scholarly journals Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene AllplexTM 2019-nCoV assay

2020 ◽  
Author(s):  
Wesley Freppel ◽  
Natacha Merindol ◽  
Fabien Rallu ◽  
Marco Bergevin
Keyword(s):  
Detection Rate ◽  
Rna Extraction ◽  
Free Water ◽  
Proteinase K ◽  
N Gene ◽  
False Negatives ◽  
Infected People ◽  
Planetary Scale ◽  
Testing Method ◽  
Viral Genes

Abstract The fight against the COVID-19 pandemic has created an urgent need to detect and isolate infected people. The challenge for clinical laboratories has been finding a high throughput, cheap, and efficient testing method in the context of extraction reagent shortages on a planetary scale. To answer this need, we studied SARS-CoV-2 detection in nasopharyngeal swabs stored in UTM (Universal Transport Media) or RNAse-free water by rRT-PCR with the Seegene Allplex TM 2019-nCoV assay without RNA extraction. Optimal results were obtained with 1/2 dilution for swabs in RNAse free water (30/30 detected) and 1/5 dilution for swabs in UTM (29/30 detected) followed by thermal lysis. In addition, a proteinase K (PK) treatment allows a significant reduction of invalid results and increases sensitivity for detection of low viral load specimens. In a panel of 90 known positives with all 3 viral genes present and N gene Ct values from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a panel of 60 low positives with only the N gene detectable at Ct values > 30, the detection rate was 76.7% with PK vs 53.3% without it and the invalid rate fell off from 18.3% to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, however false negatives become frequent above this range. Finally, we show that swabs should be stored at -70 o C rather than 4 o C when testing cannot be performed within 72 hours of collection when laboratories are overwhelmed.

scholarly journals Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Wesley Freppel ◽  
Natacha Merindol ◽  
Fabien Rallu ◽  
Marco Bergevin
Keyword(s):  
Detection Rate ◽  
High Efficiency ◽  
False Negative ◽  
Rna Extraction ◽  
Free Water ◽  
Cost Savings ◽  
Global Scale ◽  
Proteinase K ◽  
N Gene ◽  
Infected People

Abstract Background The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. Results Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at − 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. Conclusion We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

scholarly journals Critical Aspects Concerning the Development of a Pooling Approach for SARS-CoV-2 Diagnosis Using Large-Scale PCR Testing

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 902
Author(s):  
Daniel Cruceriu ◽  
Oana Baldasici ◽  
Loredana Balacescu ◽  
Stefana Gligor-Popa ◽  
Mirela Flonta ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Diagnostic Assay ◽  
Assay Sensitivity ◽  
Infected People ◽  
Multiple Parameters ◽  
Accurate Procedure ◽  
Manual Methods ◽  
Pooling Strategy ◽  
Critical Aspects

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.

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 One-step RNA extraction for RT-qPCR detection of 2019-nCoV

2020 ◽  
Author(s):  
Monica Sentmanat ◽  
Evguenia Kouranova ◽  
Xiaoxia Cui
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Virus Transmission ◽  
Extraction Methods ◽  
Viral Rna ◽  
Taqman Probe ◽  
N Gene ◽  
Structural Protein ◽  
Diagnostic Panel ◽  
Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

Hypersensitivity to Tobacco Mosaic Virus in N’-Gene Hosts: Which Viral Genes are Involved?

1990 ◽  
pp. 345-359 ◽  
Author(s):  
K. W. Mundry ◽  
W. Schaible ◽  
M. Ellwart-Tschürtz ◽  
H. Nitschko ◽  
C. Hapke
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
N Gene ◽  
Viral Genes

scholarly journals Detection and partial characterization of an isolate of Groundnut ringspot virus in Solanum sessiliflorum

