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

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.

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

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.716436 ◽

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.

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

10.1101/2020.09.25.20201921 ◽

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.

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Development and Validation of Two In-house, Low-Cost SARS-CoV-2 Detection Assays

10.1101/2020.05.18.20105510 ◽

2020 ◽

Author(s):

Fatimah Alhamlan ◽

Ahmed Alqahtani ◽

Dana Bakheet ◽

Marie Bohol ◽

Sahar Althawadi ◽

...

Keyword(s):

Low Cost ◽

False Negative ◽

Rna Extraction ◽

Melting Curve ◽

Sybr Green ◽

Heat Processing ◽

Nasopharyngeal Swab ◽

Level 2 ◽

Biosafety Level ◽

Biosafety Level 2

Background One major challenge for detecting the virus that causes COVID19 is commercial SARSCoV2 testing kit or reagent availability. To allow every laboratory or hospital access to an inhouse assay, we developed two low cost SARSCoV2 detection assay protocols using inhouse primers and reagents equipment on hand in most biology or diagnostic laboratories a SYBR Green based RTPCR and PCR assays. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated alternative RNA extraction protocols. Methods SARSCoV2 genome sequences deposited into the GISAID database were retrieved to design and synthesize inhouse primers. Forty patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocols. Both assays used TRIzol and heat-processing techniques to extract RNA from patient samples and to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. Results The sensitivity and specificity of the primers were evaluated using samples previously confirmed positive for SARSCoV2. The positive amplicons were sequenced to confirm the results. The assay protocols were developed, and the specificity of each PCR product was confirmed using melting curve analyses. The most accurate heat processing technique for primers with short amplicon lengths was 95C for 15 mins. Of 40 samples, both the SYBR Green based quantitative RTPCR assay and the PCR assay detected SARSCoV2 target genes in 28 samples, with no false positive or false-negative results. These findings were concordant with those of the diagnostic laboratory that tested the same samples using a Rotor Gene PCR cycler with an Altona Diagnostics SARSCoV2 kit (R2=0.889). Conclusions These approaches are reliable, repeatable, specific, sensitive, simple, and low cost tools for the detection of SARSCoV2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or cost ineffective.

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CONTAIN: An open-source shipping container laboratory optimised for automated COVID-19 diagnostics

10.1101/2020.05.20.106625 ◽

2020 ◽

Author(s):

Kenneth T. Walker ◽

Matthew Donora ◽

Anthony Thomas ◽

Alexander James Phillips ◽

Krishma Ramgoolam ◽

...

Keyword(s):

Open Source ◽

Diagnostic Testing ◽

Rna Extraction ◽

Urban Environments ◽

Diagnostic Systems ◽

Molecular Assays ◽

Shipping Container ◽

Liquid Handling ◽

Level 2 ◽

Biosafety Level

AbstractThe COVID-19 pandemic has challenged diagnostic systems globally. Expanding testing capabilities to conduct population-wide screening for COVID-19 requires innovation in diagnostic services at both the molecular and industrial scale. No report to-date has considered the complexity of laboratory infrastructure in conjunction with the available molecular assays to offer a standardised solution to testing. Here we present CONTAIN. A modular biosafety level 2+ laboratory optimised for automated RT-qPCR COVID-19 testing based on a standard 40ft shipping container. Using open-source liquid-handling robots and RNA extraction reagents we demonstrate a reproducible workflow for RT-qPCR COVID-19 testing. With five OT2 liquid handlers, a single CONTAIN unit reaches a maximum daily testing capacity of 2400 tests/day. We validate this workflow for automated RT-qPCR testing, using both synthetic SARS-CoV-2 samples and patient samples from a local NHS hospital. Finally, we discuss the suitability of CONTAIN and its flexibility in a range of diagnostic testing scenarios including high-density urban environments and mobile response units.Visual abstract

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Stability of SARS-CoV-2 RNA in Viral Lysis Buffer Stored at Different Temperatures

Journal of Laboratory Physicians ◽

10.1055/s-0040-1722551 ◽

2020 ◽

Vol 12 (04) ◽

pp. 268-270

Author(s):

Nagaraj Perumal ◽

Rajeev Kumar Jain ◽

Rakesh Shrivastava ◽

Jaya Lalwani ◽

Deepti Chaurasia

Keyword(s):

Diagnostic Testing ◽

Rna Extraction ◽

Delayed Diagnosis ◽

Viral Rna ◽

Molecular Diagnostic ◽

Viral Lysis ◽

Specimen Processing ◽

Different Temperatures ◽

Lysis Buffer ◽

The Stability

Abstract Objectives The present COVID-19 pandemic resulted in an increased need for molecular diagnostic testing. Delay in the specimen processing and suboptimal storage of suspected samples in laboratories leads to degradation of SARS-CoV-2 viral RNA. Viral lysis buﬀers from RNA extraction kits have the potential to stabilize RNA. Hence, this study aimed to investigate the stability of SARS-CoV-2 RNA in viral lysis buffer at different temperatures and time periods. Materials and Methods Aliquots of samples with known SARS-CoV-2 RNA were processed in viral lysis buﬀers simultaneously, stored separately at 2 to 8°C and 22 to 28°C for 24 hours, 48 hours and 72 hours. SARS-CoV-2 viral RNA was extracted from each aliquot and analyzed using multiplex real-time PCR. Results SARS-CoV-2 RNA in samples placed in viral lysis buffer was stable for 48 hours at both 2 to 8°C and 22 to 28°C temperatures. Slight decline in the viral RNA quantity was found on aliquots tested after 48 hours of both the temperatures. Conclusions Viral lysis buffer maintains the integrity of SARS-CoV-2 RNA for up to 48 hours even at room temperature and supports delayed diagnosis with an overwhelming sample load in testing laboratories.

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

10.1101/2021.04.14.439928 ◽

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.

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

PLoS ONE ◽

10.1371/journal.pone.0249149 ◽

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.

Download Full-text