scholarly journals RNA Extraction Alternative Method for SARS-CoV-2 Molecular Diagnosis

2021 ◽  
Author(s):  
Elisa Bianchini ◽  
Francesco Rossignolo ◽  
Melissa Perfranceschi ◽  
Chiara Cazzin ◽  
Ronaldo Silva ◽  
...  
Keyword(s):  
Internal Control ◽  
Extraction Method ◽  
Target Genes ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Molecular Test ◽  
Control And Prevention ◽  
Set Up

Background: the devastating outbreak of COVID-19 poses serious challenges for the diagnostics laboratories, which are often facing global shortage of reagents and equipment. With the aim of increasing the diagnostic throughput for SARS-CoV-2 molecular test, the purpose of this study was to validate an additional RNA extraction method respect to those already recommended by WHO and the US Centers for Disease Control and Prevention (CDC). Methods: a new protocol for RNA extraction from nasopharyngeal swab was set up, adapting the Qiagen RNeasy 96 plate and validated on a set of 100 clinical samples analyzed in parallel by Roche-Magnapure method (already recommended by CDC guidelines). Results: the internal control and target genes analysis showed a good agreement between the two extraction methods indicating that the two methods can be considered equivalent and that the RNeasy-adapted method can be applied for the SARS-CoV-2 diagnostics. The addition of this new extraction method resulted in a throughput increase for SARS-CoV-2 molecular test of about 2000 samples/month during the initial months of the pandemic emergency in which the lack of reagents for the extraction led to an insufficient sample processing throughput of the analysis of the swabs.

scholarly journals Evaluation of the efficacy of LAMP-based SARS-CoV-2 detection with simple RNA extraction from nasopharyngeal swabs: A prospective observational study

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260732
Author(s):  
Masaki Karino ◽  
Mizuki Harada ◽  
Chihiro Yamada ◽  
Kyoko Fukuoka ◽  
Megumi Sugo ◽  
...  
Keyword(s):  
Extraction Method ◽  
Prospective Observational Study ◽  
Medical Center ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Viral Rna ◽  
Nasopharyngeal Swab ◽  
Measurement Principle ◽  
Comparison Of The Results ◽  
Reliable Testing

The Loopamp SARS-CoV-2 Detection Kit is used for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loop-mediated isothermal amplification (LAMP) is based on a measurement principle that can be used with a relatively simple device. Detection using this kit requires viral RNA extraction from samples with the QIAGEN QIAamp Viral Mini Kit (QIAGEN extraction) or the Loopamp Viral RNA Extraction Kit (Eiken extraction), which are recommended by the manufacturer. However, the efficacy of LAMP-based SARS-CoV-2 detection using these extraction methods has not been compared. In this study, we aimed to compare the results of genome extraction and detection from nasopharyngeal swab samples using the QIAGEN and Eiken extraction kits. The present study involved patients who presented to the Rinku General Medical Center with suspected COVID-19 (25 positive and 26 negative cases). A comparison of the results obtained using each extraction method with those obtained via PCR showed that the positive, negative, and overall concordance rates between QIAGEN extraction and PCR were 96.0% (24/25 samples), 100% (26/26), and 98.0% (50/51; κ = 0.96, 95% CI = 0.69–1.00), respectively. Results with Eiken extraction were also favorable, with positive, negative, and overall concordance rates of 88.0% (22/25), 100% (26/26), and 94.1% (48/51; κ = 0.88, 95% CI = 0.61–1.00), respectively. Favorable results were obtained using both QIAGEN and Eiken extraction kits. Since Eiken extraction can be completed in a few minutes, it enables prompt and reliable testing for SARS-CoV-2 detection.

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 Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 240
Author(s):  
Alison Woodward ◽  
Alina Pandele ◽  
Salah Abdelrazig ◽  
Catherine A. Ortori ◽  
Iqbal Khan ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Extraction Method ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Serum Starvation ◽  
Extraction Techniques ◽  
Metabolic Genes ◽  
Dual Extraction

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

Rapid Virus Detection Procedure for Molecular Tracing of Shellfish Associated with Disease Outbreaks

2007 ◽  
Vol 70 (4) ◽  
pp. 967-974 ◽  
Author(s):  
ANA MARIA de RODA HUSMAN ◽  
FROUKJE LODDER-VERSCHOOR ◽  
HAROLD H. J. L. van den BERG ◽  
FRANÇOISE S. LE GUYADER ◽  
HILDE van PELT ◽  
...  
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Virus Detection ◽  
Disease Outbreaks ◽  
Extraction Methods ◽  
Virus Rna ◽  
Pathogenic Viruses ◽  
Rna Extraction Methods ◽  
Digestive Glands ◽  
Outbreak Situation

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III–spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

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 > 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 < 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.