2002 ◽  
Vol 27 (3) ◽  
pp. 249-253 ◽  
Author(s):  
ALESSANDRA J. BOARI ◽  
EUNIZE MACIEL-ZAMBOLIM ◽  
DOUGLAS D. LAU ◽  
GAUS S. A. LIMA ◽  
ELLIOT W. KITAJIMA ◽  
...  
Keyword(s):  
Amazon Basin ◽  
Rna Extraction ◽  
Northern Region ◽  
Ringspot Virus ◽  
N Gene ◽  
Electron Microscopic ◽  
Systemic Necrosis ◽  
Thin Sections ◽  
Local Lesions ◽  
Stem Necrosis

The cubiu (Solanum sessiliflorum) fruit, originating in the Amazon basin, is commonly used in that region for food, medicine, and cosmetics. In an experimental culture of cubiu, in order to evaluate its adaptation to conditions in the Northern region of the state of Rio de Janeiro, it was observed plants with mosaic symptoms. A cubiu plant was collected and analyzed to identify the etiological agent. After mechanical passage through a local lesion host, a host range test was performed. The virus induced chlorotic local lesions in Chenopodium quinoa, necrotic local lesions in Gomphrena globosa, mosaic in S. sessiliflorum, leaf and stem necrosis in tomato (Lycopersicon esculentum) 'Rutgers', mosaic and leaf distortion in Datura stramonium and Physalis floridana, and necrotic local lesions followed by systemic necrosis and plant death in four Nicotiana species. Electron microscopic observations of ultra thin sections from infected cubiu leaves showed the presence of spheroidal, membrane-bound particles typical of tospovirus species. Analysis of the nucleocapsid protein from concentrated virus particles indicated the presence of a 28 kDa protein. RT-PCR was performed after total RNA extraction from infected IPA-6 tomato leaves. A fragment of approximately 0,8 kbp corresponding to the N gene was amplified, cloned and sequenced. The N protein from the cubiu isolate was 95% homologous to the Groundnut ringspot virus (GRSV) protein, and no more than 85% homologous to those from Zucchini lethal chlorosis virus (ZLCV) and Chrysanthemun stem necrosis virus (CSNV), Tomato spotted wilt virus (TSWV), and Tomato chlorotic spot virus (TCSV). This is the first report of the occurrence of GRSV (or any other plant virus) in cubiu.

scholarly journals Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR

2018 ◽  
Vol 30 (6) ◽  
pp. 807-812 ◽  
Author(s):  
Ashley C. Weiser ◽  
Korakrit Poonsuk ◽  
Sarah A. Bade ◽  
Phillip C. Gauger ◽  
Marisa Rotolo ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
False Negative ◽  
Rna Extraction ◽  
Free Water ◽  
Negative Control ◽  
Respiratory Syndrome Virus ◽  
Sample Handling ◽  
Oral Fluids

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate–phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

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.

Automatic crater detection over the Jezero crater area from HiRISE imagery

2020 ◽  
Author(s):  
Konstantinos Servis ◽  
Anthony Lagain ◽  
Gretchen Benedix ◽  
David Flannery ◽  
Chris Norman ◽  
...  
Keyword(s):  
Detection Rate ◽  
False Negative ◽  
Detection Algorithm ◽  
Impact Craters ◽  
Reference Points ◽  
Training Dataset ◽  
False Detection ◽  
Planetary Scale ◽  
Area Of Interest ◽  
Crater Detection

