scholarly journals Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms

2021 ◽  
Vol 11 ◽  
Author(s):  
Ruth E. Thom ◽  
Lin S. Eastaugh ◽  
Lyn M. O’Brien ◽  
David O. Ulaeto ◽  
James S. Findlay ◽  
...  
Keyword(s):  
Heat Treatment ◽  
High Throughput ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Sample Type ◽  
Diagnostic Laboratory ◽  
Viral Pathogen ◽  
Infectious Dose ◽  
Viral Transport Medium ◽  
Lysis Buffer

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

scholarly journals Evaluation of the SARS-CoV-2 inactivation efficacy associated with buffers from three kits used on high-throughput RNA extraction platforms

2021 ◽  
Author(s):  
Ruth E Thom ◽  
Lin Eastaugh ◽  
Lyn O'Brien ◽  
David Ulaeto ◽  
James S Findlay ◽  
...  
Keyword(s):  
Heat Treatment ◽  
High Throughput ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Sample Type ◽  
Diagnostic Laboratory ◽  
Viral Pathogen ◽  
Viral Transport ◽  
Viral Transport Medium ◽  
Lysis Buffer

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QiaCube HT and the ThermoFisher Kingfisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type; SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer from the MagMax Pathogen RNA/DNA kit (ThermoFisher), used on the Kingfisher Flex, which included guanidinium isothiocycnate (GITC), a detergent, and isopropanol demonstrated a minimum inactivation efficacy of 1 x 105 TCID50/ml.  An alternative lysis buffer from the MagMax Viral/Pathogen Nucleic Acid kit (Thermofisher) also used on the Kingfisher Flex and the lysis buffer from QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QiaCube HT (both of which contained GITC and a detergent) reduced titres by 1 x 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 minutes, 68 °C) did not completely inactivate the virus, demonstrating a reduction of 1 x 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

scholarly journals Improved Sensitivity, Safety, and Rapidity of COVID-19 Tests by Replacing Viral Storage Solution with Lysis Buffer

2020 ◽  
Author(s):  
Oran Erster ◽  
Omer Shkedi ◽  
Gil Benedek ◽  
Eyal Zilber ◽  
Itay Varkovitzky ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Detection Threshold ◽  
Test Tube ◽  
Clinical Samples ◽  
Sample Collection ◽  
Standard Guideline ◽  
Viral Transport Medium ◽  
Two Samples ◽  
Lysis Buffer ◽  
And Control

AbstractConducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic.Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling.We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB).We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

scholarly journals Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0249149
Author(s):  
Oran Erster ◽  
Omer Shkedi ◽  
Gil Benedek ◽  
Eyal Zilber ◽  
Itay Varkovitzky ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Detection Threshold ◽  
Test Tube ◽  
Clinical Samples ◽  
Sample Collection ◽  
Standard Guideline ◽  
Viral Transport Medium ◽  
Two Samples ◽  
Lysis Buffer ◽  
And Control

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

scholarly journals Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 624 ◽  
Author(s):  
Boris Pastorino ◽  
Franck Touret ◽  
Magali Gilles ◽  
Lea Luciani ◽  
Xavier de Lamballerie ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Rna Extraction ◽  
Cell Culture Supernatant ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
European Standard ◽  
Lysis Buffer ◽  
Level 2 ◽  
Biosafety Level ◽  
Chaotropic Agents

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 min contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.

scholarly journals Rapid isothermal amplification and portable detection system for SARS-CoV-2

2020 ◽  
Vol 117 (37) ◽  
pp. 22727-22735 ◽  
Author(s):  
Anurup Ganguli ◽  
Ariana Mostafa ◽  
Jacob Berger ◽  
Mehmet Y. Aydin ◽  
Fu Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Alternative Pathway ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

scholarly journals Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

2015 ◽  
Vol 53 (10) ◽  
pp. 3148-3154 ◽  
Author(s):  
Sophie J. Smither ◽  
Simon A. Weller ◽  
Amanda Phelps ◽  
Lin Eastaugh ◽  
Sarah Ngugi ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Clinical Sample ◽  
Rna Extraction ◽  
Heat Treatments ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sample Type ◽  
Viral Inactivation ◽  
Blood Samples ◽  
Diagnostic Samples

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10850% tissue culture infective dose per milliliter [TCID50· ml−1]) and murine blood (EBOV concentration of 1 × 107TCID50· ml−1) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.

scholarly journals In-House, Rapid, and Low-Cost SARS-CoV-2 Spike Gene Sequencing Protocol to Identify Variants of Concern Using Sanger Sequencing

