón-Torres ◽  
C. Reinoso ◽  
Daniela G. Navas-León ◽  
Sarah Briceño ◽  
Gema González
Keyword(s):  
Magnetic Nanoparticles ◽  
Low Cost ◽  
Rna Extraction ◽  
International Health ◽  
Private Companies ◽  
Wide Range ◽  
Chemical Availability ◽  
Scalable Synthesis ◽  
The Cost ◽  
And Control

Abstract Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT-PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~ 10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA extraction to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~ 50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.

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