&lt;p&gt;Impact craters are used to determine the ages of planetary surfaces. Absolute dating of meteorites or in situ geochronology provide a few essential reference points, but these techniques are rare and not yet applicable at the planetary scale. Therefore, impact crater counting techniques will remain the major tool to decipher planetary surface history. This approach requires a tedious mapping and morphological inspection of a large number of circular features to distinguish true and primary impact craters. The most complete database of Martian craters includes a catalog of more than 384,000 impact structures larger than 1 km in diameter. This database is considered to be complete for this diameter range. A requirement to determine young surface ages on Mars must include smaller impact craters, typically a hundred meters in diameter, found on the area of interest.&lt;/p&gt;&lt;p&gt;To access to the crater population of this size range at a planetary scale we built a Crater Detection Algorithm (CDA) trained on THEMIS images where impact craters larger than 1 km from the Robbins &amp; Hynek database have been identified. Our model offer a true detection rate of 0.9. We then applied our CDA on the global CTX mosaic within the &amp;#177;45&amp;#186; latitudinal band leading to ~17 million of detection &gt;100m in diameter.&lt;/p&gt;&lt;p&gt;The ultimate goal of our work is now to automatically compile smaller impact craters (5m&lt;D&lt;100m) visible on HiRISE imagery dataset offering a resolution of 25cm/px. We trained our algorithm on a part of the HiRISE mosaic (NASA/JPL/MSSS/The Murray Lab) covering a part of the Jezero crater (E77-5_N18_0) where 1650 craters have been manually identified. A portion of this population of craters has then be selected in order to be sure to include the most confident impact features in the training dataset, finally resulting to 1624 craters over this entire image.&lt;/p&gt;&lt;p&gt;Our model has been applied over the entire HiRISE mosaic covering the Jezero crater where more than 27,298 craters &gt;3m have been detected. In order to validate our results, we compared the detection obtained on 30 tiles of 960px x 960px randomly chosen on a part of the mosaic (E77-25_N18-25) which have not been included into the training dataset with a manual identification, thus constituting the ground truth. For this purpose, we decided to categorize each tile according to the type of terrain mostly represented on each of them: rocky terrain, smooth terrain and dunes fields. We have also specified when the image exhibited some vertical stripes leading to the fourth category.&lt;/p&gt;&lt;p&gt;On rocky and smooth terrains, the CDA produce very good results: only 5% of detection on the average are false detection and 16% of craters on average have not been detected by the CDA. However, the CDA is less efficient on dune fields since 35% of detection are false detection and 15% of craters have not been identified. Finally, images exhibiting some vertical stripes significantly decrease the detection rate of the CDA since 56% of detection are false negative and 20% of craters have not been detected.&lt;/p&gt;

scholarly journals Comparison Between a Standard and SalivaDirect RNA Extraction Protocol for Molecular Diagnosis of SARS-CoV-2 Using Nasopharyngeal Swab and Saliva Clinical Samples

2021 ◽  
Vol 9 ◽  
Author(s):  
Sofía N. Rodríguez Flores ◽  
Luis Mario Rodríguez-Martínez ◽  
Bernardita L. Reyes-Berrones ◽  
Nadia A. Fernández-Santos ◽  
Elthon J. Sierra-Moncada ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Assay Performance ◽  
Two Samples ◽  
Oropharyngeal Swab

During the COVID-19 pandemic, a certified laboratory of Tamaulipas, Mexico has processed over 100,000 samples of COVID-19 suspected patients, working a minimum of 100 tests daily. Thus, it would be beneficial for such certified laboratories nationwide to reduce the time and cost involved in performing the diagnosis of COVID-19, from sample collection, transportation to local lab, processing of samples, and data acquisition. Here, 30 nasopharyngeal swab and saliva samples from the same COVID-19 individuals were assessed by a standard nucleic acid extraction protocol, including protein lysis with proteinase K followed by binding to column, washing, and elution, and by the SalivaDirect protocol based on protein lysis, skipping the other steps to reduce processing time and costs. The genomic RNA was amplified using a SARS-CoV-2 Real-Time PCR kit. A variation (P &gt; 0.05) in the 95% CIs = 72.6%–96.7% was noted by using the SalivaDirect protocol and saliva samples (sensitivity of 88.2%) in comparison to those of standard protocol with oropharyngeal swab samples (95% CIs = 97.5%–100%; sensitivity of 100%) as reported elsewhere. However, when using nasopharyngeal swab samples in the SalivaDirect protocol (sensitivity of 93.6%; 95% CIs = 79.2%–99.2%), it was in concordance (P &lt; 0.05) with those of the standard one. The logical explanation to this was that two samples with Ct values of 38, and 40 cycles for gene E produced two false negatives in the SalivaDirect protocol in relation to the standard one; thus, there was a reduction of the sensitivity of 6.4% in the overall assay performance.

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