scholarly journals Comparison of Different Kits for SARS-CoV-2 RNA Extraction Marketed in Brazil

2020 ◽  
Author(s):  
Ana Karolina Antunes Eisen ◽  
Meriane Demoliner ◽  
Juliana Schons Gularte ◽  
Alana Witt Hansen ◽  
Karoline Schallenberger ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Rna Extraction ◽  
Severe Pneumonia ◽  
Rna Isolation ◽  
Isolation And Purification ◽  
Silica Column ◽  
Negative Results ◽  
Mean Delay ◽  
Spanish Flu ◽  
The One

ABSTRACTDecember 2019 marked the begining of the greatest pandemic since Spanish Flu, the disease named Covid-19 that cause severe pneumonia. Until May 19, 2020 more than 4 million and 700 thousand cases were oficially notified with about 316 thousand deaths. Etiological agent of the disease was identified as being a new coronavirus, Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2). In this study we compared four different manual methods for RNA isolation and purification for detection of SARS-CoV-2 through qRT-PCR, as well as the extraction quality itself through detection of RNAse P. Magnetic beads-based (MagMax™) and silica column-based (Biopur®) methods presented the better performances. Concerning to the mean delay in CT values when compared to MagMax™, TRIzol™, Biopur® and EasyExtract presented 0,39, 0,95 and 5,23 respectively. Agreement between positive and negative results of different methods when compared with the one with better performance MagMax™ was 94,44% for silica column-based method (Biopur®), 88,89% for phenol-chroloform-based method (TRIzol™) and 77,78% for EasyExtract. We aimed to evaluate how reliable each method is for diagnostic purposes and to propose alternatives when usual methods are not available. In this regard, magnectic beads and silica column-based methods are convenient and reliable choices and phenol-chloroform-based method could also be chosen as an alternative.

Step 2: RNA extraction and RT-qPCR v1

2021 ◽  
Author(s):  
Peter H L Krijger ◽  
Tim A Hoek ◽  
Sanne Boersma ◽  
Lieke I P M Donders ◽  
Maaike M C Broeders ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Storage System ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Robotic Arms ◽  
Liquid Handling ◽  
2D Barcode ◽  
Simultaneous Processing ◽  
Multiple Sample ◽  
Liquid Handling Robot

STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. Key features of the customized Tecan Fluent robot are (1) on-board 1D and 2D barcode scanners, (2) an automated tube decapper, (3) two robotic arms for simultaneous processing of different procedural steps, (4) a newly-designed spin vessel to keep magnetic beads in solution and immediately transferable to 384-well plates, (5) a built-in magnetic deck and a 384-channel pipetting head for rapid RNA isolation, (6) a heating device for fast drying of RNA prior to elution, (7) a built-in plate sealer and (8) a plate storage system to allow processing of multiple sample plates in a single run (See Materials). Here we describe the RNA extraction and RT-qPCR protocol as currently applied in STRIP.

scholarly journals Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

2020 ◽  
Author(s):  
Diana Rose E. Ranoa ◽  
Robin L. Holland ◽  
Fadi G. Alnaji ◽  
Kelsie J. Green ◽  
Leyi Wang ◽  
...  
Keyword(s):  
Large Scale ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Clinical Samples ◽  
Molecular Testing ◽  
Short Supply ◽  
Isolation And Purification ◽  
Repeat Testing ◽  
Large Populations

AbstractConvenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations.Graphical Abstract

The First Half of the Twentieth Century

2018 ◽  
Author(s):  
Alexander Gillespie
Keyword(s):  
Habitat Management ◽  
Social Economic ◽  
Chemical Pollution ◽  
Freshwater Conservation ◽  
The Great Depression ◽  
Negative Results ◽  
The Social ◽  
The World ◽  
The One ◽  
The Welfare State

The years between 1900 and 1945 were very difficult for humanity. In this period, not only were there two world wars to survive but also some of the worst parts of the social, economic, and environmental challenges of sustainable development all began to make themselves felt. The one area in which progress was made was in the social context, in which the rights of workers and the welfare state expanded. The idea of ‘development’, especially for the developing world, also evolved in this period. In the economic arena, the world went up, and then crashed in the Great Depression, producing negative results that were unprecedented. In environmental terms, positive templates were created for some habitat management, some wildlife law, and parts of freshwater conservation. Where there was not so much success was with regard to air and chemical pollution.

scholarly journals High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

2017 ◽  
Vol 38 (4) ◽  
pp. 2201 ◽  
Author(s):  
Gabrielle Silveira de Campos ◽  
Ricardo Antônio Ayub ◽  
Rafael Mazer Etto ◽  
Carolina Weigert Galvão ◽  
Marília Aparecida Stroka ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Sodium Perchlorate ◽  
Rna Isolation ◽  
World Market ◽  
Molecular Techniques ◽  
Guanidine Thiocyanate ◽  
High Quality ◽  
Total Rna ◽  
High Concentrations ◽  
Cucumis Melo L

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

scholarly journals Patients Admitted for Variant Alpha COVID-19 Have Poorer Outcomes than Those Infected with the Old Strain

2021 ◽  
Vol 10 (16) ◽  
pp. 3550
Author(s):  
Matteo Vassallo ◽  
Sabrina Manni ◽  
Camille Klotz ◽  
Roxane Fabre ◽  
Paola Pini ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Inflammatory Markers ◽  
Clinical Characteristics ◽  
Clinical Presentation ◽  
Severe Pneumonia ◽  
Demographic Parameters ◽  
Background Characteristics ◽  
Risk Of Death ◽  
Mean Delay ◽  
Diabetes And Obesity

