scholarly journals Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Aravind Natarajan ◽  
Alvin Han ◽  
Soumaya Zlitni ◽  
Erin F Brooks ◽  
Summer E Vance ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Viral Rna ◽  
Detection Methods ◽  
Common Condition ◽  
Viral Pathogen ◽  
Standardized Protocol ◽  
Patient Health ◽  
Stool Samples ◽  
Key Features ◽  
Rna Concentration

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

scholarly journals Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aravind Natarajan ◽  
Alvin Han ◽  
Soumaya Zlitni ◽  
Erin F. Brooks ◽  
Summer E. Vance ◽  
...  
Keyword(s):  
Respiratory Infection ◽  
Viral Rna ◽  
Viral Pathogen ◽  
Patient Health ◽  
Reliable Quantification

AbstractPatients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

scholarly journals Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Aravind Natarajan ◽  
Alvin Han ◽  
Soumaya Zlitni ◽  
Erin F. Brooks ◽  
Summer E. Vance ◽  
...  
Keyword(s):  
Respiratory Infection ◽  
Fecal Sample ◽  
Rna Extraction ◽  
Viral Rna ◽  
Bovine Coronavirus ◽  
Standardized Protocol ◽  
Specialized Equipment ◽  
Ease Of Access

Abstract COVID-19 patients shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. In our benchmarking study, we recommend a standardized protocol for the preservation, extraction and detection of viral RNA from stool. This protocol includes a preservative, viral RNA extraction steps, and PCR-based quantification methods to maximize yield and detection of SARS-CoV-2 RNA. Our protocol takes advantage of commercially available reagents and equipment to maximize ease of access and consistency across studies. Additionally, we apply an attenuated bovine coronavirus vaccine as a spike-in control, and synthetic RNA standards to improve standardization and reliability of the assay. While we recommend both ddPCR and RT-qPCR-based assays, we acknowledge that ddPCR may be prohibitively expensive due to the necessity of specialized equipment and reagents. This protocol was developed with a focus on SARS-CoV-2 RNA, but may apply to other coronaviruses as well. We estimate that this protocol takes between 6 to 8 hours total to quantify the viral RNA load in a fecal sample.

scholarly journals It’s the Little Things (in Viral RNA)

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Jiří František Potužník ◽  
Hana Cahová
Keyword(s):  
Mass Spectrometry ◽  
Viral Rna ◽  
Detection Methods ◽  
Scientific Methodology ◽  
Rna Modifications ◽  
Chemical Modifications ◽  
Viral Protein Synthesis ◽  
Antibody Assays ◽  
Rna Export ◽  
Generation Sequencing

ABSTRACT Chemical modifications of viral RNA are an integral part of the viral life cycle and are present in most classes of viruses. To date, more than 170 RNA modifications have been discovered in all types of cellular RNA. Only a few, however, have been found in viral RNA, and the function of most of these has yet to be elucidated. Those few we have discovered and whose functions we understand have a varied effect on each virus. They facilitate RNA export from the nucleus, aid in viral protein synthesis, recruit host enzymes, and even interact with the host immune machinery. The most common methods for their study are mass spectrometry and antibody assays linked to next-generation sequencing. However, given that the actual amount of modified RNA can be very small, it is important to pair meticulous scientific methodology with the appropriate detection methods and to interpret the results with a grain of salt. Once discovered, RNA modifications enhance our understanding of viruses and present a potential target in combating them. This review provides a summary of the currently known chemical modifications of viral RNA, the effects they have on viral machinery, and the methods used to detect them.

scholarly journals Tracking SARS-CoV-2 in Sewage: Evidence of Changes in Virus Variant Predominance during COVID-19 Pandemic

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1144 ◽  
Author(s):  
Javier Martin ◽  
Dimitra Klapsa ◽  
Thomas Wilton ◽  
Maria Zambon ◽  
Emma Bentley ◽  
...  
Keyword(s):  
Viral Rna ◽  
Clinical Samples ◽  
Environmental Surveillance ◽  
Rna Sequences ◽  
Virus Variant ◽  
First Case ◽  
Low Levels ◽  
South East England ◽  
Wastewater Samples ◽  
Rna Concentration

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the ongoing coronavirus disease (COVID-19) pandemic, is frequently shed in faeces during infection, and viral RNA has recently been detected in sewage in some countries. We have investigated the presence of SARS-CoV-2 RNA in wastewater samples from South-East England between 14th January and 12th May 2020. A novel nested RT-PCR approach targeting five different regions of the viral genome improved the sensitivity of RT-qPCR assays and generated nucleotide sequences at sites with known sequence polymorphisms among SARS-CoV-2 isolates. We were able to detect co-circulating virus variants, some specifically prevalent in England, and to identify changes in viral RNA sequences with time consistent with the recently reported increasing global dominance of Spike protein G614 pandemic variant. Low levels of viral RNA were detected in a sample from 11th February, 3 days before the first case was reported in the sewage plant catchment area. SARS-CoV-2 RNA concentration increased in March and April, and a sharp reduction was observed in May, showing the effects of lockdown measures. We conclude that viral RNA sequences found in sewage closely resemble those from clinical samples and that environmental surveillance can be used to monitor SARS-CoV-2 transmission, tracing virus variants and detecting virus importations.

