scholarly journals An optimised protocol for detection of SARS-CoV-2 in stool

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tianqi Li ◽  
Enriqueta Garcia-Gutierrez ◽  
Daniel A. Yara ◽  
Jacob Scadden ◽  
Jade Davies ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Procedure ◽  
Nasopharyngeal Swab ◽  
Clinical Practices ◽  
Oral Transmission ◽  
Viral Particles ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Reliable Methods

Abstract Background SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples. Results Stool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool. Conclusions Here we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.

scholarly journals An optimised protocol for detection of SARS-CoV-2 in stool

2021 ◽  
Author(s):  
T Li ◽  
E Garcia-Gutierrez ◽  
J Scadden ◽  
J Davies ◽  
C Hutchins ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Procedure ◽  
Clinical Practices ◽  
Faecal Microbiota ◽  
Chain Reaction ◽  
Oral Transmission ◽  
Viral Particles ◽  
Stool Samples ◽  
Polymerase Chain

AbstractAimSARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples.MethodsStool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Debris was removed via centrifugation and supernatants were concentrated by ultrafiltration. RNA was then extracted from the concentrated material using a commercial kit and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes.ResultsThe RNA extraction procedure we used allowed the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool.ConclusionsHere we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as faecal microbiota transplant (FMT).

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals RT-RC-PCR: a novel and highly scalable next-generation sequencing method for simultaneous detection of SARS-COV-2 and typing variants of concern

2021 ◽  
Author(s):  
Christopher J Mattocks ◽  
Daniel Ward ◽  
Deborah JG Mackay
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Assay Method ◽  
Nasopharyngeal Swab ◽  
Qualitative Detection ◽  
Reverse Complement ◽  
Sequencing Method ◽  
Polymerase Chain ◽  
The Uk

We describe a novel assay method: reverse-transcription reverse-complement polymerase chain reaction (RT-RC-PCR), which rationalises reverse transcription and NGS library preparation into a single closed tube reaction. By simplifying the analytical process and cross-contamination risks, RT-RC-PCR presents disruptive scalability and economy while using NGS and LIMS infrastructure widely available across health service, institutional and commercial laboratories. We present a validation of RT-RC-PCR for the qualitative detection of SARS-CoV-2 RNA by NGS. The limit of detection is comparable to real-time RT-PCR, and no obvious difference in sensitivity was detected between extracted nasopharyngeal swab (NPS) RNA and native saliva samples. The end point measurement of RT-RC-PCR is NGS of amplified sequences within the SARS-CoV-2 genome; we demonstrated its capacity to detect different variants using amplicons containing delH69-V70 and N501Y, both of which emerged in the UK Variant of Concern B.1.1.7 in 2020. In summary, RT-RC-PCR has potential to facilitate accurate mass testing at disruptive scale and cost, with concurrent detection of variants of concern.

scholarly journals A school outbreak of Norwalk-like virus: evidence for airborne transmission

2003 ◽  
Vol 131 (1) ◽  
pp. 727-736 ◽  
Author(s):  
P. J. MARKS ◽  
I. B. VIPOND ◽  
F. M. REGAN ◽  
K. WEDGWOOD ◽  
R. E. FEY ◽  
...  
Keyword(s):  
Odds Ratio ◽  
Adjusted Odds Ratio ◽  
Nucleotide Sequence Analysis ◽  
Chain Reaction ◽  
Epidemiological Features ◽  
Positive Stool ◽  
Viral Particles ◽  
Stool Samples ◽  
Polymerase Chain ◽  
The Times

An outbreak of gastroenteritis affected a school attended by children aged 4–11 years. Epidemiological features suggested this was due to Norwalk-like virus (NLV) and this was confirmed by polymerase chain reaction (PCR). Nucleotide sequence analysis of the PCR amplicons revealed identical strains in all five positive stool samples. Pupils were significantly more likely to become ill following an episode of vomiting within their classroom (adjusted odds ratio 4·1, 95% CI 1·8–9·3). The times from exposure to illness were consistent with direct infection from aerosolized viral particles where exposure to vomiting was high.Cleaning with quaternary ammonium preparations made no impact on the course of the outbreak. However, the outbreak stopped after the school closed for 4 days and was cleaned using chlorine-based agents. This study confirms the importance of vomiting in the transmission of NLV and provides evidence that direct infection with aerosolized viral particles occurs.