Objectives: The variant alpha COVID-19 rapidly spread across Europe in early 2021. While this variant’s increased infectivity has been proven, little is known of its clinical presentation and outcomes compared to the old strain. Methods: We identified patients admitted to the Cannes General Hospital for variant alpha-related COVID-19 infection from January to April 2021. Their main demographic parameters, inflammatory markers and clinical characteristics were recorded. Patients admitted from October to December 2020 for 20E (EU1) COVID-19 were selected as controls. Differences between groups were analyzed. Results: We included 157 patients (mean age 73 years; 58% men; mean delay of symptoms 6.9 days). Comorbidities were present in 92% (mainly hypertension, diabetes and obesity or overweight). The prevalence of comorbidities did not differ between groups. In 28% of cases, patients either died or required transfer to the Intensive Care Unit (ICU). The cause of death or of transfer to the ICU was presumably associated with severe pneumonia. Variant alpha COVID-19 had 3.8-fold higher risk of death or transfer to the ICU compared to the old strain. Discussion: Patients infected with variant alpha COVID-19, despite similar background characteristics, had a higher risk of unfavorable outcomes than those infected with the old strain, suggesting increased virulence related to this variant.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals Pathways from marine protected area design and management to ecological success

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1424 ◽  
Author(s):  
Murray A. Rudd
Keyword(s):  
Marine Protected Area ◽  
Theoretic Approach ◽  
Negative Results ◽  
Complex Interactions ◽  
General Terms ◽  
Ecological Performance ◽  
Ecological Isolation ◽  
Ecological Success ◽  
The One

Using an international dataset compiled from 121 sites in 87 marine protected areas (MPAs) globally (Edgar et al., 2014), I assessed how various configurations of design and management conditions affected MPA ecological performance, measured in terms of fish species richness and biomass. The set-theoretic approach used Boolean algebra to identify pathways that combined up to five ‘NEOLI’ (No-take,Enforced,Old,Large,Isolated) conditions and that were sufficient for achieving positive, and negative, ecological outcomes. Ecological isolation was overwhelming the most important condition affecting ecological outcomes butOldandLargewere also conditions important for achieving high levels of biomass among large fishes (jacks, groupers, sharks). Solution coverage was uniformly low (<0.35) for all models of positive ecological performance suggesting the presence of numerous other conditions and pathways to ecological success that did not involve the NEOLI conditions. Solution coverage was higher (>0.50) for negative results (i.e., the absence of high biomass) among the large commercially-exploited fishes, implying asymmetries in how MPAs may rebuild populations on the one hand and, on the other, protect against further decline. The results revealed complex interactions involving MPA design, implementation, and management conditions that affect MPA ecological performance. In general terms, the presence of no-take regulations and effective enforcement were insufficient to ensure MPA effectiveness on their own. Given the central role of ecological isolation in securing ecological benefits from MPAs, site selection in the design phase appears critical for success.

scholarly journals Direct-RT-qPCR Detection of SARS-CoV-2 without RNA Extraction as Part of a COVID-19 Testing Strategy: From Sample to Result in One Hour

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 605 ◽  
Author(s):  
Eva Kriegova ◽  
Regina Fillerova ◽  
Petr Kvapil
Keyword(s):  
High Throughput Screening ◽  
High Speed ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
Ease Of Use ◽  
Diagnostic Tools ◽  
Heat Inactivation

Due to the lack of protective immunity in the general population and the absence of effective antivirals and vaccines, the Coronavirus disease 2019 (COVID-19) pandemic continues in some countries, with local epicentres emerging in others. Due to the great demand for effective COVID-19 testing programmes to control the spread of the disease, we have suggested such a testing programme that includes a rapid RT-qPCR approach without RNA extraction. The Direct-One-Step-RT-qPCR (DIOS-RT-qPCR) assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one hour while maintaining the high sensitivity and specificity required of diagnostic tools. This optimised protocol allows for the direct use of swab transfer media (14 μL) without the need for RNA extraction, achieving comparable sensitivity to the standard method that requires the time-consuming and costly step of RNA isolation. The limit of detection for DIOS-RT-qPCR was lower than seven copies/reaction, which translates to 550 virus copies/mL of swab. The speed, ease of use and low price of this assay make it suitable for high-throughput screening programmes. The use of fast enzymes allows RT-qPCR to be performed under standard laboratory conditions within one hour, making it a potential point-of-care solution on high-speed cycling instruments. This protocol also implements the heat inactivation of SARS-CoV-2 (75 °C for 10 min), which renders samples non-infectious, enabling testing in BSL-2 facilities. Moreover, we discuss the critical steps involved in developing tests for the rapid detection of COVID-19. Implementing rapid, easy, cost-effective methods can help control the worldwide spread of the COVID-19 infection.

Graphene Oxide Conjugated Magnetic Beads for RNA Extraction

2017 ◽  
Vol 12 (15) ◽  
pp. 1883-1888 ◽  
Author(s):  
Xuan-Hung Pham ◽  
Ahruem Baek ◽  
Tae Han Kim ◽  
Sang Hun Lee ◽  
Won-Yeop Rho ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Magnetic Beads ◽  
Rna Extraction

scholarly journals STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Robbert Boudewijns ◽  
Hendrik Jan Thibaut ◽  
Suzanne J. F. Kaptein ◽  
Rong Li ◽  
Valentijn Vergote ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Severe Pneumonia ◽  
Neutrophil Infiltration ◽  
Innate Immune ◽  
Micro Ct ◽  
Virus Dissemination ◽  
The One ◽  
Interferon Responses ◽  
New Strategies ◽  
Severe Lung

AbstractEmergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

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