scholarly journals SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 863 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Müller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
N Gene ◽  
Extraction Protocol ◽  
Large Scale Testing

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Spore shedding pattern of Enterocytozoon bieneusi in asymptomatic children

2005 ◽  
Vol 54 (5) ◽  
pp. 473-476 ◽  
Author(s):  
Mathirut Mungthin ◽  
Ittisak Subrungruang ◽  
Tawee Naaglor ◽  
Pote Aimpun ◽  
Wirote Areekul ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Enterocytozoon Bieneusi ◽  
Detection Methods ◽  
Asymptomatic Group ◽  
Source Of Infection ◽  
Asymptomatic Children ◽  
Immunodeficiency Virus ◽  
Spore Shedding ◽  
Stool Samples ◽  
Stool Specimens

Stool samples from seven human immunodeficiency virus (HIV)-negative and two HIV-positive children with asymptomatic Enterocytozoon bieneusi infections were daily examined to quantify spore shedding using Gram-chromotrope staining under light microscopy. The spore shedding pattern and intensity in these children was variable. Mean spore concentrations in the stool samples from these children ranged from 2.4 × 102 to 1.2 × 105 spores per gram. Light microscopy could detect spores in stool specimens for 9–33 days, while PCR was able to detect E. bieneusi in stool specimens for 3–40 days longer. This suggests that light microscopy may not detect low levels of spore shedding. Considering that the asymptomatic group are a potential source of infection, detection methods with a higher sensitivity should be used.

Longitudinal changes in viral RNA concentration in patients with chronic hepatitis C and/or HIV infection in the absence of antiviral therapy

2013 ◽  
Vol 58 (2) ◽  
pp. 391-395 ◽  
Author(s):  
Pablo Barreiro ◽  
Pablo Labarga ◽  
José V. Fernández-Montero ◽  
Eva Poveda ◽  
Carmen de Mendoza ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Hiv Infection ◽  
Antiviral Therapy ◽  
Chronic Hepatitis C ◽  
Viral Rna ◽  
Longitudinal Changes ◽  
Rna Concentration

scholarly journals SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP

2020 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Mueller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Large Scale ◽  
Magnetic Beads ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Extraction Protocol ◽  
Large Scale Testing ◽  
Commercial Kits

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and alternative, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric RT-LAMP using N primers, as well as RT-qPCR using E gene primers showing that the here presented RNA extraction protocol can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

scholarly journals Individual variability in patterns and dynamics of fecal gluten immunogenic peptides excretion after low gluten intake

2022 ◽  
Author(s):  
Laura Coto ◽  
Carolina Sousa ◽  
Angel Cebolla
Keyword(s):  
Interindividual Variability ◽  
Elisa Test ◽  
Individual Variability ◽  
High Variability ◽  
Detection Methods ◽  
Lateral Flow Immunoassay ◽  
Gluten Free ◽  
Immunogenic Peptides ◽  
Stool Samples

Abstract Purpose Determination of Gluten Immunogenic Peptides (GIP) in feces is a direct tool for gluten exposure detection. The sensitivity of GIP detection methods for cases of unintentional low gluten intakes is unknown. We studied the interindividual variability in the kinetic of excretion under homogeneously controlled dietary conditions, and the sensitivity of fecal GIP tests after low amounts of punctual gluten ingestions. Methods Participants (n = 20) followed the same gluten-free menu for 12 days in which two separated doses of gluten (50 mg and 2 g) were ingested and all the depositions were collected. GIP from stool samples were analyzed by ELISA and lateral flow immunoassay (LFIA) tests. Results Most participants had detectable GIP after 50 mg and 2 g gluten ingestions using ELISA test (72.2% and 95%, respectively), whereas the LFIA test showed less sensitivity (22.2% and 80%, respectively). GIP were detected at higher either frequency or concentration in the range of 12–36 h after 50 mg intake, and 12–84 h after 2 g consumption. Considering this period, diagnostic sensitivity of GIP detection after a single 50 mg ingestion may be significatively increased analyzing three stool samples per individual. High variability among participants was found in the time and amount of GIP excretion; however, some individuals showed common patterns for both gluten intakes. Conclusion Sporadic gluten exposure detection may require several fecal samples to achieve level of sensitivity above 90%. Interindividual variability in the dynamic of GIP excretion may suggest patterns of gluten metabolism.

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