2021 ◽  
Author(s):  
Fatimah Alhamlan ◽  
Dana Bakheet ◽  
Marie Bohol ◽  
Madain Alsanea ◽  
Basma Alahaideb ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Rna Extraction ◽  
Whole Genome ◽  
Diagnostic Laboratory ◽  
Spike Gene

Background: The need for active genomic sequencing surveillance to rapidly identify circulating SARS-CoV-2 variants of concern (VOCs) is critical. However, increased global demand has led to a shortage of commercial SARS-CoV-2 sequencing kits, and not every country has the technological capability or the funds for high-throughput sequencing platforms. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol that uses supplies, equipment, and methodologic expertise available in standard molecular or diagnostic laboratories to identify circulating SARS-CoV-2 variants of concern. Methods: Sets of primers flanking the SARS-CoV-2 spike gene were designed using SARS-CoV-2 genome sequences retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID) Database and synthesized in-house. Primer specificity and final sequences were verified using online prediction analyses with BLAST. The primers were validated using 282 nasopharyngeal samples collected from patients assessed as positive for SARS-CoV-2 at the diagnostic laboratory of the hospital using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit. The patient samples were subjected to RNA extraction followed by cDNA synthesis, conventional polymerase chain reaction, and Sanger sequencing. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole genome sequencing of the same samples. Results: Sanger sequencing using the newly designed primers and next-generation whole genome sequencing of 282 patient samples indicated identical variants of concern results: 123 samples contained the alpha variant (B.1.1.7); 78, beta (B.1.351), 0, gamma (P.1), and 13, delta (B.1.617.2). Moreover, the remaining samples were non-VOC that belonged to none of these variants and had 99.97% identity with the reference genome. Only four samples had poor sequence quality by Sanger sequencing owing to a low viral count (Ct value >38). Therefore, mutation calls were >98% accurate. Conclusions: Sanger sequencing method using in-house primers is an alternative approach that can be used in facilities with existing equipment to mitigate limitations in high throughput supplies required to identify SARS-CoV-2 variants of concern during the COVID-19 pandemic. This protocol is easily adaptable for detection of emerging variants.

scholarly journals Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

2020 ◽  
Author(s):  
Julien Fassy ◽  
Caroline Lacoux ◽  
Sylvie Leroy ◽  
Latifa Noussair ◽  
Sylvain Hubac ◽  
...  
Keyword(s):  
High Throughput ◽  
Cellular Response ◽  
Rna Extraction ◽  
Reference Method ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Rna Purification ◽  
Large Populations ◽  
Reaction Volumes

AbstractThe emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several procedures, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

scholarly journals Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0243333
Author(s):  
Julien Fassy ◽  
Caroline Lacoux ◽  
Sylvie Leroy ◽  
Latifa Noussair ◽  
Sylvain Hubac ◽  
...  
Keyword(s):  
High Throughput ◽  
Cellular Response ◽  
Rna Extraction ◽  
Reference Method ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Extraction Step ◽  
Large Populations ◽  
Reaction Volumes

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

scholarly journals COVIDFast: A high-throughput and RNA extraction-free method for SARS-CoV-2 detection in swab (SwabFAST) or saliva (SalivaFAST)

2022 ◽  
Author(s):  
Yue Qiu ◽  
Ling Lu ◽  
Amanda Halven ◽  
Rachel Terrio ◽  
Sydney Yuldelson ◽  
...  
Keyword(s):  
High Throughput ◽  
Nasal Swab ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Diagnostic Process ◽  
Conventional Pcr ◽  
Extraction Step ◽  
Working Together ◽  
Bottle Neck

There is an urgent need of having a rapid, high throughput, yet accurate SARS-COV-2 PCR testing to control the COVID19 pandemic. However, the RNA extraction step in conventional PCR creates a major bottle neck in the diagnostic process. In this paper we modified the CDC COVID-19 assay and developed an RNA-extraction free RT-qPCR assay for SARS-CoV-2, i.e. COVIDFast. Depending on sample types, the assay is further divided into SwabFAST, which uses anterior nares nasal swab, and SalivaFAST, which uses saliva. By utilizing the proprietary buffer for either swab or saliva samples, the performance of SwabFAST or SalivaFAST is equivalent to RNA-extraction SARS-CoV-2 RT-qPCR in both contrived and clinical samples. The limit of detection of either assay is 4 copies/uL. We further developed a semi-automatic system, which is easy to adapt by clinical lab for implementation of a high-throughput SARS-CoV-2 test. Working together with the COVIDCheck Colorado, we have tested over 400,000 samples using COVIDFast (83.62% SwabFAST and 16.38% SalivaFAST) in less than a year, resulting in significant clinical contribution in the battle against COVID-19 during the pandemic.

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