scholarly journals A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing

2020 ◽  
Author(s):  
Devon Chandler-Brown ◽  
Anna M. Bueno ◽  
Oguzhan Atay ◽  
David S. Tsao
Keyword(s):  
Test Performance ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Unmet Need ◽  
Molecular Diagnostic ◽  
Sequencing Data ◽  
Viral Particles ◽  
The Us ◽  
Sequencing Data Analysis

AbstractThere is currently an urgent unmet need to increase coronavirus disease 2019 (COVID-19) testing capability to effectively respond to the COVID-19 pandemic. However, the current shortage in RNA extraction reagents as well as limitations in qPCR protocols have resulted in bottlenecks in testing capacity. Herein, we describe a novel molecular diagnostic for COVID-19 based on Sanger sequencing. This assay uses the addition of a frame-shifted spike-in, a modified PCR master mix, and custom Sanger sequencing data analysis to detect and quantify SARS-CoV-2 RNA at a limit of detection comparable to existing qPCR-based assays, at 10-20 genome copy equivalents. Crucially, our assay was able to detect SARS-CoV-2 RNA from viral particles suspended in transport media that was directly added to the PCR master mix, suggesting that RNA extraction can be skipped entirely without any degradation of test performance. Since Sanger sequencing instruments are widespread in clinical laboratories and commonly have built-in liquid handling automation to support up to 3840 samples per instrument per day, the widespread adoption of qSanger COVID-19 diagnostics can unlock more than 1,000,000 tests per day in the US.

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab and saliva patient samples

2020 ◽  
Author(s):  
Dawn M Dudley ◽  
Christina M. Newman ◽  
Andrea M Weiler ◽  
Mitchell D. Ramuta ◽  
Ceclia G. Shortreed ◽  
...  
Keyword(s):  
Infectious Virus ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Lower Sensitivity ◽  
Acid Extraction ◽  
Short Supply ◽  
Screening Programs ◽  
Qrt Pcr ◽  
The Us

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed samples, albeit with approximately 100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is sufficient to detect samples from which infectious virus can be cultured. Therefore, samples that test positive in this assay contain levels of virus that are most likely to perpetuate transmission. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR and we provide a revised method to work directly with saliva as starting material. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2020 ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Transport ◽  
Extraction Step ◽  
Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

scholarly journals Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing.

2020 ◽  
Author(s):  
Nikhil Shri Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Arvind Kothandaraman ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Performance Metrics ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Limited Resource ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Pcr Assay

The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA-extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65°C for 15 minutes; 80°C for 5 minutes; 90°C for 5 minutes or 80°C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90°C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be ~40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.

scholarly journals Ultrasensitive measurement of both SARS-CoV2 RNA and serology from saliva

2021 ◽  
Author(s):  
Dmitry Ter-Ovanesyan ◽  
Tal Gilboa ◽  
Roey Lazarovits ◽  
Alexandra Rosenthal ◽  
Xu G. Yu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Saliva Sample ◽  
Rna Extraction ◽  
Viral Rna ◽  
Nasopharyngeal Swab ◽  
Rna Detection ◽  
Viral Particles ◽  
Highly Sensitive ◽  
Nasal Swabs ◽  
Serology Test

AbstractTests for COVID-19 generally measure SARS-CoV2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and serology from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive serology test based on Single Molecule Array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with serology identifies more patients as infected than either method alone. Our results suggest the utility of combining viral RNA and serology testing from saliva, a single easily accessible biofluid.

scholarly journals Multiplexed and extraction-free amplification for simplified SARS-CoV-2 RT-PCR tests

2020 ◽  
Author(s):  
Samantha A. Byrnes ◽  
Ryan Gallagher ◽  
Amy Steadman ◽  
Crissa Bennett ◽  
Rafael Rivera ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
Rapid Onset ◽  
Technical Expertise ◽  
Sample Collection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rna Purification ◽  
Polymerase Chain ◽  
Multiple Challenges

The rapid onset of the global COVID-19 pandemic has led to multiple challenges for accurately diagnosing the infection. One of the main bottlenecks for COVID-19 detection is reagent and material shortages for sample collection, preservation, and purification prior to testing. Currently, most authorized diagnostic tests require RNA extraction from patient samples and detection by reverse transcription polymerase chain reaction (RT-PCR). However, RNA purification is expensive, time consuming, and requires technical expertise to perform. Additionally, there have been reported shortages of the RNA purification kits needed for most tests. With these challenges in mind, we report on extraction-free amplification of SARS-CoV-2 RNA directly from patient samples. In addition, we have developed a multiplex RT-PCR using the CDC singleplex targets. This multiplex has a limit of detection of 2 copies/μL. We have demonstrated these improvements to the current diagnostic workflow, which reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

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