Development of an automated RNA-based nuclease protection assay for the detection of expressed EML-ALK fusions, EGFR, and KRAS mutations in NSCLC.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22185-e22185
Author(s):  
Matthew Rounseville ◽  
Krishna Maddula ◽  
Bruce Seligmann ◽  
Ihab Botros ◽  
Vijay Modur
Keyword(s):  
Kras Mutation ◽  
Target Genes ◽  
Rna Extraction ◽  
Point Mutations ◽  
Gene Mutations ◽  
Extraction Methods ◽  
Protection Assay ◽  
Kras Mutations ◽  
Nuclease Protection Assay

e22185 Background: The management of patients for NSCLC requires appropriate molecular pathology assessment to determine treatment. However current methods require the use of more than one technology including sequencing for EGFR and KRAS mutation and in situ hybridization for EML-ALK fusion, resulting in slow turnaround, and technical as well as logistical complexities. Here, we report the development of an automated microarray based quantitative nuclease protection assay that determines gene fusions, deletions and point mutations in target genes such as EML4/ALK, EGFR and KRAS from expressed mRNA directly from FFPE tissue without prior RNA extraction. Methods: We designed mutation and fusion specific probes for nuclease protection and measured the sensitivity, specificity, and reproducibility of gene mutations and fusions using in vitro transcribed RNA and cell lysates known to contain the target mutations. In addition to synthetic samples, we also screened 88 lung cancer specimen for EML-ALK fusions. Results: Mutations in KRAS (G12D, G12C, G12V); EGFR (D761Y, T790M, L858R); EML-ALK fusions (v1 v2, v3a, v3b-3, v4, v5a, v5b-3 and v6) were detected at concentrations of 3 copies/cell in 15,000 cells even when present as low as 5% of wild type IVT RNA. The sensitivity corresponds to an amount of FFPE sample ranging from 0.3 to 0.6 square cm of a 5mm FFPE section per replicate assay making it suitable for needle biopsy specimen. Six samples with probable EML4-ALK fusions were identified in the 88 adenomatous NSCLC samples and will be subjected to further confirmation. Additional clinical validation of all the assays will be presented in our poster. Conclusions: An automated, multiplexed, EGFR and KRAS mutation and EML-ALK gene fusion from expressed mRNA is feasible. The assay can be run directly on FFPE sections, does not require RNA extraction or amplification, and is completed within 24 hours.

scholarly journals Molecular detection of SARS-CoV-2 using a reagent-free approach

2020 ◽  
Author(s):  
Ronan Calvez ◽  
Andrew Taylor ◽  
Leo Calvo-Bado ◽  
Colin Fink
Keyword(s):  
Internal Control ◽  
Extraction Method ◽  
Molecular Detection ◽  
Rna Extraction ◽  
Heat Processing ◽  
Virus Transport ◽  
Validation Process ◽  
Treatment Conditions ◽  
Different Temperatures ◽  
Alternative Approaches

AbstractShortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Fomsgaard et al 20205 recently presented results using heat-processing of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost.Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this technique. We first investigated various treatments using different temperatures, incubation times and sample volumes based on the above study to optimise the heat-treatment conditions. Although the initial data confirmed the published results, further investigations revealed unexpected inhibitory properties of some commonly used virus transport media (VTMs) on some commercially available RT-qPCR mixes, emphasising the critical importance of a thorough validation process to determine the most adapted reagents to be used depending on the sample types to be tested.In conclusion, although the method works, with very consistent Ct values and an excellent sensitivity when compared to a conventional RNA extraction method, it is critical to include an internal control to check each sample for potential inhibition.

scholarly journals Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247792
Author(s):  
Valeria Genoud ◽  
Martin Stortz ◽  
Ariel Waisman ◽  
Bruno G. Berardino ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Standard Technique ◽  
Proteinase K ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Nasopharyngeal Swab ◽  
Reverse Transcription Pcr ◽  
Extraction Step ◽  
Commercial Kits ◽  
Inexpensive Procedure

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

Environmental Swabs as a Tool in Norovirus Outbreak Investigation, Including Outbreaks on Cruise Ships

2009 ◽  
Vol 72 (1) ◽  
pp. 111-119 ◽  
Author(s):  
INGEBORG L. A. BOXMAN ◽  
REMCO DIJKMAN ◽  
NATHALIE A. J. M. te LOEKE ◽  
GEKE HÄGELE ◽  
JEROEN J. H. C. TILBURG ◽  
...  
Keyword(s):  
Detection Rate ◽  
Clinical Symptoms ◽  
Rna Extraction ◽  
Viral Rna ◽  
Food Preparation ◽  
Clinical Samples ◽  
Viral Gastroenteritis ◽  
Clinical Specimens ◽  
Cruise Ships ◽  
Set Up

In this study, we investigated whether environmental swabs can be used to demonstrate the presence of norovirus in outbreak settings. First, a procedure was set up based on viral RNA extraction using guanidium isothiocyanate buffer and binding of nucleic acids to silica. Subsequently, environmental swabs were taken at 23 Dutch restaurants and four cruise ships involved in outbreaks of gastroenteritis. Outbreaks were selected based on clinical symptoms consistent with viral gastroenteritis and time between consumption of suspected food and onset of clinical symptoms (>12 h). Norovirus RNA was demonstrated by real-time reverse transcriptase PCR in 51 of 86 (59%) clinical specimens from 12 of 14 outbreaks (86%), in 13 of 90 (14%) food specimens from 4 of 18 outbreaks (22%), and in 48 of 119 (40%) swab specimens taken from 14 of 27 outbreaks (52%). Positive swab samples agreed with positive clinical samples in seven outbreaks, showing identical sequences. Furthermore, norovirus was detected on swabs taken from kitchen and bathroom surfaces in five outbreaks in which no clinical samples were collected and two outbreaks with negative fecal samples. The detection rate was highest for outbreaks associated with catered meals and lowest for restaurant-associated outbreaks. The use of environmental swabs may be a useful tool in addition to testing of food and clinical specimens, particularly when viral RNA is detected on surfaces used for food preparation.

scholarly journals Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Duane S. Juang ◽  
Terry D. Juang ◽  
Dawn M. Dudley ◽  
Christina M. Newman ◽  
Molly A. Accola ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Large Scale ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Total Analysis System ◽  
Total Analysis ◽  
Analysis System ◽  
Disease Testing

AbstractThe COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

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